HEPATIC METABOLISM DURING SEPSIS Paulo Roberto Leitao de
Transcrição
HEPATIC METABOLISM DURING SEPSIS Paulo Roberto Leitao de
HEPATIC METABOLISM DURING SEPSIS A thesis presented in part fufilment of the the requirements for the degree of Doctor of Philosophy of the University of Oxford by Paulo Roberto Leitao de Vasconcelos Trinity Term 1987 Green College Oxford This thesis is dedicated to my wife Dione, to my daughter Raquel, to my sons Paulo Roberto and Marcelo, and to my parents Acknowledgements I am extremely grateful to both my biochemical and clinical supervisors. I will always be indebted to Dr. Williamson, who made possible the realization of the experimental work, when, in his kind way, he allowed me to work in the Metabolic Research Laboratory. I would consider myself very gifted if I could keep with me a tiny part of Dr Williamson's insight into scientific thinking. I am also very grateful for his constant encouragement, sense of humour, and skilled supervision, both at the bench throughout the project, and also during the period of writing. I am profoundly grateful to Mr. Kettlewell for all his guidance during the clinical studies. He accepted me to do research in Oxford and has always been a fund of encouragement, enthusiasm, and constructive ideas, as well as an example of a surgeon concerned with the day to day metabolic status of his patients. I also would like to thank him for having taught me how to clinically assess and parenterally feed a surgical patient in need, and to ask many questions about this unphysiological, but sometimes life-saving, form of nutrition. Mr. Kettlewell has always made sharp scientific criticisms during his supervision of the clinical and written work. I would like to thank Mrs. Vera Ilic for introducing me into the world of enzymatic analysis, and for doing so many insulin assays. I am grateful to Mr. Reginald Hems for teaching me the art of preparing isolated hepatocytes. I am also grateful to Dr. Geoffrey Gibbons for his supervision and assitance in the studies involving cholesterol metabolism. My thanks to Mrs. Claudia Oller do Nascimento who has very kindly performed the lipoprotein lipase assays. I am very grateful to Miss Jane Clarke and Mr. Mike Dixon for helping me with the clinical studies. Ms. Mellany Burnnet from the Diabetes Research Laboratory has kindly measured the plasma insulin from parenterally fed patients. I also would like to express my thanks to the consultant surgeons of the John Radcliffe Hospital who allowed me to study their patients, and mostly to the patients who agreed to be involved in the parenteral nutrition studies. I am sincerely indebted to C.A.P.E.S. (Brazil) for having granted me with their scholarship, and for giving support to this project. I would like to thank Dr. Alexandre Kalache and Dr. Haroldo Juaçaba, in Brazil, for all their encouragement and support about my decision to come to Oxford. I would like to acknowledge skillful computer assistance from Dr. Manoel Odorico Moraes Filho. I am very grateful to Dr. Richard Dawking for allowing me to use his laser printer. Finally, I would like to thank my wife Dione, who has been so supportive and understanding all over these four years, always encouraging me to persevere. HEPATIC METABOLISM DURING SEPSIS Thesis submitted to the University of Oxford for the Degree of Doctor of Philosophy Paulo R. L. de Vasconcelos Green College Trinity Term, 1987 Oxford Abstract Sepsis is an important clinical problem causing morbidity and mortality. Survival depends on the metabolic adaptation, which is part of a concerted physiological and immunological response. Time, nutritional status, the hormonal milieu, and the severity of infection modulate the metabolic response. The liver has a central role in intermediary metabolism during sepsis. The time-course (12, 24, and 48h) of changes in hepatic metabolism during sepsis was studied. A rat peritonitis model, which mimics human abdominal sepsis, was used. Moderate sepsis was induced by caecal-ligation-and- puncture. Blood and hepatic metabolite concentrations, and gluconeogenesis in isolatedhepatocytes were measured. Sepsis increased blood and liver gluconeogenic precursors, but caused no change in blood glucose or plasma non-esterified fatty-acids; blood and hepatic ketonebodies decreased. These changes were accompanied by increased plasma insulin in septic-rats. Gluconeogenesis from all precursors (alanine, lactate, pyruvate, glutamine, dihydroxyacetone) was decreased by sepsis (48h). Sepsis impaired ketogenesis from long-chain fatty-acids (oleate) in hepatocytes from septic rats at all time-points, whereas when short-chain fatty-acids (butyrate) were the precursors ketogenesis was unchanged. This may be due to decreased entry of long-chain acylCoA into the mitochondria in the liver of septic-rats. Sepsis stimulated in vivo hepatic lipogenesis (24, 48h) and cholesterogenesis (12, 24, 48h). The increased cholesterogenesis was accompanied by increase in total hydroxymethylglutaryl-CoA reductase activity, but no change in the active form of the enzyme was observed. The key factor responsible for these metabolic changes may be the elevated plasma insulin concentrations. Treatment of septic-rats, with mannoheptulose or streptozotocin, to lower their plasma insulin reversed many of the metabolic changes to the profile seen in control-rats, supporting the view that hyperinsulinaemia is an important regulatory-factor in hepatic metabolism during sepsis. The hepatic metabolic changes induced by sepsis were compared to those found in parenterally-fed patients. Many metabolic changes were common, and the hyperinsulinaemia seen in both sepsis and total-parenteral-nutrition was again implicated. TABLE OF CONTENTS CHAPTER 1 Page Introduction 01 Factors determining the development of infection 02 Factors increasing susceptibility to infection 04 The relation of infection and injury with malnutrition 05 and immunity Historical Aspects and the importance of infection 09 The metabolic effects of starvation and injury 12 General description of the metabolic response to sepsis 15 Multiple organ failure 19 Sepsis and endotoxin 22 The effect of gram-positive bacterial components during 24 infection The role of the phagocytic cells in infection 25 The role of the macrophage during sepsis 26 The role of interleukin-1 in the metabolic response to 30 sepsis The importance of cachectin during sepsis 32 The role of corticosteroids in the response to sepsis 34 The counter-regulatory hormones and the regulation of 37 the metabolic response to sepsis Sepsis and the effects of insulin 38 Biological response modifiers during sepsis 40 Objective of the study 45 TABLE OF CONTENTS CHAPTER 2 Materials and Methods Page Biochemicals and Enzymes 47 Animals 47 Sepsis induced by caecal-ligation and puncture 47 Sham-operated rats 49 Bile duct-ligated rats 50 Preparation of blood, plasma and tisue samples 51 Determination of metabolites 52 Calculations of blood and liver metabolites 66 Insulin determination 66 Measurements of lipogenesis and cholesterogenesis 67 Preparation of isolated hepatocytes 69 Incubation procedure 72 Preparation of microsomes 74 Determination of hepatic enzyme activity 74 Determination of lipoprotein lipase activity 78 Measurements of radioactivity 79 Statistical Analysis 80 TABLE OF CONTENTS Time course of changes in blood and liver metabolites and plasma insulin after induction of sepsis Page CHAPTER 3 Introduction 81 Changes in blood and plasma metabolites 81 Changes in metabolite concentrations in the liver 83 Aims of the study 84 Results 85 Time course of changes in the concentrations of blood glucose, 85 lactate, pyruvate and alanine Time course of changes in plasma insulin concentration 87 Time course of changes in plasma triacylglycerols and non- 88 esterified fatty acid concentrations Time course of changes in the concentrations of blood ketone 89 bodies Changes with time in the hepatic concentration of lactate, 90 pyruvate, alanine, glutamine and glutamate Time course of changes in hepatic concentrations of glucose 93 Changes in glycogen concentration in the liver after induction 93 of sepsis Time course of changes in the concentrations of hepatic 94 gluconeogenic intermediates in vivo Time course of changes in hepatic concentrations of ketone 96 bodies in vivo Time course of changes in hepatic concentrations of acetyl-CoA 97 and ATP in vivo Discussion 98 TABLE OF CONTENTS Time course of changes in rates of gluconeogenesis in isolated hepatocytes during the induction of sepsis CHAPTER 4 Page Introduction 102 Design of the study 105 Results 106 Discussion 112 TABLE OF CONTENTS Time course of changes in the metabolism of [1-14C]oleate and the rate of ketogenesis in isolated liver cells after induction of sepsis Page CHAPTER 5 Introduction 116 Metabolism of long- and short-chain fatty acids 116 Changes in plasma non-esterified fatty acids and blood 118 hepatic ketone body concentration during sepsis Design of the studies Results 120 121 Time course of rates of ketogenesis from long- and short- 121 chain fatty acids in isolated hepatocytes The metabolism of [1-14C]oleate during sepsis 123 Discussion 125 TABLE OF CONTENTS Time course of changes in rates of lipogenesis and hepatic cholesterogenesis in vivo after the induction of sepsis CHAPTER 6 Page Introduction 129 Experimental design 134 Results 135 Effects of sepsis on hepatic lipogenesis 135 Effects of sepsis on squalene and cholesterol synthesis 137 in vivo and hydroxymethylglutaryl-CoA reductase activity in the liver Effects of sepsis on lipoprotein lipase activity and rates of 140 lipogenesis in vivo in adipose tissue The effect of the complete interruption of the entero- 142 hepatic circulation of bile acids accompainied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver in vivo Discussion 143 TABLE OF CONTENTS The effects of modulation of plasma insulin on hepatic metabolism during the induction of sepsis CHAPTER 7 Page Introduction 147 Experimental design 150 Results 151 The effects of mannoheptulose or streptozotocin treatment 151 on changes in concentrations of blood glucose, lactate, pyruvate and alanine during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 154 on the concentrations of blood ketone bodies during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 155 on plasma insulin concentration during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 156 on hepatic concentration of lactate, pyruvate, alanine and glucose during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 158 on the hepatic concentraton of metabolites involved in gluconeogenesis during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 163 on the changes in hepatic concentrations of ketone bodies, acetyl-CoA and ATP during the induction of sepsis The effects of mannoheptulose or streptozotocin treatment 165 on the changes in rates of lipogenesis and cholesterogenesis in vivo during the induction of sepsis The effects of mannoheptulose treatment on the metabolic 169 changes of sham-operated rats The effects of insulin replacement, given to septic rats treated 172 with mannoheptulose, on the metabolic changes induced by sepsis Discussion 174 TABLE OF CONTENTS General discussion CHAPTER 8 Page General discussion 181 The regulation of the metabolic response to sepsis 182 Is the metabolic response to sepsis adaptive? 184 Manipulation of the metabolic response to sepsis 185 TABLE OF CONTENTS Common features of the changes in hepatic metabolism induced by sepsis and by total parenteral nutrition CHAPTER 9 Page Introduction 189 Cyclical Parenteral Nutrition 192 Ezperimental Design 197 Study on liver dysfunction developing in parenterally fed 197 patients during sepsis Randomized controlled study on standard versus tailored total 199 parenteral nutrition (TPN) Controlled study on continuous versus cyclical total parenteral 203 nutrition (TPN) Statistical analysis 206 Results 207 The effects of sepsis on liver function tests in patients 207 receiving total parenteral nutrition (TPN) Tailored glucose versus standard glucose TPN regimen 210 Standard versus tailored TPN - Effect on nitrogen balance 212 Continuous versus cyclical TPN - Changes in blood metabolites 213 and plasma insulin over 24 h Changes in liver function during standard versus tailored total parenteral nutrition (TPN) 222 Changes in liver function during continuous versus cyclical 224 total parenteral nutrition (TPN) Discussion 225 TABLE OF CONTENTS Page References 232 Publications arising from this Thesis 267 LIST OF FIGURES Page Fig. 1.1. Changes in the determinats of sepsis 03 Fig. 1.2. The relationship between sepsis/injury and malnutrition 06 Fig. 1.3. Summary of the metabolic changes in starvation and injury 12 Fig. 1.4. Phases of the metabolic response to injury/sepsis 14 Fig. 1.5. Metabolic changes in response to sepsis 18 Fig. 1.6. Some clinical aspects of sepsis and endotoxin treament 23 Fig. 1.7. Role of the macrophage in initiating the immunological 28 response to sepsis Fig. 2.1. Caecal-ligation 48 Fig. 2.2. Caecal-ligation and puncture 48 Fig. 2.3. Sham-operation 50 Fig. 2.4. Liver perfusion apparatus for hepatocyte isolation 71 Fig. 3.1. Blood glucose 85 Fig. 3.2. Blood lactate 86 Fig. 3.3. Blood pyruvate 86 Fig. 3.4. Blood alanine 87 Fig. 3.5. Insulin concentration 88 Fig. 3.6. Plasma triacylglycerol concentration 88 Fig. 3.7. Non-esterified fatty acids 89 Fig. 3.8. Blood ketone bodies 89 Fig. 3.9. Liver lactate 90 Fig. 3.10. Liver pyruvate 91 Fig. 3.11. Liver alanine 91 Fig. 3.12. Liver glutamine concentration 92 Fig. 3.13. Hepatic glutamate concentration 92 Fig. 3.14. Liver glucose 93 Fig. 3.15. Metabolites involved in hepatic gluconeogenesis (24h) 95 Fig. 3.16. Metabolites involved in hepatic gluconeogenesis (48h) 95 Fig. 3.17. Liver ketone bodies 96 Fig. 3.18. Acetyl-CoA concentration in the liver 97 Fig. 3.19. ATP concentration in the liver 97 Fig. 4.1. Gluconeogenesis and glycolysis 103 Fig. 4.2. Design of the study (Chapter 4) 105 Fig. 4.3. Rate of gluconeogenesis - no substrate 106 Fig. 4.4 Rate of gluconeogenesis from lactate [5mM] 107 LIST OF FIGURES Page Fig. 4.5. Rates of gluconeogenesis from lactate 108 Fig. 4.6. Rate of gluconeogenesis from alanine [5mM] 108 Fig. 4.7. Rate of gluconeogenesis from pyruvate [5mM] 109 Fig. 4.8. Rate of gluconeogenesis from glutamine [5mM] 110 Fig. 4.9. Rate of gluconeogenesis from dihydroxyacetone [5mM] 111 Fig. 5.1. Simplified scheme on the hepatic metabolism of long- and 117 shor-chain fatty acids Fig. 5.2. Design of the studies (Chapter 5) 120 Fig. 5.3. Rate of ketone body formation - no added substrate 121 Fig. 5.4. Rate of ketogenesis from butyrate [5mM] 122 Fig. 5.5 Rate of ketogenesis from oleate [2mM] 122 Fig. 5.6. Metabolism of [1-14C]oleate 124 Fig. 6.1. Simplified scheme of lipid metabolism in the liver 131 Fig. 6.2. 134 Fig. 6.7. Experimental design (Chapter 6) Rate of 3H2O incorporation into fatty acids Rate of 3H2O incorporation into non-saponifiable lipids Rate of 3H2O incorporation into lipids Rate of 3H2O incorporation into squalene Rate of 3H2O incorporation into cholesterol Fig. 6.9. HMG-CoA reductase activity 139 Fig. 6.10. Rate of lipogenesis in white adipose tissue 140 Fig. 6.11. Rate of lipogenesis in brown adipose tissue 141 Fig. 6.12. Hepatic cholesterol synthesis and bile duct ligation 142 Fig. 7.1. D-mannoheptulose 149 Fig. 6.3. Fig. 6.4. Fig. 6.5. Fig. 6.6. 135 136 136 137 138 Fig. 7.2. Experimental design (Chapter 7) 150 Fig. 7.3. Blood glucose concentration 151 Fig. 7.4. Blood lactate concentration 152 Fig. 7.5. Blood pyruvate concentration 153 Fig. 7.6. Blood alanine concentration 153 Fig. 7.7. Blood concentration of ketone bodies 154 Fig. 7.8. Plasma insulin concentration 155 Fig. 7.9. Hepatic lactate concentration 156 Fig. 7.10. Hepatic pyruvate concentration 157 Fig. 7.11. Hepatic alanine concentration 157 Fig. 7.12. Hepatic glucose concentration 158 Fig. 7.13. Metabolites involved in gluconeogenesis 160 LIST OF FIGURES Page Fig. 7.14. Metabolites involved in gluconeogenesis 161 Fig. 7.15. Metabolites involved in gluconeogenesis 162 Fig. 7.16. Hepatic concentration of ketone bodies 163 Fig. 7.17. Hepatic concentration of acetyl-CoA 164 Fig. 7.18. Fig. 7.19. 164 165 Fig. 7.22. Hepatic concentration of ATP Rate of 3H2O incorporation into fatty acids Rate of 3H2O incorporation into non-saponifiable lipids Rate of 3H2O incorporation into squalene Rate of 3H2O incorporation into cholesterol 167 Fig. 7.23. Fig. 7.24. HMG-CoA reductase activity Rate of 3H2O incorporation into fatty acids 168 171 Fig. 7.25. Rate of 3H2O incorporation into cholesterol 171 Fig. 7.26. Metabolic changes in septic rats after treatment to lower 174 Fig. 7.20. Fig. 7.21. 166 167 plasma insulin Fig. 7.27. Possible effects of sepsis and treatment to lower plasma insulin on the hepatic glucose 6-phosphate/glucose cycle 177 Fig. 8.1. Possible changes in the metabolic response to sepsis 182 determined in part by the severity of infection Fig. 8.2. Possible cascade of events leading to the hepatic metabolic 186 response to sepsis Fig. 9.1. Some common metabolic features shared by sepsis and TPN 189 Fig. 9.2. Criteria for selection of patients to receive cyclical TPN 193 Fig. 9.4. Principal indications for home TPN 194 Fig. 9.5. Continuous versus Cyclical TPN - Study design 203 Fig. 9.6. Patients with abnormal albumin during sepsis and TPN 207 Fig. 9.7. Patients with abnormal bilirubin during sepsis and TPN Fig. 9.8. Patients with abnormal alkaline phosphatase during 208 sepsis and TPN Fig. 9.9. Patients with abnormal AST during sepsis and TPN 209 Fig. 9.10. Standard versus tailored TPN - Blood metabolites 211 Fig. 9.11. Standard versus tailored TPN - Blood metabolites 211 Fig. 9.12. Standard versus tailored TPN - Blood ketone bodies 212 Fig. 9.13. Tailored versus standard TPN - Effect on nitrogen balance 213 Fig. 9.14. Blood glucose 213 Fig. 9.15. Blood pyruvate 214 LIST OF FIGURES Page Fig. 9.16. Blood glutamate 215 Fig. 9.17. Blood lactate 215 Fig. 9.18. Blood alanine 216 Fig. 9.19. Plasma insulin 216 Fig. 9.20. Blood ketone bodies 217 Fig. 9.21. Effects of cyclical TPN on blood pyruvate 218 Fig. 9.22. Effects of cyclical TPN on blood glutamate 218 Fig. 9.23. Effects of cyclical TPN on blood lactate 219 Fig. 9.24. Effects of cyclical TPN on blood alanine 219 Fig. 9.25. Effects of cyclical TPN on blood glucose 220 Fig. 9.26. Effects of cyclical TPN on plasma insulin 220 Fig. 9.27. Effects of cyclical TPN on blood ketone bodies 221 Fig. 9.28. Continuous versus cyclical TPN - Effect on nitrogen balance 221 Fig. 9.29. Changes with time in albumin during TPN 222 Fig. 9.30. Changes with time in bilirubin during TPN 222 Fig. 9.31. Changes with time in alkaline phosphatase during TPN 223 Fig. 9.32. Changes with time in aspartate transaminase during TPN 223 List of Tables Page Table 3.1. Time course of changes in the concentration of hepatic 94 gluconeogenic intermediated in vivo Table 5.1. Effects of sepsis on metabolism of [1-14C]oleate in isolated 124 hepatocytes from 48 h starved rats Table 7.1. The effects of mannoheptulose or streptozotocin treatment 159 on the hepatic concentration of metabolites involved in gluconeogenesis after the induction of sepsis invivo Table 7.2. The effects of mannoheptulose treatment on the metabolic 170 changes of sham-operated rats in vivo Table 7.3. Th e effects of insulin administration on the metabolic 173 changes of septic rats treated with mannoheptulose in vivo at 48 h after the operation Table 9.1. Patients fed on tailored or standard TPN regimen 200 Table 9.2. Main indication for TPN 200 Table 9.3. The Harris-Benedict equation 201 Table 9.4. Patients who received cyclical parenteral nutrition 201 Table 9.5. Nitrogen and energy intake 210 Table 9.6. Nitrogen and energy according to the body weight 210 Table 9.7. Tailored versus Standard TPN - Effects on liver function 224 tests Table 9.8. Continuous versus cyclical TPN - Effects on liver function Table 7.9. A comparison between blood metabolite values obtained during the 'post-absorptive' cyclical TPN ("OFF" or infusion free period) and those found in normal controls after an overnight fast 224 CHAPTER 1 INTRODUCTION FACTORS DETERMINING SUSCEPTIBILITY TO INFECTION Enviromental and genetic factors play and important role in determining susceptibility to infection, as well as, its severity. They do so by interfering in the immunological defence action and the overall metabolic response. Age: Age influences not only susceptibility to infection, but also its clinical course. Many infectious diseases show a characteristic age distritubion. Infections with ubiquitous organisms are seen firstly in infants, shortly after they have lost the maternal antibody transfered across the placenta, and have a peak incidence in early childhood. Maturation of the immunological system, associated with gradual acquisition of protective immunity, due to successive exposures to infection, appear to promote a more adapted response to infectious threats in adulthood. With ageing immunity declines increasing susceptibility to infection again. Sex and hormonal factors : Not only hormonal but social factors are implicated in the variation in the sex distribution of infectious diseases. Sex has a role in defining occupation and, therefore, can influence how close men and women come into contact with the source of infection. Pregnancy can predispose to various infections, including hepatitis, pneumoccocal infections, amaebiasis and malaria, possibly reflecting changes in immunological function and in metabolism that occur at that time (Greenwood , 1983). The presence of conditions leading to excess glucocorticosteroids, such as adrenal hyperplasia, adrenal tumors, or steroid therapy, may increase susceptibility to infections, e.g., infections with herpes simplex and herpes zoster. Corticosteroids probably exert their action on the immune and metabolic response ( ). Patients with diabetes, especially if the lattter in not very well controlled, show increased susceptibility to infections, with the common development of abscesses, boils and urinary tract infections. Diabetic patients also present with impaired cellmediated immune reactions and chemotaxis by polymorphonuclear lymphocytes ( ). Immunity, Injury and malnutrition : A number of clinical and experimental studies have shown that defficiency of protein, of individual vitamins, and of trace elements increase susceptibility to infection ( ). In developing countries, most attention ought to be paid to children with severe protein-calorie malnutrition, as these children frequently die from infection ( ). The clear association of nutrition and infection could recently be seen during the drought in Ethiopia and other African countries that led to wide-spread profound malnutrition as the predisposing factor in the death by infectious disease of tens of thousands of people ( ). In indrustrialized countries, severe malnutrition is encounted as a complication of conditions, such as generalized malignant disease, cirrhosis, and intestinal malabsortion due to inflammatory bowel disease, more frequently than as a result of pure dietary deficiency. However, considerable attention has been given to the fact that malnutrition is also widely prevalent in surgical patients, and in close association with mobidity and even mortality (Bistrian et al , 1974; Hill et al , 1977 and 1979). Today, careful assessment of the nutritional status, associated with administration of nutritional support to the malnourished hospitalized patient is well incorporated into modern clinical care practice (Elwyn, 1980; Kettlewell, 1982; Winters & Greene, 1983; Silk, 1983; Phillips & Odgers, 1986). Drugs : The problem of infection in patients receiving cytotoxic or immuno supressive drugs is well recognized ( ). Alcohol in excess increases susceptibility to many infections, especially when liver failure supervenes ( ) . The neglect of simple hygienic precautions when administering their intravenous injections in hard-line drug addicts leads to septicaemia as a common terminal event, or alternatively, to the contamination with the Acquired Immunodeficiency Syndrome virus ( ) . Smoking also predisposes to respiratory infection due to the damage of the epitheleum of the respiratory tract ( ) . Malignant disease : Immune responsiveness is impaired in patients with dissiminated malignant disease ( ) . The frequent association of cancer and malnutrition, added to the harmful effects of radiotherapy or treatment with cytotoxic drugs further increases the risk of developing infection in these patients. Whether the provision of nutritional support, with the aim to correct malnutrition, will have a beneficial effect in diminishing such risk is still debatable (Dudrick ? ; Copeland, 1986; ). OBJECTIVE OF THE STUDY The aim of the work described in this thesis was to study the important changes in hepatic metabolism induced by sepsis. Clinical studies of sepsis are difficult to perform, not only due to the usual restrictions on the actual scope of clinical studies, based on ethical grounds, but because of the difficulty in establishing comparable groups of patients. There is usually variability on the severity and type of the septic insult, which is often associated with variable degrees of trauma, the latter being commonly accompanied by different nutritional and hormonal status. Furthermore, it is also not unusual to find variation on the time the observations are made; and time appears to have a very important role on the intensity and nature of the changes brought about by sepsis. Interpretation of the results under these circunstances may, therefore, be very difficult, for they may reflect, in part, the effects of the different conditions of experimentation. In view of the difficulties with patients, we decided to study the time course of the effects of an experimental model of peritonitis on the hepatic metabolism of the rat. The particular septic model used has proved to be very reproducible and clinically sound (Wichterman et al , 1980). In addition, there appears to be no information on the time course of the changes in hepatic metabolism in the same experimental model of sepsis. The results of the present work may, therefore, help to resolve some of the apparent discrepancies about hepatic metabolism during sepsis, found in the review publications in the literature, in both experimental and clinical sepsis (Blackburn, 1977; Beisel & Wannemacher, 1980; Cerra, 1982; Neufeld et al , 1982; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986), as illustrated by Fig. 1.1. Fig. 1.1. The general term 'sepsis' has caused considerable confusion in clinical and experimental studies (Wichterman et al , 1980) because it refers to different conditions in different studies. Some experimental septic models induce a low-grade smoldering infection which can last for days or weeks, whereas other models describe a very aggressive process that produces death whithin hours. For the purpose of the work described in this thesis, 'sepsis' is regarded as an acute invasive infection in an animal, which causes the development of a toxic state (weakness, anorexia, pile erection, lethargia, etc.), leading to the eventual development of circulatory colapse - septic shock. A wide range of microbial organisms can cause sepsis, especially gram-positive and gram-negative bacteria. While the term 'bacteraemia' refers to bacteria in the blood, irrespective of whether they do or do not induce the toxic state of sepsis, 'septicaemia' refers to a toxic state produced by an acute infectious process in the blood. It must be emphasized, however, that animals dying of infection, with circulatory colapse, with established septic shock were excluded from the present studies. THE IMPORTANCE OF INFECTION Infection was the major factor in worldwide population control for mankind until only recently. Before the 17th century, epidemics presumably of smallpox, plague diphtheria, measles, cholera and infectious diarrheal diseases, were common episodic events causing death of more than 50% and sometimes as much as 90% within a region (McNeil, 1977). Better hygiene and improved nutrition increased the average life expectancy, and contributed to the five-fold increase in the world population over the last 150 years (Alexander, 1986). Over this period, the explosion in scientific methods, associated with the constant search for controlling and understanding the mechanisms of infection, have had a tremendous effect on diseases. Smallpox, one of the most common causes of death in the world a few hundred years ago, is now extinct (Barnes & Robertson, 1981), whereas e.g. to achieve the control and eventually the cure for the Acquired Immuno Defficiency Syndrome, caused by an infection with the human imno-deficiency virus, poses itselsf as a difficult challenge to the scientific community today (Anon, 1986). Infection may be part of a primary disease process, for example pneumonia, or may occur as a complication of surgery, such as a pelvic abscess following abdominal surgery. However, until the 19th century, the formation of pus was considered a normal part of healing - pus bonum et laudbile . John Hunter [1728-1793] recorded in his 'Treatise on the Blood, Inflamation and Gun Shot Wounds', published in 1794, that 'inflamation is not only the cause of diseases but it is often the mode of cure'. Louis Pasteur [1822-1825] caused a dramatic change the surgical opinion and atitude by showing that fermentation was caused by living multiplying matter and, therefore, that pus formation and wound infection had to be also caused by minute organisms from the enviroment. Joseph Lister [1827-1912] became aware of Pasteur's research and by the use of carbolic established the principles of antisepsis. This was a major revolution in surgery which progressed to the use of aseptic surgical technique and led to a great reduction in surgical morbidity and mortality. Since Thomas Latta (1832) first reported in the Lancet the prevention of death from cholera by the intravenous injection of 6 pints of almost normal saline containing 3 drachms of salt and 2 scruples of sodium bicarbonate, or about 2.8 litres of 89 mM Na+, 78 mM Cl-, and 11 mM bicarbonate, the use of fluid and electrolyte therapy has become such a familiar part of medicine that it is rarely considered today (Veech R L, 1986). The successiful clinical use of antibiotics, described for the first time by the Oxford group (Abraham et al , 1941) was a gigantic step towards the fight against infection. However, despite the introduction of asepsis and antisepsis, fluid and electrolyte therapy, specific antibiotics, careful monitoring, aggressive operative intervention and intravenous nutritional support, infection still remains a very important clinical problem. Studies have indicated that about one third of surgical patients suffer from infection at some stage of their stay in hospital (Altemier et al, 1976; Green et al , 1977; Cruse & Foord, 1980). Surgical sepsis plays an important role in the morbidity and mortality encountered in an intensive care unit and constitutes a major impediment in the resolution of critical ilness, with septic patients presenting a three-fold mortality rate as compared with non-septic patients (McLean & Boulanger, 1985). The increasing use of immunosuppressive drugs and corticosteroids enhances susceptibility to infection. Despite severely injured patients, transplant patients, patients with cancer and diabetes melitus are surviving longer now, these patients have an increased risk of infection. Invasive techniques such as indwelling intravenous catheters, especially long-term catheters used for intravenous feeding, and bladder catheters also increase the risk of sepsis. The magnitude of the stress imposed by sepsis is a function of the size of the infectious process, the number of invading organisms and their virulence. Survival depends not only on the adequacy of the immunological reactions to contain and eliminate invading microbes, but also on a series of integrated physiological and metabolic responses necessary to maintain energy production and cells function thoughout the body (Clowes et al, 1985). It is encouraging, however, that the complex pathophysiology of bacterial sepsis is gradually becoming better understood. The mechanisms of how sepsis becomes a frequent cause of ventilatory or renal failure , or multiple organ failure, on the other hand, are yet to be completely clarified. GENERAL DESCRIPTION OF THE METABOLIC RESPONSE TO SEPSIS The course of the metabolic response to trauma, as ilustrated by Fig. 1.2., was first described by Sir David Curthbertson (1930) more than 50 years ago. These changes will be discussed briefly as they are similar to those found in the response to sepsis (For review see: Silk, 1983; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986). Fig. 1.2. The ebb phase This phase is characterized by a rapid fuel mobilization, haemodynamic instability and increased plasma catecolamine concentrations (Coward et al , 1966). Insulin plasma concentration is variable (Frayn, 1986) despite a possible inhibition of its release by the action of the higher catecolamines on the pancreatic ß-cell (Porte & Robertson, 1973). Glucocorticoids and glucagon are also reported to be elevated (Forse & Kinney, 1985). Glycogen is mobilised for immediate energy production but the metabolic rate falls (O'Donnell, 1976). Although there is some argument about the need, and how adaptive such a massive fuel mobilization is (Frayn, 1986), maintenance of volaemia and tissue perfusion appear to be aim during this phase. It is during the ebb phase, or when conditions similar to those of the ebb phase are evoked, that septic shock may develop. The shock of the ebb phase of injury/trauma may be different from that of sepsis. In the former, there is decreased energy production associated with hypovolaemia, whereas in the ebb phase of sepsis, the 'septic shock' may present in a hyperdynamic state, with increased energy production, hyperventilation, increased central venous pressure, increased cardiac output, oliguria, decreased peripheral resistance, hypotension and lactic acidosis (Siegel et al , 1967; Forse & Kinney, 1985). This hyperdynamic septic state then evolves to the hypodynamic septic state with low central venous pressure, hypotention, low cardiac output and increased peripheral resistance (MacLean, 1981; Forse & Kinney, 1985). This suppports the view of a bimodal response to severe acute sepsis, with an initial hypermetabolic hyperdynamic period followed by a hypometabolic hypodynamic late septic state (Chaudry et al , 1979; Wichterman et al , 1980). The flow phase If death does not occur as a result of hypovolaemia or direct damage to vital organs during the ebb phase, a more prolonged period follows (the flow phase), which is characterized by an increase in the metabolic rate, breakdown of body tissue and weight loss. Increased plasma concentrations of insulin and glucagon have been reported during this phase (Biesel & Wannemacher, 1980; Neufeld et al , 1982). Generally this phase of the metabolic response can be regarded as a balance between the action of insulin and those of the counterregulatory hormones, namely, glucocorticoids, glucagon and catecholamines (Forse & Kinney, 1985). Another characteristic feature of this phase is the presence of hypermetabolism (Cuthbertson, 1979), which appears to be proportional to the degree of stress (Elwyn et al , 1981) or the severity of infection. So major infections such as peritonitis are associated with increases of up to 60% above the resting energy expenditure (Elwyn, 1980). The aim of this hypermetabolic catabolic phase appears to be to maintain energy production and cell function thoughout the body, at a time dietary intake may be scarce and/or not absorbable. If this is acheived and the immunological and metabolic responses take a successiful course, the changes leading to net catabolism are gradually replaced by those leading to net anabolism, merging into the 'anabolic' or 'convalescent' phase. The anabolic phase The transition from the catabolic flow phase to the anabolic period of convalesce can occur quickly, within a few days, as with soft tissue injury or can occur over several days as with extensive trauma (Moore & Brennan, 1975). The distinct metabolic changes that occur during anabolism aim to replace the tissue lost over the catabolic phase, whereas resting energy expenditure may fall from the elevated levels of the flow phase to subnormal levels, similar to those found in depleted patients (Moore & Brennan, 1975; Forse & Kinney, 1985). However, while the pattern of the response to injury shows a defined time-sequence, from the trauma itself until recovery, the course of the response to sepsis is not so predictable, because of fluctuations in the nature and intensity of the metabolic changes, which are linked with the severity, the type of infection, and variation in the nutritional and hormonal state. Take, e.g., a patient with intra-abdominal sepsis proceeding well during the flow phase. If part of the avascular fibrinous wall of a previously isolated-off abscess is eroded bacteraemia can develop, which in turn, will precipitate events of the ebb phase leading or not to the establishment of a 'septic shock' state. SEPSIS AND ENDOTOXAEMIA Endotoxin is a lipoprotein-carbohydrate complex (LPS) found in the cell wall of all gram-negative bacteria. Andre Boivin (1941) was the first to isolate endotoxins from gram-negative bacteria, and some ten years latter endotoxins were implicated as the cause of human gram-negative shock (Borden & Hall, 1951; Braude, 1953). This hypothesis has led to numerous studies on the physiological and biochemical alterations associated with endotoxin inocculation into various laboratory animals over the last three decades. Furthermore, some of the results of such studies are usually incorporated into the descriptions of the pathophysiology of clinical sepsis and septic shock, and in their treatement (Wichterman et al , 1980). Some of the clinical aspects that result from gram-negative sepsis and endotoxin desagree (Fig. 1.3.), as described by Wichterman et al (1980). Nevertheless, both endotoxin and sepsis can activate the macrophages to produce interleukin-1 and/or its by products, which appear to be important mediators of the metabolic changes brought about by sepsis and/or endotoxin (Fig. 1.4. - Cohn, 1978; Clowes et al , 1983), and can cause similar hormonal and metabolic changes, such as, hyperinsulinaemia and hyperglucagonaemia (Yelich & Filkins, 1980; Beisel & Wannemacher, 1980), and the inhibition of the hyperketonaemia in response to starvation (Neufeld et al , 1982). The development of multiple organ failure syndrome also appears to follow both endotoxaemia (Tracey et al , 1986) and sepsis (Duff, 1985). Therefore, data concerning endotoxaemia, relevant, will be included in this manuscript. Fig. 1.3. whenever considered On one hand, there appears to be no evidence that endotoxin has a direct active role in human shock (Baue et al , 1969). In addition, very few septic patients have endotoxin identified in their blood, and if so, at concentrations which are quite low (Levin et al , 1972; Postel et al , 1975). On the other hand, recent studies support the view endotoxin acts in an indirect way via stimulation of polymorphonuclear leucocytes (Tracey et al , 1986; Beutler & Cerami, 1987; Christou et al , 1987), which may help to explain some of the differences in the clinical findings. MULTIPLE ORGAN FAILURE The failure of vital organs in the septic and/or injured patient has received the attention of a number of recent studies (for review see: Polk, 1982; Duff, 1985). It is clear now that multiple organs are often involved, usually remote from the site of infection. In addition, the clinical manifestations of their involvement show a sequential pattern. Failure of the renal, respiratory and cardiovascular systems often occur first, and, if this is not followed by recovery, there is failure of the brain, liver and gut (Duff, 1985). The lungs The earliest recognized form of organ failure was that involving the lung. Adult respiratory distress syndrome (ARDS), 'shock lung', 'Viet Nam lung' and 'lung lesion of sepsis' all describe a type of respiratory failure, with each name being related with their initiating event: shock, trauma and/or sepsis (Polk, 1982). The predominat characteristic of adult respiratory distress syndrome appears to be the associataion with infection ( Horovitz & Shires, 1974; Eiseman & Narton, 1975; Fulton & Jones, 1975). Fulton & Jones (1975) showed that 85% of their patients had clinically identifiable sepsis, before or along with the establishment of pulmonary dysfunction. The pathophysiology of lung failure is not clear, but there is some evidence from clinical and experimental studies that the development of the pulmonary oedema is a result of increased permeability of pulmonary capilaries. Increased capillary parmeability has been reported in sheep injected with endotoxin or bacteria, as judged by the increase in the rate of appearance and the concentration of protein in the lung limph (Brigham et al , 1979). Adult patients with ARDS showed increased rate of incorporation of blood radiolabelled albumin into the broncheoalveolar fluid (Sibbald et al, 1979). The mechanism of cappilary injury is not fully explained, but the effects of complement activation may play an important role (Creddock et al, 1977; Jacob et al , 1980). This endothelial cell injury could also be the result of the action of the polymorphonuclear leucocytes (or their products) which are konwn to be aggregated to activated complement. To support this view there is evidence that drug-induced neutropoenia can diminish or prevent the effects of infusion of activated complement (Craccock et al, 1977). Other causative factors are also likely to be involved, as experimental lung lesions caused by infusion of complement are never as extensive as those observed in septic patients (Hyers, 1981). Using a radioaerosol lung scanning technique, Tennemberg et al (1987) studied patients with trama and sepsis, and reached the conclusion that complement-mediated neutrophil activation alone appeared to be unable of initiating lung injury or precipitating ARDS. Complement-mediated neutrophil activation may, therefore, serve as an early, potentiating mechanism for other polymorphonuclear triggering mediators. It is also well known that the lungs, in addition to their gas exchange and filter function, and the fact they are target-organs of circulatory factors, they clear many substances from the circulation such as 5hydroxytryptamine. The lungs also synthesise and release various agents, that may act on smooth muscle and, therefore, interfere with the function of other organs; the lungs being thus regarded in a way analogous to an "endocrine gland" (Aun & Birolini, 1985). The kidneys The pathophysiology of the injuries leading to renal failure is not fully understood. Acute tubular necrosis may or may not be present. Decreased renal blood flow, endotoxin action and vasocontriction of renal arteries have been implicated as possible mechanisms (Duff, 1985). Fortunately renal failure can be treated with success with haemodialysis until recovery of function returns, but the latter is usually linked to the recovery of function of other organs, especially the liver. The liver Severe sepsis causes liver damage and liver function is impaired in the multiorgan failure syndrome. Liver function tests show a pattern suggestive of cholestasis with a rise in serum bilirubin and alkaline phosphatase, accompanied by moderate elevation of SGPO and SGPT (Royle et al , 1978). Jaundice is the clinical sign which reflects such changes, and appears at a later stage, as compared to the biochemical changes. Changes in blood amino acid concentrations include elevations of phenylalanine, taurine, cystine and methionine, which are metabolized in the liver (Freund et al , 1978). Macroscopic changes in livers of monkeys, injected with live E. coli bacteria, include intravascular sequestration of degranulating leucocytes and fibrinous deposits of platelet agregates in synusoids and the spaces of Disse (Balis et al , 1979). The post-morten of patients who died with multi-organ failure reveals hapatic changes such as, focal necrosis, fatty accumulation , congestion and enlargement of synusoids, and cholestasis (Champion, 1976). Gastrointestinal system The gastrointestinal (G.I.) system may fail due to ulceration, loss of barrier function or malabsorption. In patients dying from multi-organ failure, the post-morten shows massive denudation of the gastrointestinal tract mucosa, which suggests a failure in the gastrointestinal barrirer function, enhancing abnormal absorption of bacteria and/or bacterial products (toxins) (Duff, 1978). Pathophysiological mechanisms of multiorgan failure syndrome A common mechanism used to explain the development of multiorgan failure is found on the action of free radical (O2-, OH-, H2O2). As a result of sepsis, or sepsis associated with trauma (usually represented by the operation to treat localized sepsis), there is activation of the complement cascade through the alternative pathway, and release of factor C5a (a polypeptide of 74 residues and a molecular weight of 15,000). This peptide would interact with circulating neutrophils to increase their adherence and probably to degranulate when exposed to suitable stimuli (Simmons & Solomkin, 1985). The agregation of granulocytes would, in turn, release free radicals in ammounts sufficient to cause cellular damage, dysfunction and eventualy organ failure (Duff, 1985). Recent evidence, however, appears to indicate a very important role of the macrophage in the response to sepsis. Activated macrophages by bacteria or bacterial products, e.g. endotoxin [lipopolysaccharide (LPS)], can release a polypeptide hormone (cachetin) which might initiate tissue injuries, the latter being not different from those caused by endotoxin inoculation (Tracey et al , 1986; Beutler et al, 1987). THE ROLE OF PHAGOCYTIC CELLS Phagocytic cells, including neutrophils, monocytes and macrophages, are responsible for the process of phagocytosis. At the begining of this century Metchnidoff (1905) described phagocytosis, namely as the ingestion and subsequent killing of micro-organisms (endocytosis), and believed this was a very important process in both the prevention and fight against infection. Latter on, however, it became clear that an important contribution of phagocytes to the inflamatory response is the exocytosis of enzymes and other proteins, some of them possibly carriers of important signals for the development of this response (inflammatory or humoral mediators). Exocytosis, on the other hand, can occur independetly from endocytosis, in response to different stimuli from those of the latter (Bentwood & Henson, 1980). These proteins released by the inflammatory cells are capable of various actions, such as making accessible the site of inflammation from the adjunct vascular space (e.g. collagenase and elastase); regulating local inflammatory mediators (e.g. carboxypeptidase); controlling the concentration of metabolites involved in bacterial growth (e.g. lactoferrin and vitamin B12); and enhancing an extracelluar environment adverse to micro-organisms through the action of acid hydrolases, cationic proteins and lysozymes (Simmons & Solomkin, 1985). THE MACROPHAGE Macrophages are distributed throughout the body and have developed specific characteristics in various organs, from which they acquired their respective names (Kuppfer's cells within the liver,alveolar macrophages in the lung, messengial cells in the kidney, and glial cells (which are thought to be derived from macrophages) in the brain. It appears to be clear that macrophages have adapted for local defence purposes, and in addition, macrophage function can also vary at different sites (For reviwe sse: Takemura & Werb, 1984). The three primary functions of macrophaghes, as described by Cristou et al (1987), are listed bellow: 1. Immune function Antigen presentation 2. Phagocytic-microbicidal function Phagocytosis Destruction of viral infected cells Oxidative killing mechanisms Chemotactic factors Lysozyme relase Destruction of intracellular parasites 3. Secretory function Proteases Procoagulant activity Interleukin 1 (IL-1) Colony stimulating factor Angiogenesis factor Plasminogen activating factor Prosaglandins/leukotrienes Interferon Tumor necrosis factor (TNF)/cachetin "Hepatocyte modulating factor(s)" The differences in macrophage function could also be understood in terms of degree of macrophage activation, namely, how 'stimulated' the macrophage is. A spectrum of stimulation can be recognized depending on the potency of the stimulating agent. Thus, macrophages which are stimulated with e.g. interferon or complement are reffered to as "inflammatory" or 'elicited" macrophages, whereas macrophages stimulated, for example, by endotoxin (Lipopolysaccharide - LPS) are fully "activated" macrophages (Cohn, 1978). The activation of macrophages results in enhanced phagocytosis (Cohn, 1978), increased production of toxic oxygen radicals (Tsunawaki et al , 1974) and the release of various proteases and hydrolytic enzymes (Takomura & Werb, 1984). There is also release (Fig. 1.4.) of various monokines (Takamura & Werb, 1984; Dinarello et al, 1986; Aggarwal et al , 1985; Beutler et al , 1986; Kovaks et al , 1985; ). Fig. 1.4. Release of oxygen radicals by activated macrophages (Babion et al, 1973; Craddock et al, 1977) has also been reported in sepsis. Interleukin 1 can be produced by virtually all activated macrophages (Dinarello & Mier, 1986) and has been implicated as an important mediator in the hypercatabolism of proteins in response to sepsis (Baraco et al, 1983; Clowes et al, 1983 and 1985). Macrophages treated with endotoxin (LPS) in vitro show altered secretion of various proteins (Largen et al, 1986). An important monokine released by endotoxin stimulation of macrophages is the Tumor Necrosis Factor (cachectin), which appears to be an important humoral mediator of the systemic metabolic changes during sepsis (Butler et al, 1985; Cerami et al, 1986). Studies using co-culture of hepatocytes with Kupffer's cells or macrophages, in the presence or absence of inflammatory stimulation, showed that there was a significant depression of hepatocytes protein synthesis when endotoxin (LPS) was added to the co-culture medium. However, in the abscence of either macrophages or Kupffer's cells, endotoxin had no direct effect on hepatocytes protein synthesis (West et al, 1985 and 1985). More recent evidence suggests the macrophagemediated inhibition of protein synthesis in isolated liver cells to occur via a monokine mediator yet to be identified, but not Interleukin 1 or cachectin (Christou et al ,1987). The importance of macrophages in response to sepsis can also be demonstrated by the important role of resident macrophages in the peritoneal cavity to fight bacteria and avoid peritonitis. Within 20 min of bacterial injection into the peritonial cavity, 35 to 45% of the bacteria inoculated will be associated with macrophages (Dunn et al, 1987). Baker and Kupper (1985) using the caecal-ligation and puncture (19gauge needle) model in two strains of mice, showed 100% mortality in septic endotoxin-sensitive mice (C3H/HeN), whereas the moratlity of septic endotoxin-insensitive mice (C3H/HeJ) was only 25%, within the same period of time after the induction of sepsis (4 days). These two strains of mice differ only in one gene, which controls some aspects of macrophage function (Adams et al, 1981). Therefore, although macrophages are necessary for local protection against sepsis, infection-stimulated macrophage secretory products may also be responsible for the induction of some of the irreversible changes of the response to sepsis, e.g. multiple organ failure. THE ROLE OF INTERLEUKIN 1 Interleukin 1 is critically important in the normal immune response by allowing T-helper cells to differentiate and secrete Interleukin 2, which is required for numerous lymphocyte effector functions (Fig. 1.4.). Interleukin 1, or its by-products, have been suggested as the link between the immunological and the metabolic response to sepsis (Clowes et al, 1986). Interleukin 1 is a polypeptide secreted by macrophages (molecular weight 14,000 - for review see: Dinarello & Wolff, 1982; Dinarello & Mier, 1986). Because the biochemical nature of every macrophage-derived factor is not yet well defined, some of these factors, initially described by their performance in different functions, may actually be the same molecule. There is some evidence now Interleukin 1 plays multiple roles (Takemura & Werb, 1984). It can act as a potent chemotaxin for neutrophils and enhance endothelial adhesiveness. It has also effects on fibroblast proliferation and may be used in the wound healing (Dinarello, 1984). Endogenous pyrogen, which acts on the hypotalamus to change the set point of body temperature (Baracos et al, 1983) and acute phase protein syntesis promoting factor (Stein et al, 1981) seem to be Interleukin 1. Muscle proteolysis induced by sepsis appears to be stimulated by a circulating clevage product of Interleukin-1, namely, proteolysis inducing factor (Clowes et al, 1983 and 1985). It has been suggested leucocytic pyrogen (interleukin-1) or proteolysis-inducing factor may act in the muscle by increasing the production of prostaglanding E2 (PGE2) which, in turn, would activate lysosomal proteases by a calcium depending mechanism (Rodeman et al , 1982; Freund et al, 1986). PGE2 is derived from arachinodate via the cyclooxygenase reaction (Rodeman et al , 1983). Therefore, cyclooxygenase inhibitiors, such as indomethacin and aspirin, might be useful in atenuating or decreasing muscle proteolysis induced by sepsis. Haemodynamic abnormalities and survival rates have been reported to be improved by the use of indomethacin in various endotoxin and septic models (Fletcher et al , 1976 and 1977; Short et al , 1981; Fletcher, 1982 and 1983). However, there is evidence that administration of indomethacin to rats, subjected to caecal-ligation and puncture, did not affect the increased muscle proteolytic rate found in these septic rats. When indomethacin was added in vitro release of PGE2 by septic and normal muscles, incubated in the presence of septic plasma, was reduced by 50%, but the increased proteolytic rate in these muscles remained unchanged (Hasselgren et al , 1985). On the other hand, Freund et al (1986) showed that whole body protein degradation was significantly lower in indomethacin-treated septic rats, but this was associated with lower plasma albumin and decreased protein synthesis in muscle and liver, at a time new protein production is essential, therefore not supporting the use of indomethacin in sepsis. Miccolo et al (1986) using pharmacological dosage of aspirin, given to septic patients, and measuring urea production rate through the incorporation of 15N into the plasma pool, showed no effect of aspirin on the protein breakdown rate in septic man. Crude leucocytic endogenous mediator (LEM) consists of substances derived from polymorphonuclear leukocytes, which include endogenous pyrogen (interleukin-1), and is reponsible for inducing acute-phase reactant production in the liver and changing the plasma concentrations of certain divalent cations, namely, iron and zinc downwards and copper upwards (Dinarello, 1984; Kampschmidit, 1984). LEM is also able to induce hyperinsulinaemia, hyperglucagonaemia, glycogen depression, and other metabolic changes similar to those found in sepsis (George et al , 1977). Interleukin-1, in vitro , did not affect the inhibited muscle amino acid uptake in response to sepsis (Hassselgren et al, 1986). However, interleukin-1 injected into the rat in vivo , had a stimulatory effect on hepatic metabolism, in isolated liver cells, in a time-dependent pattern (Roh et al, 1986). These changes included stimulation of gluconeogenesis from alanine[0.5mM], elevation in non-secretory protein synthesis and increase in oxygen consumption. If interleukin-1 was added to the hepatocyte incubation medium, however, no stimulatory effect was obtained, suggesting the effects of interleukin-1 upon isolated hepatocytes are indirect. This view of indirect action of interleukin-1 in the liver, in vitro, is further supported by the fact that interleukin-1, when added to hepatocytes' culture alone, or to hepatocytes co-cultured with Kuppfer's cells, had no effect on rates of protein synthesis (Christou et al ,1987). CACHECTIN Cachetin (or Tumor Necrosis Factor) is a polypeptide hormone secreted by the macrophage, originally isolated in the course of studies of the mechanisms reponsible for cachexia in chronic disease, and known for its capacity to modulate adipocyte metabolism, to lyse tumor cells in vitro , and to induce hemorrhagic necrosis of certain transplantable tumors in vivo (for review see: Tracey et al , 1986; Beutler & Cerami, 1987). It has a sub-unit size of approximately 17 kilodaltons and constitutes between 1 and 2 % of the total secretory protein produced by endotoxin-activated macrophages in vitro (Beutler et al , 1985). The role of cachetin as a mediator of endotoxic shock was suggested by the fact mice passively immunized against the hormone were found to be protected against the lethal effect of endotoxin (lipopolysaccharide), as reported by Beutler et al (1985). Tracey et al (1986) infused cachectin into rats, in quantities similar to those produced endogenously in response to endotoxin, causing piloerection and diarrhea. Haemoconcentration, shock, metabolic acidosis, transient hyperglycaemia followed by hypoglycaemia, and hyperkalaemia were also observed. Gross and microscopic examination demonstrated severe damage to target organs. Adrenal and pancreatic hemorrhages were commonly observed. Polymorphonuclear leukocytes thrombi were deposited into the major arteries of the lungs, and this was associated with severe pneumonitis. Administration of low doses of cachectin (100 to 200 µg/kg body wt. in rats) induces renal tubular necrosis, and ischemic and hemorrhagic lesions in the gastreointestinal tract (Beutler & Cerami, 1987). To minimize inadvertent release of cachetin, its synthesis appears to be tightly regulated as both transcriptional and post-transcriptional activation must occur to allow its production (Beutler et al , 1986). Glucocorticoids strongly antagonize the effects of endotoxin when administered before the endotoxic insult. If glucocorticoids are added to macrophage cultures cachectin synthesis will be completed inhibited. However, glucocorticoids will only promote this action if added to the culture before macrophages are activated by endotoxin. Therefore, no inhibiting effect occurs if dexametasone is added after lipossacaride induced-macrophage activation. It has been shown cachectin appears to act by suppressing biosynthesis of several adipocyte-specific mRNA molecules (Torti et al ,1985). Cachectin can also induce the synthesis and/or release of specific proteins, including interleukin-1 by monocytes and endothelial cells (Dinarello et al , 1986; Nawroth et al , 1986). Cachectin can activate polymorphonuclear leukocytes stimulating their adhesion to endothelialcell surfaces and enhancing their phagocytic activity (Gamble et al , 1985; Shalaby et al , 1985). Some other effects that may explain the action of cachectin in vivo include stimulation of the production of procoagulant activity altering the properties of the vascular endothelium and alteration of patterns of antigenic expresion (Beutler & Cerami, 1987). Specific neutralization of cachectin and related cytokines may offer new therapeutic tools to treat sepsis. It has been shown that mice treated with a polyclonal antiserum directed against mouse cachectin become resistant to the lethal effect of endotoxin (Beutler & Cerami, 1985). Therefore, it would seem possible that neutralizing monoclonal antibodies directed against human cachectin may prove to be useful in the treatment of sepsis, particularly in its early stages. THE ROLE OF STEROIDS The complex balance between the anti-inflammatory properties and the physiological requirement for steroids may explain the conflicting conclusions about steroid therapy in sepsis (Hess & Manson, 1983; Nohr & Meakins, 1985). Although controversial, the clinical use of steroids in septic shock is common practice and large doses of steroids (50-150 mg/kg body wt.) have been given (Forse and Kinney, 1985). The rationale for the use of these agents in sepsis has included the ability of these drugs to stabilize lysosomal and cell membranes, to inhibit complement-induced granulocyte aggregation, to improve myocardial performance (possibly due to inhibition of oxygen-free radical generation), to modulate the release of endogenous opiates, to decrease arachidonate metabolies, and to promote a shift in the oxyheomoglobin dissociation curve to the right (Karakusis, 1986). Corticosteroids are important in maintaining normal physiology and their concentration may or may not be increased in response to sepsis (Sibbald et al , 1977). Elevation in steroid levels during sepsis may be a protective response to decrease tissue damage by modulating inflammation. The effects of steroids on specific immune processes are also complex. Leucocyte traffic is altered and specific antibody responses are either not affected or enhanced by steroids (Nohr & Meakins, 1985). In the liver, pharmacological doses of steroids have been shown to diminish the disruptive effects of gram-negative bacteraemia and endotoxin on the reticuloendothelial system (Balis et al , 1979). Steroids inhibit margination and degranulation of polymorphonuclear leucocytes in the microcirculation of the liver, lung and other organs, and may therefore be able to inhibit complement activation and the release of factors which decrease capillary permeablity, the latter being implicated with the pathophysiology of multiple organ failure syndrome (Duff, 1985). Steroids block the synthesis of prostaglandins by inhibiting the release of arachidonic acid from membrane phospholipids, and this may be one mechanism by which steroids exert their beneficial effect in septic and shock (Carlson et al , 1977; Fletcher, 1982; Halla-Angeras et al , 1986). As arachidonate is the substrate of both the cyclooxygenase and the lipoxygenase pathways, steroids also may inhibit the synthesis of leukotrienes (Samuelsson, 1983). Recent evidence has shown a protective action of dietylcarbamazine, an inhibitor of leukotriene biosynthesis, against the lethal effect of endotoxin in mice (Hagmann et al , 1984), given support to the view of leukotriene inhibition as another possible alternative mechanism steroids may use to promote their beneficial actions during endotoxin and/or infection. Hall-Angeras et al (1986) have shown that survival rate of rats, subjected to laparotomy and intravenous infusion of live E. coli, was significantly improved by methylpredinisolone or diethylcarbamazine. However, this protective effect appeared not be related to inhibited prostaglandin synthesis, but might have been due to inhibited production of leukotrienes. Glucocorticoids also act as important positive modulators of hepatic and renal gluconeogenic enzymes, as they play a permissive role in the induction of enzymes by other hormones, such as glucagon and epinephrine (Shackleford et al , 1986). There is evidence that sepsis or endotoxin treatment are able to inhibit gluconeogenesis (Filkins & Cornell, 1974; Guillem et al , 1982; Clemens et al , 1984). Investigations into the cause of impaired gluconeogenesis have shown a blockage in the glucocorticoid induction of important gluconeogenic enzymes, including glucose-6-phosphatase, tryptophan oxygenase and phosphoenolpyruvate carboxykinase (Ripe and Berry, 1972; Guckian, 1973; Cannonico et al , 1977; Shackleford et al , 1986). Therefore, interest has been growing on the mechanisms of the endotoxinglucocorticoid antagonism. Moore et al (1976, 1985) have determined that inhibition is elicited by a protein mediator released from macrophages, named "glucocorticoid-antagonizing factor" . Glucocorticoids are known to act via intracellular receptors to which they bind with high affinity after diffusion through the cell membrane (Higgins & Gehring, 1978). These steroid-receptor complexes suffer conformational change ("activation"), and then bind to acceptor sites in the nucleus, resulting in enhanced transcription of a small percentage of genes (Schmidt & Litwack, 1982). Shackleford et al (1986), studying the hormonal regulation of phosphoenolpyruvate carboxykinase in endotoxin treated mice, have demonstrated no effect of endotoxaemia on binding, and on affinity or number of glucocorticoid receptor binding sites, rulling out down-regulation of receptors as the mechanism responsible for the inhibition of induction. They reached the conclusion that endotoxin does not act at the early events in the glucocorticoid induction process, but might act at a subsequent step. Very recent evidence shows glucocorticoids are able to completely inhibit cachetin, a hormone released by endotoxin-activated macrophages, provided they are applied to macrophage cultures in advance of activation (Beutler & Cerami, 1987). THE COUNTER-REGULATORY HORMONES There are difficulties in understanding hormonal mechanisms involved in producing the responses of flow phase of injury / sepsis, and in fitting the time-course of the action of a specific hormone, or group of hormones, to the time-sequence of the metabolic changes (Frayn, 1986). Because of their known general catabolic effects, the counterregulatory hormones, including adrenaline, cortisol, glucagon and growth hormone (Cryer, 1981), have received considerable attention in the study of sepsis. The action of these hormone, in concert, can produce stimulation of lipolysis, glycogenolysis and gluconeogenesis (Frayn, 1986). Infusion of catecolamines associated with glucagon and cortisol has been shown to mimic most of the metabolic features of the flow phase after injury, such as increased metabolic rate with elevation of glucose turnover, increased gluconeogenesis and negative nitrogen balance (Bessey et al , 1984; Gelfand et al , 1984). However, contrary to the view of the counter-regulatory hormones being the main regulators of the changes of the flow phase, is the fact that neither the concentrations, or the time course of changes in the concentrations of these hormones in injured patients agree with those described in the infusion studies (Frayn, 1986). Watters et al (1986) infusing hydrocortisone, glucagon and epinephrine, associated or not with the injection of an inflammatory agent (etiocholanolone), to healthy volunteers reached to the conclusion that both inflarmmatory and endocrine mediators are necessary to stimulate the responses to sepsis. SEPSIS AND INSULIN SECRETION Insulin is perhaps the most studied, but the least understood homone in its role during the acute response to sepsis. Many of the early features of the response characterized by fuel mobilization, such as, complete use of glycogen reserves and mobilization of triacylglycerol might be explained by a decrease of insulin secretion (Frayn, 1986), possibly due to supression of insulin release through the effect of higher concentrations of catecolamines, found in sepsis (Biesel & Wannemacher, 1980), on the pancreatic ß-cell (Porte & Robertson, 1973). However, although plasma insulin concentration has been reported to be decreased in experimental sepsis (Kelleher et al , 1982; Lang et al , 1984) and in the septic man (Clowes et al , 1966), it has also been shown to be increased during experimental sepsis (Neufeld et al, 1980 and 1982; Biesel & Wannemacher, 1980) and in the septic man (Long et al ,1985; Shaw et al, 1985). Higher concentrations of insulin have also been reported during endotoxaemia (Yelich & Filkins, 1980; Neufeld et al , 1982). This variability in insulin concentration may be explained by differences in the experimental model of sepsis used, the changes in the type and source of sepsis, nutritional status and the timepoint observations were made. Despite this variability, the metabolic responses seem to be relatively uniform and insulin, therefore, appears to act ineffectively during infection being unable to produce its anabolic effects. Insulin Resistance One unified way of looking at the endocrine control of the metabolic changes after injury/sepsis is the presence of an unresponsiveness of the 'net storage' or the anabolic processes to the elevation in insulin concentration. Therefore, glucose production in vivo appears to be enhanced while glucose storage is reduced in the presence of high insulin (Black et al , 1982). Despite insulin to promote normal fat storage, net fat mobilization and oxidation are increased (Stoner, 1983; Frayn et al , 1984). To further ilustrate the failure of proper insulin action is the finding of a striking positive relationship between the time course of the increase in plasma insulin with the amount of nitrogen excretion, in injured patients (Frayn, 1986). Despite the general concept of insulin resistance during injury/sepsis has been supported for many years (Butterfield, 1955; Hinton et al , 1971; Beisel & Wannemacher, 1980) there is a lack of understanding of the insulin action at the tissue level, from receptor through possible intermediates to target pathways. Black et al (1982) using a modification of the euglycaemic insulin clamp technique to study injured patients, suggested that the sensitivity of the insulin receptor to circulating insulin is normal, and that insulin resistance in peripheral tissues (probably skeletal muscle) may be caused by a postreceptor defect. Clemmens et al (1984) studying isolated perfused livers showed a resistance to the effects of insulin on gluconeogenesis in the livers of septic rats (caecal-ligated and punctured rats), as compared to those found in the livers of shamoperated rats. Hasselgren et al (1987) reported some evidence for insulin resistance of protein breakdown in septic muscle, while the response to the hormone of amino acid transport and protein synthesis was not altered in sepsis. BIOLOGICAL RESPONSE MODIFIERS All treatments, procedures and agents used to restore homaeostasis and maintain normal anatomy and physiology are regarded as biological response modifiers (For review see: Meakins, 1981; Nohr & Meakins, 1985). Despite the difficulties in selecting the appropriate modifier, as well as, in evaluating their action, they appear to hold much promise in the modulation of the metabolic response to injury/sepsis. A list of some of the biological response modifiers is shown below (Nohr & Meakins, 1985). Biological agents Endogenous Tissues and cells Hormones Passive immunization Target-directed monoclonal antibodies Miscellaneous Exogenous Specific: active immunization Non-specific: adjuvants Drugs Corticosteroids Free radical sacvengers Iron binding compounds Prostaglanding related drugs Levamisole Opiate antagonists Insulin release inhibitors Others General Methods Restore anatomy and physiology Surgery Nutrition Plasmapheresis Fever Some of these modifiers will be briefly considered. Thymic extracts, namely a derivative of thymopoetin, TP5, increases survival in a septic burned animal model (Waymak et al , 1984). The role of some hormones (steroids and counterregulatory hormones) has already been mentioned in this introductory chapter. The important role of endotoxin (LPS) in the pathogenesis of sepsis suggests that antibody to LPS may be therapeutically useful as an antitoxin. However, the most immunologically accessible poetion of LPS (the O antigen side chain) varies among gram-negative bacteria. Nevertheless, animal models and a human trial using human anti-E coli J5 antiserum have shown protection against the lethal effects of gramnegative sepsis (Dunn & Ferguson, 1982; Ziegler et al , 1982). It has also been demonstrated (Beutler et al , 1985) that mice treated with a polyclonal antiserum directed against mouse cachectin become resistant to the lethal effect of lipopolysaccahride (LPS). Thus the use of monoclonal antibodies may also have potential therapeutic applicability. Fibronectin is an opsonic glycoprotein important in promoting phagocytosis by cells of the reticuloendothelial system. Plasma levels of this glycoproetin are decreased in septic patients. Scovill et al (1978) have shown that the administration of fibronectin as cryoprecipitate can restore some aspects of host physiology towards normal. Zymosan, a cell wall preparation of Saccharomyces cerevisiae which can attract and activate leucocytes enhancing their phagocytic properties (Williams et al , 1982), has been used as a pre-treatment (injected intraperitoneally) to rodents and successifully decreased mortality due to peritonitis (Joyce et al ,1978). On the other hand, Goris et al (1986) advocate the use of zymosan, also intraperitoneally, to induce multiple organ failure and 'sepsis without bacteria'. The role of prostaglanding inhibitors in the modulation of the metabolic response to sepsis has also being discussed previously. Opiate receptors in the central nervous system may mediate some effects of septic shock, possibly in conjunction with opiate receptors in the periphery (Nohr & Meakins, 1985). Administration of the opiate antagonist, naloxone hydrochloride, can alleviate the hypotension of endotoxin shock to varying degrees, to increase mean and pulse blood pressure, elevate respiratory rate, prevent acidosis, attenuate hypoglycaemia, stabilize lysosomal membranes and elevate white blood cell and platelet concentrations (Groeger et al , 1983; Hinshaw et al , 1984; Ipp et al , 1984). However, some of the reasons to question their value as a therapeutic intervention for septic shock include the fact that studies evaluating survival time have shown no long-term survival benefits; naloxone cardiovascular's effects have been mostly assessed only during the early phase of shock; the effects on cardiovascular parameters due to naloxone are often short-lived and minimally beneficial; despite increasing arterial pressure naloxan may also present adverse actions, such as regional vascular resistance; species variability has been reported with regard to some of naloxone's vascular actions; and finally, naloxone stimulates the sympathoadrenal system which may impair perfusion of the microcirculation (Hinshaw et al , 1984). Hyperinsulinaemia is a common event as part or the metabolic response to sepsis (Biesel & Wannemacher, 1980). One unified view of the metabolic changes during sepsis is the lack of response to the anabolic action of insulin (Frayn , 1986). Hyperinsulinaemia, itself, has been implicated as the cause, rather than the product of insulin resistance (Marangou et al , 1986). Therefore, drugs modulating insulin release can also be used not only to assess a possible reversal in insulin sensitivity during sepsis, but also to try to reverse some of the metabolic changes mediated under the conditions of elevated plasma insulin. Diazoxide (Henquin et al , 1982), mannoheptulose (Simon et al , 1972) and streptozotocin (Bolaffi et al , 1986) are examples of such drugs and their action during sepsis will be discussed in the following Chapters of this thesis. Surgery Although surgery itself is immuno-suppressive, delayed type hypersensitivity responses can be improved by the drainage of visceral abscesses (Meakins et al , 1979). In addition, the drainage of an intraabdominal abscess can also restore the function of 'failed' organs (Polk & Shields, 1977), so that organ failure should be regarded as a potentially reversible 'organ dysfunction' (Bohnen et al, 1983). However, the use of percutaneous drainage under computerized tomography guidance may be a reasonable choice, as the risks of a negative laparotomy in multiple organ failure require clarification. Nutrition Refeeding via oral or intravenous route has been shown valuable in correcting several abnormalities in immunological function. Total parenteral nutrition has been reported as capable of restoring skin test reactivity, restoring lymphocyte transformation, correcting serum immunoglobulin levels and restoring specific antibody responses and neutrophil chemotaxis (Nohr & Meakins, 1985). However, the choice of the best method of nutritional support, wether by the use of enteral or parenteal nutrition, is still controversial. The best nutritional regimen and the timing of feeding after injury or the establishment of sepsis are also to be defined. Despite the beneficial effects, rigorous documentation of a survival benefit due to nutritional support is still not available. CHAPTER 1 INTRODUCTION Sepsis remains an important cause of death and morbidity (Altemeier, 1980; Meakins, 1985). It frequently complicates surgical operations and delays the resolution of major illness (Cerra, 1982; McGowan & Gorbach, 1983; Wilmore et al , 1983; Meakins, 1985). Infection occurs despite surgical advances supported by modern and safe anaesthesia, potent and specific antibiotics, better monitoring of the clinical and metabolic course, effective nutritional support, and a more clear understanding and application of the principles of physiology. The general term 'sepsis' has caused considerable confusion in clinical and experimental studies (Wichterman et al , 1980) because it refers to different conditions in different studies. Some experimental septic models induce a lowgrade "smoldering" infection which can last for days or weeks, whereas other models describe a very aggressive process that causes death within hours. While infection is a localized focus of invading mircroorganisms confined to a tissue space, and microorganisms within the blood stream are objective evidence of systemic invasion, a septic state can be regarded as the addition of the local and the systemic effects produced by a focus of infection. For the purpose of the work described in this thesis, 'sepsis' is regarded as an acute invasive infection, which involves stimulation of phagocytosis with mobilization and activation of white blood cells, associated with important changes in intermediate metabolism, causing the development of a toxic state (weakness, anorexia, pile erection, lethargy, etc.), leading to the eventual failure of most organs and/or circulatory collapse (septic shock). A wide range of microbial organisms can cause sepsis, especially grampositive and gram-negative bacteria. While the term 'bacteraemia' refers to bacteria in the blood, irrespective of whether they do or do not induce the toxic state of sepsis, 'septicaemia' refers to a toxic state produced by viable proliferating bacteria in the blood. Infection in the critically ill patient can be classified into two main types, according to the nature of the contaminating flora and some clinical characteristics, as described by Meakins (1985): Type 1. Usually monomicrobial Monomicrobial infection occurs when an organ or structure becomes infected with a single strain of organism, for example, streptococcal endocarditis, lobar pneumonia, tuberculosis, etc. It can also occur associated with therapeutic manipulation, for example, line infection, urinary tract infections, etc. Type 2. Usually polymicrobial Polymicrobial infection, in contrast, occurs when a body space becomes contaminated with normal intestinal flora, for example, diverticulitis, perforated colon, etc. It can be related to the surgical procedure required to manage the primary disease, or to extrinsic complications from surgery, such as peritonitis, empyema, enteric fistulae. It can also occur as a result of civilian or military injuries. FACTORS DETERMINING THE DEVELOPMENT INFECTION The development of infection depends on three major determinants: the causative organism, for example, the bacteria; the environmental factors; and the general defence mechanisms, which are designed to contain and resolve the established infection. Bacterial factors involve virulence, pathogenicity, number of microorganisms and antibiotic resistance. Enviromental factors are the changes that influence the milieu, including the contact with the infectious source, and the local barriers to colonization, which lead eventually to tissue invasion. Defence mechanisms involve the systemic response to the bacterial invasion of tissues, including changes in the immune system, which are associated with the physiological and metabolic response (Meakins, 1985). Fig. 1.1. shows the interrelation of these determinants in the normal subject and in the septic patient. Fig. 1.1. These determinants are basic to the generation of sepsis. In the homeostatic or normal state (Fig. 1.1. - top illustration), the intersection of the quadrangles represents the relationship between the determinants and the probability of infection, which is very low indeed. However, all three determinants alter their relationship in patients at high risk of developing infection and in the already infected patient, as represented by the area of intersection in the bottom illustration (Fig. 1.1.). FACTORS INCREASING SUSCEPTIBILITY TO INFECTION Some of the environmental and genetic factors play an important role in determining susceptibility to infection, as well as its severity. Age Age influences not only susceptibility to infection, but also its clinical course. Many infectious diseases show a characteristic age distribution. Infections with ubiquitous organisms are seen firstly in infants, shortly after they have lost the maternal antibody transferred across the placenta, and have a peak incidence in early childhood. Maturation of the immunological system, associated with gradual acquisition of protective immunity due to successive exposures to infection, appear to promote a more adapted response to infectious threats in adulthood. With old age immunity declines increasing susceptibility to infection again. Sex and hormonal factors Not only hormonal but social factors are implicated in the variation in the sex distribution of infectious diseases. Sex has a role in defining occupation and, therefore, can influence how close men and women come into contact with the source of infection. Pregnancy can predispose to various infections, including hepatitis, pneumoccocal infections, amaebiasis and malaria, possibly reflecting changes in immunological function and in metabolism that occur at that time (Greenwood , 1983). The presence of conditions leading to excess glucocorticosteroids, such as adrenal hyperplasia, adrenal tumors, or steroid therapy, may also increase susceptibility to infections, for example, infections with herpes simplex, and herpes zoster. Corticosteroids probably exert their action via the immune and metabolic responses (Karakusis, 1986). Patients with diabetes, especially if poorly controlled, show increased susceptibility to infections, with the common development of abscesses, boils and urinary tract infections. Diabetic patients also present with impaired cell-mediated immune reactions and chemotaxis by polymorphonuclear leucocytes (Bagdada et al , 1974; Greenwood, 1983). The relation of infection and/or injury with malnutrition and immunity A number of clinical and experimental studies have shown that defficiency of protein, of individual vitamins, and of trace elements increase susceptibility to infection (Chandra, 1979; Hessov, 1981). The work with protein energy malnutrition in developing countries, where many children suffer from undernutrition and frequently die from infection, has revealed a close relationship between nutritional status, immune response and infection ( Chandra & Newbourne, 1977). The clear association of nutrition and infection could be seen recently during the famine in Ethiopia and other African countries which led to wide-spread profound malnutrition as the main predisposing factor in the death by infection of tens of thousands of people (Alexander, 1986). In industrialized countries, severe malnutrition is encountered as a complication of other conditions, such as generalized malignant disease, cirrhosis, and intestinal malabsortion due to inflammatory bowel disease, more frequently than as a result of pure dietary deficiency. However, considerable attention has been given to the fact that malnutrition is also widely prevalent in surgical patients, and bears a close association with morbidity and even mortality (Bistrian et al , 1974; Hill et al , 1977; Hessov, 1981). Spanier et al (1976), in body composition studies, directly related the depletion of body cell mass to immunocompetence and repletion of body cell mass with restoration of immune function. Malnourished patients may present delayed hypersensitivity (Meakins et al , 1977). Anergy occurs in prolonged starvation and appears to be reversed by refeeding (Spanier et al , 1976). However, the relationship between nutritional state and immunocompetence is far more complex, for other factors beside malnutrition, such as injury, surgery and infection itself, may also intervene in the imunne response (Hill, 1981). Infection and/or injury may in turn contribute to the protein-calorie malnutrition, possibly by causing anorexia and simultaneously increasing the energy requirements. It is, therefore, probable that the malnourished patient who becomes infected, with or without associated injury, enters a vicious circle with a potential spiral descent to innanition, anergy and death due to overwhelming infection (Fig. 1.2.) (Kettlewell et al , 1979). Fig. 1.2. In his classical metabolic balance studies, in patients with long bone fractures, Cuthbertson (1930) emphasized the significance of proper nutrition in reducing the protein loss following injury or surgery. Today, careful assessment of the nutritional status leading to administration of nutritional support to the malnourished hospitalized patient, is well incorporated into modern clinical care practice (Dudrick et al , 1971; Elwyn, 1980; Kettlewell, 1982; Winters & Greene, 1983; Silk, 1983; Phillips & Odgers, 1986). Some studies have reported that the survival of some groups of patients, such as those with intestinal fistulae, severe sepsis, multiple injuries and after radical oesophagogastric surgery has improved in recent years, and this has been attributed, in part, to the role of nutritional support, either as enteral or parenteral nutrition (Elwyn, 1980; Mullen et al , 1980; Yamada et al , 1983; Philips & Odgers, 1986). Drugs Malnutrition and infection in cancer patients receiving cytotoxic or immunosupressive drugs is well recognized, and has led to the use of intravenous feeding as adjuvant therapy (Fischer, 1984; Copeland, 1986). Alcohol, in excess, increases susceptibility to many infections, especially when liver failure supervenes. Among other factors decreasing the resistance of alcoholics to infection is impaired cell mediated immunity, in particular reduced mobilization of granulocytes and impaired clearing function of the macrophage- monocyte system, loss of delayed hypersensitivity and a decrease in t lymphocytes in peripheral blood. (Lieber & DeCarli, 1977). The neglect of simple hygienic precautions when administering their intravenous injections in hard-line drug addicts leads to septicaemia as a common terminal event, or alternatively, to the contamination with the Acquired Immunodeficiency Syndrome virus (Anon, 1986). Smoking also predisposes to respiratory infection due to the damage of the epitheleum of the respiratory tract (Greenwood, 1983) . Malignant disease Immune responsiveness is impaired in patients with dessiminated malignant disease (Kettlewell, 1979). The frequent association of cancer and malnutrition, added to the harmful effects of radiotherapy or treatment with cytotoxic drugs, further increases the risk of developing infection in these patients. The provision of nutritional support, with the aim to correct malnutrition, has been largely used in such patients, although whether this contributes to diminishing the infection risk is still to be established clearly (Fischer, 1984; Copeland, 1986). HISTORICAL ASPECTS AND THE IMPORTANCE OF INFECTION Infection was the major factor in worldwide population control of mankind until very recently. Before the 17th century, epidemics of smallpox, plague diphtheria, measles, cholera and infectious diarrhoeal diseases, were common episodic events causing death of more than 50% and sometimes as much as 90% within a region (McNeil, 1977). Better hygiene and improved nutrition increased the average life expectancy, and contributed to the five-fold increase in the world population over the last 150 years (Alexander, 1986). Over this period, the explosion in scientific methods, associated with the constant search for controlling and understanding the mechanisms of infection, have had a tremendous effect on infectious diseases. Smallpox, one of the most common causes of death in the world a few hundred years ago, is now extinct (Barnes & Robertson, 1981). By contrast, the control and eventual cure of the potentially catastrophic Acquired Immuno Defficiency Syndrome, caused by an infection with the human immuno-deficiency virus, poses a similar challenge to the scientific community today (Anon, 1986). Infection may be part of a primary disease process, for example pneumonia, or may occur as a complication of treatment, for example pelvic abscess following abdominal surgery or line infection during intravenous feeding. However, until the 19th century, the formation of pus was considered a normal part of healing - pus bonum et laudbile . John Hunter [1728-1793] recorded in his "Treatise on the Blood, Inflamation and Gun Shot Wounds", published in 1794, that "inflamation is not only the cause of diseases but it is often the mode of cure". Louis Pasteur [1822-1825] caused a dramatic change in the surgical attitude towards infection by showing that fermentation was caused by living multiplying matter and, therefore, that pus formation had to be also caused by minute organisms from the enviroment. Joseph Lister [1827-1912] by the use of carbolic established the principles of antisepsis and revolutionized surgical practice. This major revolution in surgery, which progressed to the use of aseptic surgical technique, led to a great reduction in surgical morbidity and mortality. Lister's first scientific analysis of the septic state is found in his "On the early stages of inflammation" (1858) when he thought "The effects of irritation of the tissues are twofold. Firstly, there is a dilatation of the arteries which is produced through the nervous system. Secondly, there is an alteration in the tissues on which the irritant acts directly. This alteration imparted an adhesiveness to both the red and the white corpuscles, making them prone to stick to one another and to the wall of the vessels, and so giving rise to stagnation of blood and ultimately to obstruction" (Singer & Underwood, 1962). Since Thomas Latta (1832) first reported in the Lancet the prevention of death from cholera by the intravenous injection of 6 pints of almost normal saline containing 3 drachms of salt and 2 scruples of sodium bicarbonate, or about 2.8 litres of 89 mM Na+, 78 mM Cl-, and 11 mM bicarbonate, the use of fluid and electrolyte therapy has become such a familiar part of medicine that it is rarely considered today (Veech R L, 1986). The successful clinical use of antibiotics, described for the first time by the Oxford group (Abraham et al , 1941) was a gigantic step in the fight against infection. However, the introduction of asepsis and antisepsis, fluid and electrolyte therapy, specific antibiotics, careful monitoring, aggressive operative intervention and nutritional support, has not eliminated infection as an important clinical problem today. Studies have indicated that approximately one third of surgical patients suffer from infection at some stage of their stay in hospital. Around 10% of operated patients may develop wound infections, which will lengthen the hospital stay by a week on average (Altemier et al , 1976; Green et al , 1977; Altemier, 1980; Cruse & Foord, 1980). Surgical sepsis plays an important role in the morbidity and mortality encountered in intensive care units and constitutes a major impediment to the resolution of critical illness, with septic patients presenting a three-fold mortality rate as compared with non-septic patients (McLean & Boulanger, 1985). The increasing use of immunosuppressive drugs and corticosteroids increases the susceptibility to infection (Cupps & Fauci, 1982; Greenwood, 1983; Copeland, 1986). More severely injured patients, transplant patients, patients with cancer and diabetes melitus are surviving longer, so increasing the number of patients at increased risk from infection. In addition, invasive techniques such as indwelling intravenous catheters, especially longterm catheters used for intravenous feeding, and bladder catheters also increase the risk of sepsis (Platt et al , 1982; Pinilla et al , 1983). The magnitude of the stress imposed by sepsis is a function of the size of the infective process, the number of invading organisms and their virulence. Survival depends not only on the adequacy of the immunological reactions to contain and eliminate invading microbes, but also on a series of integrated physiological and metabolic responses necessary to maintain energy production and cell function thoughout the body (Clowes et al , 1985). It is encouraging, however, that the complex pathophysiology of bacterial sepsis is gradually becoming better understood. THE METABOLIC EFFECTS OF STARVATION AND INJURY Sepsis is frequently associated with starvation and/or injury. A summary of the metabolic effects brought about by these latter conditions is shown in Fig. 1.3. Fig. 1.3 . Metabolic changes in starvation In the transition from the fed to the starved state, a sequence of metabolic alterations occurs as the body maintains glucose homeostasis in the early phase, and then preserves body protein mass in the later phases of starvation. In early starvation, glycogen stores are largely exhausted within 24 h, and to avoid hypoglycaemia and maintain the demand for glucose by the brain, red blood cells and renal medulla, gluconeogenesis is activated. As a result of diminished or abolished absorption of glucose from the gut, plasma insulin concentrations fall enabling the increased release of amino acids (alanine and glutamine) from protein, to be used mainly as gluconeogenic precursors. The low concentrations of insulin also allow the release of free fatty acids from adipocytes to meet the major portion of the body's energy requirements, either through their total oxidation, or via the increased utilization of their partial oxidation products, namely, ketone bodies. The elevated rate of protein degradation, necessary to provide precursors for the increased gluconeogenic demand, is progressively reduced as starvation proceeds, leading to a decrease in the basal metabolic rate. A major factor in this protein-sparing adaptation, essential for survival during prolonged periods of starvation, is the gradual change from oxidation of glucose to oxidation of ketone bodies by tissues such as the brain, which normally metabolizes glucose when ketone bodies are not available. A more detailed discussion of the metabolic adaptation to starvation can be found in Felig et al (1969), Cahill (1976), and Newsholme & Leech (1983). Metabolic changes in injury Whereas the metabolic changes in starvation are geared towards conserving energy output, the metabolic response to injury is directed towards coping with the increased energy demands, and to the support of the healing wound. These responses are proportional to the severity of the injury and are characterised by three distinct phases (Fig. 1.4.): the early ebb or shock phase, the subsequent flow or hypermetabolic phase, and the recovery or anabolic phase (Cuthbertson, 1930). The changes presented in these phases have many similarities when compared to those found in the response to sepsis. (For review see: Silk, 1983; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986). Fig. 1.4. The ebb phase The ebb phase is characterized by a rapid fuel mobilization, haemodynamic instability and increased plasma catecholamine concentrations (Coward et al , 1966). The metabolic rate falls during this phase (O'Donnell, 1976). Insulin plasma concentration is variable (Frayn, 1986) despite a possible inhibition of its release by the action of the higher catecholamines on the pancreatic ß-cell (Porte & Robertson, 1973). Glucocorticoids and glucagon are also reported to be elevated (Forse & Kinney, 1985). Glycogen and triacylglycerol stores are mobilised for immediate energy production, although there is some argument about the need, and how adaptive such a massive fuel mobilization is (Frayn, 1986). Maintenance of circulatory volume and tissue perfusion appear to be aim during this phase. The flow phase If death does not occur as a result of hypovolaemia or direct damage to vital organs during the ebb phase, a more prolonged period follows (the flow phase), which is characterized by an increase in the metabolic rate, breakdown of body tissue and weight loss. Increased plasma concentrations of insulin and glucagon have been reported during this phase (Biesel & Wannemacher, 1980; Neufeld et al , 1982). Generally this phase of the metabolic response can be regarded as a balance between the action of insulin and those of the counterregulatory hormones, namely, glucocorticoids, glucagon and catecholamines (Forse & Kinney, 1985). Another characteristic feature of this phase is the presence of hypermetabolism (Cuthbertson, 1979), which appears to be proportional to the degree of stress (Elwyn et al , 1981). The function of this hypermetabolic catabolic phase appears to be to maintain energy production and cellular activity thoughout the body, at a time dietary intake may be scarce and/or not absorbable. If this is achieved and the immunological and metabolic responses take a successful course, the changes leading to net catabolism are gradually replaced by those leading to net anabolism, merging into the 'anabolic' or 'convalescent' phase. The anabolic phase The transition from the catabolic flow phase to the anabolic period of convalesce can occur quickly, for example within a few days, as with soft tissue injury, or can occur over several days as with extensive trauma (Moore & Brennan, 1975). The distinct metabolic changes that occur during anabolism aim to replace the tissue lost over the catabolic phase, whereas resting energy expenditure may fall from the elevated levels of the flow phase to subnormal levels, similar to those found in depleted patients (Moore & Brennan, 1975; Forse & Kinney, 1985). GENERAL DESCRIPTION OF THE METABOLIC RESPONSE TO SEPSIS The metabolic response to sepsis is not only directed to maintainance of energy production and cellular function thoughout the body (Clowes et al , 1985), but also to the containment and resolution of the established infection. Many of the changes in the metabolic response to injury are shared by the metabolic response to sepsis. However, while the pattern of the response to injury shows a clear time-sequence, from the moment of trauma until recovery, the course of the response to sepsis is less predictable, because of fluctuations in the nature and intensity of the metabolic changes, which in turn, are linked to the severity and type of infection, variations in the nutritional and hormonal state, or failure in the defence mechanisms. Take, for example a patient with intraabdominal sepsis proceeding well during the flow phase. If part of the avascular fibrinous wall of a previously isolated-off abscess is eroded bacteraemia can develop, which in turn, will precipitate events of the ebb phase, which in turn, may lead to 'septic shock'. The ebb phase of injury/trauma may be different from that of sepsis. In the former, there is decreased energy production associated with hypovolaemia, whereas in the ebb phase of sepsis, the patient in 'septic shock' may present in a hyperdynamic state, with increased energy production, hyperventilation, increased central venous pressure, increased cardiac output, oliguria, decreased peripheral resistance, hypotension and lactic acidosis (Siegel et al , 1967; Forse & Kinney, 1985). This hyperdynamic septic state then evolves to the hypodynamic septic state with low central venous pressure, hypotention, low cardiac output and increased peripheral resistance (MacLean, 1981; Forse & Kinney, 1985). This suppports the view of a bimodal response to severe acute sepsis, with an initial hypermetabolic hyperdynamic period followed by a hypometabolic hypodynamic late septic state (Chaudry et al , 1979; Wichterman et al , 1980). The hypermetabolism of the flow phase of sepsis, as in that of injury, appears to be proportional to the severity of infection, so that during major infections, such as peritonitis, there may be increases of up to 60% of the resting energy expenditure (Elwyn, 1980). It is during this phase that the septic process appears to enhance a progressive inability of insulin to promote its anabolic actions, leading sometimes to the development of hyperglycaemia and glucose intolerance (Clemens et al , 1982). The hormonal changes include increases in insulin and glucagon and glucocorticosteroids (Beisel & Wannemacher, 1980). Amino acid catabolism is enhanced, as well, as the formation of lactate, alanine and glutamine in muscle (O'Donnell et al , 1976; Hasselgren, 1986). The efflux of substrates from muscle to the liver associated with the hormonal changes promotes alterations in hepatic glucose and fat metabolism. Part of the glutamine released from muscle enters the kidney where it is converted to glucose and ammonia (produced to enable sufficient protons to be excreted and so prevent acidosis), part is transformed into alanine in the intestine, and part contributes to the synthesis of acute phase proteins, necessary for host resistance (Phillips & Odgers, 1986). The increased utilization of aminoacids in the liver leads to increased hepatic protein synthesis (Hasselgren et al, 1984), and to increased ureagenesis (Beisel & Wannemacher, 1980), especially from those aminoacids that cannot be catabolised in muscle (e.g. phenylalanine and tryptophan). The mobilization of non-esterified fatty acids is variable, however net whole body utilization of fat continues during the septic process as judged by measurements using indirect calorimetry (Askanasi et al , 1979). The elevation of ketone bodies during starvation is inhibited by sepsis (Neufeld et al , 1982). Hepatic function is altered (Royle & Kettlewell, 1980) and lipid accumulation in the liver may occur (Guckian et al , 1973; Champion, 1976). Eventually, the metabolic alterations may lead to multiple organ failure and death (Duff, 1985). However, the reason for the proteolysis, the regulation of the changes in hepatic metabolism, and the mechanism of development of the multiple organ failure syndrome are still not completely understood. Two interdependent hypotheses, namely "substrate deficiency" and "endocrine activation" have been put foward to attempt to explain the catabolism of injury/sepsis. In the former, the increased metabolic requirements would be met be proteolysis, lipolysis, and gluconeogenesis. In the endocrine activation hypothesis, the increased production of hormones, such as catecholamines, glucagon and corticosteroids, would lead to the catabolism of injury/sepsis (Moore, 1983). Among many of the metabolic studies of sepsis, there is usually variability in the severity and type of the septic insult, which is often associated with variable degrees of trauma, the latter being commonly accompanied by different nutritional and hormonal status. Furthermore, it is also not unusual to find variation in the time the observations are made; and time plays a very important role in the progress and nature of the metabolic changes brought about by sepsis. Interpretation or comparison of the results under these circunstances, therefore, may be misleading, for they may reflect, in part, the effects of the different experimental conditions. This may account for some of the apparent discrepancies found in the published reviews, in both experimental and clinical sepsis (Blackburn, 1977; Beisel & Wannemacher, 1980; Cerra, 1982; Neufeld et al , 1982; Wilmore et al , 1983; Forse & Kinney, 1985; Frayn, 1986), as illustrated by Fig. 1.5. Fig. 1.5. More detailed discussion of changes in metabolism of carbohydrate and fat in response to sepsis will be included in the experimental Chapters. MULTIPLE ORGAN FAILURE The failure of vital organs in the septic and/or injured patient has received the attention of a number of recent studies (for review see: Polk, 1982; Duff, 1985). It is now clear that several organs are often involved, usually remote from the site of infection. In addition, the clinical manifestations of their involvement show a sequential pattern. Failure of the renal, respiratory and cardiovascular systems often occur first, and, if this is not followed by recovery, there is failure of the brain, liver and gut (Duff, 1985). The lungs The earliest recognized form of organ failure was that involving the lung. Adult respiratory distress syndrome (ARDS), 'shock lung', 'Viet-Nam lung' and 'lung lesion of sepsis' all describe a type of respiratory failure, with each name being related with their initiating event: shock, trauma and/or sepsis (Polk, 1982). The predominant characteristic of adult respiratory distress syndrome appears to be the association with infection ( Horovitz & Shires, 1974; Eiseman & Narton, 1975; Fulton & Jones, 1975). The pathophysiology of lung failure is not clear, but there is some evidence from clinical and experimental studies that the pulmonary oedema is a result of increased permeability of pulmonary capillaries. Increased capillary permeability has been reported in sheep injected with endotoxin or bacteria, as judged by the increase in the rate of appearance and the concentration of protein in the lung lymph (Brigham et al , 1979). Adult patients with ARDS showed increased rate of incorporation of radiolabelled plasma albumin into the bronchoalveolar fluid (Sibbald et al , 1979). The mechanism of capillary injury is not fully explained, but the effects of complement activation may play an important role (Craddock et al , 1977; Jacob et al , 1980). This endothelial cell injury could also be the result of the action of the polymorphonuclear leucocytes (or their products) which are known to be aggregated by activated complement. To support this view there is evidence that drug-induced neutropoenia can diminish or prevent the effects of infusion of activated complement (Craddock et al, 1977). Other causative factors are also likely to be involved, as experimental lung lesions caused by infusion of complement are never as extensive as those observed in septic patients (Hyers, 1981). Using a radioaerosol lung scanning technique, Tennemberg et al (1987) studied patients with trauma and sepsis, and reached the conclusion that complement-mediated neutrophil activation, alone, appeared to be unable of initiating lung injury or precipitating ARDS. Complement-mediated neutrophil activation may, therefore, serve as an early, potentiating mechanism for other polymorphonuclear triggering mediators. It is also well known that the lungs, in addition to their gas exchange and filter function, and the fact they are target-organs of circulatory factors, they clear many substances from the circulation such as 5-hydroxytryptamine. The lungs also synthesise and release various agents, that may act on smooth muscle and, therefore, interfere with the function of other organs; the lungs being thus regarded in a way analogous to an "endocrine gland" (Aun & Birolini, 1985). The kidneys The pathophysiology of the injuries leading to renal failure is not fully understood. Acute tubular necrosis may or may not be present. Decreased renal blood flow, endotoxin action and vasoconstriction of renal arteries have been implicated as possible mechanisms (Duff, 1985). Fortunately renal failure can be treated successfully with haemodialysis until function returns, but the latter is usually linked to the recovery of function of other organs, especially the liver. The liver Liver function is almost always impaired in sepsis whether accompanied by the multiorgan failure syndrome or not. Liver function tests show a pattern suggestive of cholestasis with a rise in serum bilirubin and alkaline phosphatase, accompanied by moderate elevation of SGPO and SGPT (Royle & Kettlewell, 1980; Duff, 1985). Jaundice is the clinical sign which reflects such changes, and appears at a later stage in the septic process, as compared to the biochemical changes. Changes in blood amino acid concentrations include elevations of phenylalanine, taurine, cystine and methionine, which are metabolized in the liver (Freund et al , 1978). Macroscopic changes in livers of monkeys, injected with live E. coli bacteria, include intravascular sequestration of degranulating leucocytes and fibrinous deposits of platelet aggregates in sinusoids and the spaces of Disse (Balis et al , 1979). The post-morten of patients who died with multi-organ failure reveals hepatic changes such as, focal necrosis, fat accumulation, congestion and enlargement of sinusoids, and cholestasis (Champion, 1976). Gastrointestinal system The gastrointestinal (G.I.) system may fail due to ulceration, loss of barrier function or malabsorption. In patients dying from multi-organ failure, the post-morten shows massive denudation of the gastrointestinal tract mucosa, which suggests a failure in the gastrointestinal barrirer function, enhancing abnormal absorption of bacteria and/or bacterial products (toxins) (Duff, 1978 and 1985). Pathophysiological mechanisms of multiorgan failure syndrome A common mechanism used to explain the development of multiorgan failure is based on the action of free radicals (O2-, OH-, H2O2). As a result of sepsis, or sepsis associated with trauma (usually an operation to treat localized infection), there is activation of the complement cascade through the alternative pathway, and release of factor C5a (a polypeptide of 74 residues and a molecular weight of 15,000). This peptide interacts with circulating neutrophils to increase their adherence and potential to degranulate when exposed to suitable stimuli (Simmons & Solomkin, 1985). The aggregation of granulocytes, in turn, releases free radicals in amounts sufficient to cause cellular damage, dysfunction and eventualy organ failure (Duff, 1985). Recent evidence, however, appears to indicate a very important role of the macrophage in the response to sepsis. Macrophages activated by bacteria or bacterial products, such as endotoxin [lipopolysaccharide (LPS)], can release a polypeptide "hormone" called Cachectin which might initiate tissue injuries, which are the same as those caused by endotoxin inoculation (Tracey et al , 1986; Beutler et al , 1987). SEPSIS AND ENDOTOXIN Endotoxin is a lipoprotein-carbohydrate complex (lipopolysaccharide LPS) found in the cell wall of all gram-negative bacteria. Andre Boivin (1941) was the first to isolate endotoxins from gram-negative bacteria, and some ten years latter endotoxins were implicated as the cause of human gram-negative shock (Borden & Hall, 1951; Braude, 1953). This hypothesis has led to numerous studies on the physiological and biochemical alterations associated with endotoxin inocculation into various laboratory animals over the last four decades. Furthermore, some of the results of such studies are usually incorporated into the descriptions of the pathophysiology of clinical sepsis and septic shock, and in their treatment (Wichterman et al , 1980). Very few septic patients have endotoxin identified in their blood, and if they do, the concentrations are quite low (Levin et al , 1972; Postel et al , 1975). Recent studies, however, support the view that endotoxin acts indirectly, via stimulation of polymorphonuclear leucocytes (Tracey et al , 1986; Beutler & Cerami, 1987; Christou et al , 1987). There are differences in some of the clinical events that result from sepsis as compared to those induced by endotoxin inoculation (Fig. 1.6.), as described by Wichterman et al (1980). Fig. 1.6. Nevertheless, both endotoxin and sepsis can activate the macrophages to produce interleukin-1 and/or its by products, which appear to be important mediators of the metabolic changes brought about by sepsis and/or endotoxin (Cohn, 1978; Clowes et al , 1983). Both sepsis and endotoxin can also cause similar hormonal and metabolic changes, such as, hyperinsulinaemia and hyperglucagonaemia (Yelich & Filkins, 1980; Beisel & Wannemacher, 1980), and the inhibition of the hyperketonaemia in response to starvation (Neufeld et al , 1982). The development of multiple organ failure syndrome also appears to follow both endotoxaemia (Tracey et al , 1986) and sepsis (Duff, 1985). These findings suggest the metabolic response to LPS is similar to that of sepsis, although variation in the time-course of the resulting clinical events may occur. These differences may be due to the endotoxin dosage, or to the time necessary to activate the immunological cascade by endotoxin inoculation, as compared to that with the various ways clinical sepsis behaves. Therefore, data concerning endotoxin treament, whenever considered relevant, will be included in this Thesis. THE EFFECT OF GRAM-POSITIVE BACTERIAL COMPONENTS DURING INFECTION The major irritative components of gram-positive bacteria are peptidoglycan and teichoic acid. Peptidoglycan is efficient at causing an abscess: 10µg in the skin of man is an adequate dose (Murphy, 1983). It appears to be unable to fix complement via the alternative pathway. However, since all normal sera contain anti-bodies against peptidoglycan, it does fix complement by the classical pathway. Peptidoglycan is able to activate macrophages, but concentrations of 10-100 ng/ml are required, as compared to the concentration of 1ng/ml required for maximal stimulation by endotoxin. The active component appears to be N-acetyl muramyl-lala D isoglutamine (MDP), which is about 100 times more active than peptidoglycan when coupled to an inert carrier. Macrophages activated by MDP secrete the same substances as the macrophages stimulated by endotoxin. Techoic acids are efficient activators of complement via both the alternative and classical pathways. THE ROLE OF PHAGOCYTIC CELLS IN INFECTION What happens during an infection is a function of how far the invading organisms are successful in multiplying. Single organisms are disposed of by phagocytosis, without any further response. Even a few thousand bacteria can cause microscopic inflammation. A few million organism induce the generation of substances which cause enough vasodilataion, increased capillary permeability, and chemotaxis to be clinically visible as inflammation. Many millions of organisms are required to cause a local abscess. Tissue destruction is actually caused by proteolytic enzymes liberated from dead and dying phagocytic cells. Phagocytic cells, including neutrophils, monocytes and macrophages, are responsible for the process of phagocytosis. At the beginning of this century Metchnikoff (1905) described phagocytosis as the ingestion and subsequent killing of micro-organisms (endocytosis), and believed this was a very important process in both the prevention and fight against infection. Latter on, however, it became clear that an important contribution of phagocytes to the inflamatory response is the exocytosis of enzymes and other proteins, some of them possibly carriers of important signals for the development of this response (inflammatory or humoral mediators). Exocytosis, on the other hand, can occur independently from endocytosis and in response to different stimuli from those of the latter (Bentwood & Henson, 1980). These proteins released by the inflammatory cells are capable of various actions, such as making the site of inflammation accessible to an adjacent vascular space (e.g. collagenase and elastase); regulating local inflammatory mediators (e.g. carboxypeptidase); controlling the concentration of metabolites involved in bacterial growth (e.g. lactoferrin and vitamin B12); and enhancing an extracelluar environment adverse to microorganisms through the action of acid hydrolases, cationic proteins and lysozymes (Simmons & Solomkin, 1985). THE ROLE OF THE MACROPHAGE DURING SEPSIS In bacterial infections, cell wall polymers are recognized as foreign by processes which do not require antibody. In viral infections, the initial insult is usually the death of infected cells, which liberate endogenous inflammatory mediators. Immune responses, mediated either by antigen-antibody complexes or by sentitized lymphocytes become increasingly important in the inflammatory response to chronic infection. Each of these mechanisms activates the same enzymatic cascades; the complement, kinin, coagulation and fibrinolytic products. The crucial cell which is activated appears to be the macrophage. Macrophages are distributed throughout the body and have developed specific characteristics in various organs, from which they acquired their respective names (Kuppfer's cells within the liver, alveolar macrophages in the lung, mesangial cells in the kidney, and glial cells (which are thought to be derived from macrophages) in the brain. It appears to be clear that macrophages have adapted for local defence purposes, and in addition, macrophage function can also vary at different sites (For review sse: Takemura & Werb, 1984). The three primary functions of macrophages, described by Cristou et al (1987), are listed bellow: 1. Immune function Antigen presentation 2. Phagocytic-microbicidal function Phagocytosis Destruction of viral infected cells Oxidative killing mechanisms Chemotactic factors Lysozyme release Destruction of intracellular parasites 3. Secretory function Proteases Procoagulant activity Interleukin 1 (IL-1) Colony stimulating factor Angiogenesis factor Plasminogen activating factor Prostaglandins Leukotrienes Interferon Tumor necrosis factor (TNF)/Cachectin The differences in macrophage function could also be understood in terms of degree of macrophage activation, namely, how 'stimulated' the macrophage is. A spectrum of stimulation can be recognized depending on the potency of the stimulating agent. Thus, macrophages which are stimulated with e.g. interferon or complement are refered to as "inflammatory" or 'elicited" macrophages, whereas macrophages stimulated, for example, by endotoxin (Lipopolysaccharide - LPS) are fully "activated" macrophages (Cohn, 1978). The activation of macrophages results in enhanced phagocytosis (Cohn, 1978), increased production of toxic oxygen radicals (Tsunawaki et al , 1974) and the release of various proteases and hydrolytic enzymes (Takemura & Werb, 1984). There is also release (Fig. 1.7) of various monokines (Takemura & Werb, 1984; Dinarello et al , 1986; Kovaks et al , 1985). Fig. 1.7. Aggarwal et al , 1985; Beutler et al , 1986; Release of oxygen radicals by activated macrophages (Babion et al, 1973; Craddock et al, 1977) has also been reported in sepsis.Interleukin 1 can be produced by virtually all activated macrophages (Dinarello & Mier, 1986) and has been implicated as an important mediator in the hypercatabolism of proteins in response to sepsis (Baraco et al, 1983; Clowes et al, 1983 and 1985). Macrophages treated with endotoxin (LPS), in vitro , show altered secretion of various proteins (Largen et al, 1986). An important monokine released by endotoxin stimulation of macrophages is the Tumor Necrosis Factor (Cachectin), which appears to be an important humoral mediator of the systemic metabolic changes during sepsis (Butler et al, 1985; Cerami et al, 1986). Studies using coculture of hepatocytes with Kupffer's cells or macrophages, in the presence or absence of inflammatory stimulation, showed that there was a significant depression of hepatocytes' protein synthesis when endotoxin (LPS) was added to the co-culture medium. However, in the absence of either macrophages or Kupffer's cells, endotoxin had no direct effect on hepatocytes' protein synthesis (West et al , 1985 and 1985). More recent evidence suggests that the macrophage-mediated inhibition of protein synthesis in isolated liver cells occurs via a monokine mediator yet to be identified, but neither Interleukin-1 or Cachectin (Christou et al ,1987). The importance of macrophages in the response to sepsis can also be demonstrated by the important role of resident macrophages in the peritoneal cavity to fight bacteria and avoid peritonitis. Within 20 min of bacterial injection into the peritoneal cavity, approximately 40% of the bacteria inoculated will be associated with macrophages (Dunn et al , 1987). Baker and Kupper (1985) using the caecal-ligation and puncture (19-gauge needle) model in two strains of mice, showed 100% mortality in septic endotoxin-sensitive mice (C3H/HeN), whereas the mortality of septic endotoxin-insensitive mice (C3H/HeJ) was only 25%, within the same period of time after the induction of sepsis (4 days). These two strains of mice differ only in one gene, which controls some aspects of macrophage function (Adams et al , 1981). Therefore, although macrophages are necessary for local protection against sepsis, infection-stimulated macrophage secretory products may also be responsible for the induction of some of the irreversible changes of the response to sepsis, for example multiple organ failure. THE ROLE OF INTERLEUKIN-1 IN THE METABOLIC RESPONSE TO SEPSIS Interleukin-1 is critically important in the normal immune response by allowing T-helper cells to differentiate and secrete Interleukin 2, which is required for numerous lymphocyte effector functions (Fig. 1.4.). Interleukin-1, or its by-products, have been suggested as the link between the immunological and the metabolic response to sepsis (Clowes et al , 1986). Interleukin-1 is a polypeptide secreted by macrophages (molecular weight 14,000 - for review see: Dinarello & Wolff, 1982; Dinarello & Mier, 1986). Because the biochemical nature of every macrophage-derived factor is not yet well defined, some of these factors, initially described by their performance in different functions, may actually be the same molecule. There is some evidence now that Interleukin-1 plays multiple roles (Takemura & Werb, 1984). It can act as a potent chemotaxin for neutrophils and enhance endothelial adhesiveness. It has also effects on fibroblast proliferation and may be important in wound healing (Dinarello, 1984). Endogenous pyrogen, which acts on the hypothalamus to change the setting of body temperature (Baracos et al , 1983) and acute phase protein synthesis promoting factor (Stein et al , 1981) all seem to be Interleukin-1. Muscle proteolysis induced by sepsis appears to be stimulated by a circulating cleavage product of Interleukin-1, namely, proteolysis inducing factor (Clowes et al, 1983 and 1985). It has been suggested that leucocytic pyrogen (interleukin-1) or proteolysis-inducing factor may act in the muscle by increasing the production of prostaglandin E2 (PGE2) which, in turn, activates lysosomal proteases by a calcium dependent mechanism (Rodeman et al , 1982; Freund et al , 1986). PGE2 is derived from arachinodate via the cyclooxygenase reaction (Rodeman et al , 1983). Therefore, cyclooxygenase inhibitors, such as indomethacin and aspirin, might be useful in attenuating or decreasing muscle proteolysis induced by sepsis. Haemodynamic abnormalities and survival rates have been reported to be improved by the use of indomethacin in various endotoxin and septic models (Fletcher et al , 1976 and 1977; Short et al , 1981; Fletcher, 1982 and 1983). However, administration of indomethacin to rats, subjected to caecal-ligation and puncture, did not alter the increased muscle proteolytic rate found in these septic rats. When indomethacin was added in vitro the release of PGE2 by septic and normal muscles, incubated in the presence of septic plasma, was reduced by 50%, but the increased proteolytic rate in these muscles remained unchanged (Hasselgren et al , 1985). On the other hand, Freund et al (1986) showed that whole body protein degradation was significantly lower in indomethacin-treated septic rats, but this was associated with lower plasma albumin and decreased protein synthesis by muscle and liver, at a time when new protein production is essential. Miccolo et al (1986) using a pharmacological dose of aspirin inseptic patients, and measuring urea production rate through the incorporation of 15N into the plasma pool, showed no effect of aspirin on the protein breakdown rate in septic man. Crude leucocytic endogenous mediator (LEM) consists of substances derived from polymorphonuclear leucocytes, which includes endogenous pyrogen (interleukin-1), and is reponsible for inducing acute-phase reactant production in the liver and changing the plasma concentrations of certain divalent cations, namely, iron and zinc downwards and copper upwards (Dinarello, 1984; Kampschmidit, 1984). LEM is also able to induce hyperinsulinaemia, hyperglucagonaemia, glycogen depletion, and other metabolic changes similar to those found in sepsis (George et al , 1977). Interleukin-1, in vitro , does not affect the inhibited muscle amino acid uptake in response to sepsis (Hassselgren et al , 1986). However, interleukin-1 injected into the rat in vivo , had a stimulatory effect on hepatic metabolism, in isolated liver cells, in a time-dependent pattern (Roh et al , 1986). These changes included stimulation of gluconeogenesis from alanine[0.5mM], elevation in non-secretory protein synthesis and increased oxygen consumption. If interleukin-1 was added to the hepatocyte incubation medium, however, no stimulatory effect was obtained, suggesting the effects of interleukin-1 upon isolated hepatocytes are indirect. This view of the indirect action of interleukin-1 in the liver, in vitro , is further supported by the fact that interleukin-1 added to hepatocytes' culture alone, or to hepatocytes co-cultured with Kuppfer's cells, had no effect on rates of protein synthesis (Christou et al ,1987). THE IMPORTANCE OF CACHECTIN DURING SEPSIS Cachectin (or Tumor Necrosis Factor) is a polypeptide hormone secreted by the macrophage, originally isolated in the course of studies of the mechanisms reponsible for cachexia in chronic disease, and known for its capacity to modulate adipocyte metabolism, to lyse tumor cells in vitro , and to induce haemorrhagic necrosis of certain transplantable tumors in vivo (for review see: Tracey et al , 1986; Beutler & Cerami, 1987). It has a sub-unit size of approximately 17 kilodaltons and constitutes between 1 and 2 % of the total secretory protein produced by endotoxin-activated macrophages in vitro (Beutler et al , 1985). The role of cachectin as a mediator of endotoxic shock was suggested by the fact that mice passively immunized against this 'hormone' were found to be protected against the lethal effect of endotoxin (lipopolysaccharide), as reported by Beutler et al (1985). Tracey et al (1986) infused cachectin into rats, in quantities similar to those produced endogenously in response to endotoxin, causing piloerection and diarrhoea. Haemoconcentration, shock, metabolic acidosis, transient hyperglycaemia followed by hypoglycaemia, and hyperkalaemia were also observed. Gross and microscopic examination demonstrated severe damage to target organs. Adrenal and pancreatic haemorrhages were commonly observed. Polymorphonuclear leukocyte thrombi were deposited in the major arteries of the lungs, and this was associated with severe pneumonitis. Administration of low doses of cachectin (100 to 200 µg/kg body wt. in rats) induces renal tubular necrosis, and ischaemic and haemorrhagic lesions in the gastrointestinal tract (Beutler & Cerami, 1987). To minimize inadvertent release of cachectin, its synthesis appears to be tightly regulated as both transcriptional and post-transcriptional activation must occur to allow its production (Beutler et al , 1986). Glucocorticoids strongly antagonize the effects of endotoxin when administered before the endotoxic insult. If glucocorticoids are added to macrophage cultures cachectin synthesis will be completely inhibited. However, glucocorticoids will only promote this action if added to the culture before the macrophages are activated by endotoxin. Therefore, no inhibiting effect occurs if dexamethasone is added after lipopolysaccharide induced-macrophage activation. Cachectin appears to act by suppressing biosynthesis of several adipocyte-specific mRNA molecules (Torti et al ,1985). Cachectin can also induce the synthesis and/or release of specific proteins, including interleukin-1 by monocytes and endothelial cells (Dinarello et al , 1986; Nawroth et al , 1986). Cachectin can activate polymorphonuclear leucocytes stimulating their adhesion to endothelial-cell surfaces and enhancing their phagocytic activity (Gamble et al , 1985; Shalaby et al , 1985). Some other effects that may explain the action of cachectin in vivo include stimulation of the production of procoagulant activity, altering the properties of the vascular endothelium and alteration of patterns of antigenic expresion (Beutler & Cerami, 1987). Specific neutralization of cachectin and related cytokines may offer new therapeutic tools to treat sepsis. It has been shown that mice treated with a polyclonal antiserum directed against mouse cachectin become resistant to the lethal effect of endotoxin (Beutler & Cerami, 1985). Therefore, it would seem possible that neutralizing monoclonal antibodies directed against human cachectin may prove to be useful in the treatment of sepsis, particularly in its early stages. THE ROLE OF CORTICOSTEROIDS IN THE RESPONSE TO SEPSIS The complex balance between the anti-inflammatory properties and the physiological requirement for steroids may explain the conflicting conclusions about steroid therapy in sepsis (Hess & Manson, 1983; Nohr & Meakins, 1985). Although controversial, the clinical use of steroids in septic shock is common practice and large doses of steroids (50-150 mg/kg body wt.) have been given (Forse & Kinney, 1985). The rationale for the use of these agents in sepsis has included the ability of these drugs to stabilize lysosomal and cell membranes, to inhibit complement-induced granulocyte aggregation, to improve myocardial performance (possibly due to inhibition of oxygen-free radical generation), to modulate the release of endogenous opiates, to decrease arachidonate metabolites, and to promote a shift in the oxyhaemoglobin dissociation curve to the right (Karakusis, 1986). Corticosteroids are important in maintaining normal physiology and their concentration may or may not be increased in response to sepsis (Sibbald et al , 1977). Elevation in steroid levels during sepsis may be a protective response to decrease tissue damage by modulating inflammation. The effects of steroids on specific immune processes are also complex. Leucocyte traffic is altered and specific antibody responses are either not affected or enhanced by steroids (Nohr & Meakins, 1985). In the liver, pharmacological doses of steroids have been shown to diminish the disruptive effects of gram-negative bacteraemia and endotoxin on the reticuloendothelial system (Balis et al , 1979). Steroids inhibit margination and degranulation of polymorphonuclear leucocytes in the microcirculation of the liver, lung and other organs, and may therefore be able to inhibit complement activation and the release of factors which increase capillary permeablity, the latter being implicated with the pathophysiology of multiple organ failure syndrome (Duff, 1985). Steroids block the synthesis of prostaglandins by inhibiting the release of arachidonic acid from membrane phospholipids, and this may be one mechanism by which steroids exert their beneficial effect in sepsis (Carlson et al , 1977; Fletcher, 1982; Halla-Angeras et al , 1986). As arachidonate is the substrate of both the cyclooxygenase and the lipoxygenase pathways, steroids also may inhibit the synthesis of leukotrienes (Neeldeman et al , 1986; Samuelsson, 1983). Recent evidence has shown a protective action of diethylcarbamazine, an inhibitor of leukotriene biosynthesis, against the lethal effect of endotoxin in mice (Hagmann et al , 1984). This supports the concept that leukotriene inhibition is a possible alternative for the beneficial action of steroids during endotoxin and/or infection. Hall-Angeras et al (1986) have shown that the survival rate of rats subjected to laparotomy and intravenous infusion of live E. coli , was significantly improved by methylpredinisolone or diethylcarbamazine. However this protective effect appeared not to be related to inhibition of prostaglandin synthesis, but might have been due to inhibition of production of leukotrienes. Glucocorticoids also act as important positive modulators of hepatic and renal gluconeogenic enzymes, as they play a permissive role in the induction of enzymes by other hormones, such as glucagon and epinephrine (Shackleford et al , 1986). There is evidence that sepsis or endotoxin treatment are able to inhibit gluconeogenesis (Filkins & Cornell, 1974; Guillem et al , 1982; Clemens et al , 1984). Investigations into the cause of impaired gluconeogenesis have shown an inhibition of the glucocorticoid induction of important gluconeogenic enzymes, including glucose-6-phosphatase, tryptophan oxygenase and phosphoenolpyruvate carboxykinase (Ripe and Berry, 1972; Guckian, 1973; Cannonico et al , 1977; Shackleford et al , 1986). Therefore, interest has been growing in the mechanisms of the endotoxin-glucocorticoid antagonism. Moore et al (1976, 1985) have determined that inhibition is elicited by a protein mediator released from macrophages, named "glucocorticoid-antagonizing factor" . Glucocorticoids are known to act via intracellular receptors to which they bind with high affinity after diffusion through the cell membrane (Higgins & Gehring, 1978). These steroid-receptor complexes suffer conformational change ("activation"), and then bind to acceptor sites in the nucleus, resulting in enhanced transcription of a small percentage of genes (Schmidt & Litwack, 1982). Shackleford et al (1986), studying the hormonal regulation of phosphoenolpyruvate carboxykinase in endotoxin treated mice, have demonstrated no effect of endotoxaemia on binding, and on affinity or number of glucocorticoid receptor binding sites, ruling out down-regulation of receptors as the mechanism responsible for the inhibition of induction. They reached the conclusion that endotoxin does not act at the early events in the glucocorticoid induction process, but might act at a subsequent step. Very recent evidence shows glucocorticoids are able to completely inhibit cachectin production, a hormone released by endotoxin-activated macrophages, provided they are applied to macrophage cultures in advance of activation by LPS (Beutler & Cerami, 1987). THE COUNTER-REGULATORY HORMONES AND THE REGULATION OF THE METABOLIC RESPONSE TO SEPSIS There are difficulties in understanding hormonal mechanisms involved in producing the responses of the flow phase of injury or sepsis, and in fitting the time-course of the action of a specific hormone, or group of hormones, to the time-sequence of the metabolic changes (Frayn, 1986). Because of their known general catabolic effects, the counter-regulatory hormones, including adrenaline, cortisol, glucagon and growth hormone (Cryer, 1981), have received considerable attention in the study of sepsis. The action of these hormones, in concert, can stimulate lipolysis, glycogenolysis and gluconeogenesis (Frayn, 1986). Infusion of catecholamines with glucagon and cortisol has been shown to mimic most of the metabolic features of the flow phase after injury, such as increased metabolic rate with elevation of glucose turnover, increased gluconeogenesis and negative nitrogen balance (Bessey et al , 1984; Gelfand et al , 1984). However, contrary to the concept that counter-regulatory hormones are the main regulators of the changes of the flow phase, is the fact that neither the concentrations, nor the time course of changes in the concentrations of these hormones in injured patients agree with those described in the infusion studies (Frayn, 1986). Watters et al (1986) infusing hydrocortisone, glucagon and epinephrine, with or without the inflammatory agent etiocholanolone, in healthy volunteers, reached the conclusion that both inflammatory and endocrine mediators are necessary to stimulate the responses to sepsis. SEPSIS AND THE EFFECTS OF INSULIN Insulin is perhaps the most studied, but the least understood homone in its role during the acute response to sepsis. Many of the early features of the response characterized by fuel mobilization, such as, depletion of glycogen reserves and mobilization of triacylglycerol might be explained by a decrease of insulin secretion (Frayn, 1986), possibly due to supression of insulin release by the higher concentrations of catecholamines found in sepsis (Porte & Robertson, 1973; Biesel & Wannemacher, 1980). However, although plasma insulin concentration has been reported to be decreased in experimental sepsis (Kelleher et al , 1982; Lang et al , 1984) and in the septic man (Clowes et al , 1966), it has also been shown to be increased during experimental sepsis (Neufeld et al ,1980 and 1982; Biesel & Wannemacher, 1980) and in the septic man (Long et al ,1985; Shaw et al , 1985). Higher concentrations of insulin have also been reported during endotoxin treatment (Yelich & Filkins, 1980; Neufeld et al , 1982). This variability in insulin concentration may be explained by differences in the experimental model of sepsis used, the changes in the type and source of sepsis, nutritional status and the time-point at which the observations were made. Despite this variability, the metabolic responses seem to be relatively uniform and insulin, therefore, appears to act inefficiently during infection and is unable to produce its anabolic effects. Insulin Resistance One unified way of looking at the endocrine control of the metabolic changes after injury/sepsis is the unresponsiveness of the 'net storage' or the anabolic processes to the elevation in insulin concentration. Therefore, glucose production in vivo appears to be enhanced while glucose storage is reduced in the presence of high insulin (Black et al , 1982). Despite insulin's to promotion of normal fat storage, net fat mobilization and oxidation are increased (Stoner, 1983; Frayn et al , 1984). The striking positive relationship between the time course of the increase in plasma insulin with the amount of nitrogen excretion, further illustrates the failure of normal insulin action in injured patients (Frayn, 1986). Perhaps, a possible explanation to this finding is the fact that the same factors (interleukin1/proteolysis-inducing factor) may be involved in the stimulation of both insulin secretion (George et al , 1977) and protein degradation (Clowes et al , 1983 and 1986). Despite the fact that the general concept of insulin resistance during injury/sepsis has been established for many years (Butterfield, 1955; Hinton et al , 1971; Beisel & Wannemacher, 1980), there is a lack of understanding of the action of insulin at the tissue level, from receptor through possible "signals" to target pathways. Black et al (1982) using a modification of the euglycaemic insulin clamp technique to study injured patients, suggested that the sensitivity of the insulin receptor to circulating insulin is normal, and that insulin resistance in peripheral tissues (probably skeletal muscle) may be caused by a postreceptor defect. Clemmens et al (1984) studying isolated perfused livers showed a resistance to the effects of insulin on gluconeogenesis in the livers of septic rats (caecal-ligated and punctured rats), as compared to the livers of sham-operated rats. Hasselgren et al (1987) reported some evidence for insulin resistance of protein breakdown in septic muscle, while the response of amino acid transport and protein synthesis to the hormone was not altered in sepsis. BIOLOGICAL RESPONSE MODIFIERS DURING SEPSIS All treatments, procedures and agents used to restore homeostasis and maintain normal anatomy and physiology are regarded as biological response modifiers (For review see: Meakins, 1981; Nohr & Meakins, 1985). Despite the difficulties in selecting the appropriate modifier, and evaluating their action, they appear to hold some promise in modifying the metabolic response to injury/sepsis. A list of some of the biological response modifiers is shown below (Nohr & Meakins, 1985). Biological agents Endogenous Tissues and cells Hormones Passive immunization Target-directed monoclonal antibodies Exogenous Specific: active immunization Non-specific: adjuvants Bacillus Calmette-Guerin (BCG) Muramyl dipeptide (MDP) Zymosan Drugs Corticosteroids Free radical scavengers Iron-binding compounds Prostaglandin related drugs Levamisole Opiate antagonists Insulin release inhibitors Others General Methods Restore anatomy and physiology Surgery Nutrition Plasmapheresis Fever Some of these modifiers will be briefly considered. Thymic extracts, namely a derivative of thymopoetin, TP5, increases survival in a septic burned animal model (Waymak et al , 1984). The role of some hormones (steroids and counterregulatory hormones) has already been mentioned. The important role of endotoxin (LPS) in the pathogenesis of sepsis suggests that antibody to LPS may be therapeutically useful as an anti-toxin. However, the most immunologically accessible portion of LPS (the O antigen side chain) varies among gram-negative bacteria. Nevertheless, animal models and a human trial using human anti-E. coli J5 antiserum have shown protection against the lethal effects of gram-negative sepsis (Dunn & Ferguson, 1982; Ziegler et al , 1982). It has also been demonstrated (Beutler et al , 1985) that mice treated with a polyclonal antiserum directed against mouse cachectin become resistant to the lethal effects of lipopolysaccahride (LPS). Thus the use of monoclonal antibodies may also have potential therapeutic applicability. Fibronectin is an opsonic glycoprotein important in promoting phagocytosis by cells of the reticuloendothelial system. Plasma levels of this glycoprotein are decreased in septic patients. Scovill et al (1978) have shown that the administration of fibronectin as cryoprecipitate can restore some aspects of host physiology towards normal. Zymosan, a cell wall preparation of Saccharomyces cerevisiae , which can attract and activate leucocytes enhancing their phagocytic properties (Williams et al , 1982), has been used as a pre-treatment (injected intraperitoneally) in rodents and successfully decreased mortality due to peritonitis (Joyce et al ,1978). On the other hand, Goris et al (1986) advocate the use of zymosan, also intraperitoneally, to induce multiple organ failure and 'sepsis without bacteria'. The role of prostaglandin inhibitors in the modification of the metabolic response to sepsis has also being discussed previously. Opiate receptors in the central nervous system may mediate some effects of septic shock, possibly in conjunction with opiate receptors in the periphery (Nohr & Meakins, 1985). Administration of the opiate antagonist, naloxone hydrochloride, can reverse the hypotension of endotoxin shock to varying degrees, to increase mean and pulse blood pressure, elevate respiratory rate, prevent acidosis, attenuate hypoglycaemia, stabilize lysosomal membranes and elevate white blood cell and platelet concentrations (Groeger et al , 1983; Hinshaw et al , 1984; Ipp et al , 1984). However, some of the reasons to question their therapeutic value for septic shock include studies evaluating survival time, which have shown no long-term survival benefits; naloxone's cardiovascular effects have mostly been assessed during the early phase of shock; the effects on cardiovascular parameters due to naloxone are often short-lived and minimally beneficial; despite increasing arterial pressure naloxone may also produce adverse actions, such as regional vascular resistance. Species variability has been reported for some of naloxone's vascular actions; and finally, naloxone stimulates the sympathoadrenal system which may impair perfusion of the microcirculation (Hinshaw et al , 1984). Hyperinsulinaemia is a common event as part or the metabolic response to sepsis (Biesel & Wannemacher, 1980). One view is that the metabolic changes of sepsis is due to the lack of response to the anabolic action of insulin (Frayn , 1986). Hyperinsulinaemia, itself, has been implicated as the cause, rather than the product of insulin resistance (Marangou et al , 1986). Therefore, drugs modulating insulin release can also be used not only to assess a possible reversal in insulin sensitivity during sepsis, but also to try to reverse some of the metabolic changes mediated under the conditions of elevated plasma insulin. Diazoxide (Henquin et al , 1982), mannoheptulose (Simon et al , 1972) and streptozotocin (Bolaffi et al , 1986) are examples of such drugs and their action during sepsis will be discussed in later Chapters. Fever It has been observed clinically that the absence of hyperthermia during sepsis may be associated with a poor outcome. Fever can increase leucocyte capacity to mobilise, to kill and to react to bacterial antigens as well as promoting the bacteriostatic effects of serum ( Kluger, 1981; Macrowiak & Marling-Casan, 1983; Nohr & Meakins, 1985). Surgery Although surgery itself is immuno-suppressive, delayed type hypersensitivity responses can be improved by the drainage of visceral abscesses (Meakins et al , 1979). In addition, the drainage of an intra-abdominal abscess can also restore the function of 'failed' organs (Polk & Shields, 1977), so that organ failure should be regarded as a potentially reversible 'organ dysfunction' (Bohnen et al , 1983). Nutrition Refeeding via oral or intravenous route has been shown to be valuable in correcting several abnormalities in immunological function. Total parenteral nutrition is capable of restoring skin test reactivity, restoring lymphocyte transformation, correcting serum immunoglobulin levels and restoring specific antibody responses and neutrophil chemotaxis (Nohr & Meakins, 1985). However, the choice of the best method of nutritional support during sepsis, whether by the use of enteral or parenteral route, is still controversial (Elwyn, 1980; Kettlewell, 1982). While a number of studies have claimed excess carbohydrate intake may serve as a physiological stress rather than a nutritional support, others have questioned the effect of intravenous fat in imparing the reticuloendothelial system function (Jastrand et al , 1978; Carpentier et al , 1979; ). Despite controversial results, amino acid solutions enriched with branched-chain amino acids have been advocated in trying to correct the amino acid inbalance caused by sepsis (Freund et al , 1978; Cerra et al , 1985; Harry et al , 1986). The infusion of carnitineindependent fats (short and medium chain fatty acids) as energy source appears to be promising (Denninson et al , 1986). Nevertheless, the aim of nutritional support in the hypermetabolic septic patient should be to provide appropriate proportions of the substrates based on the observed metabolic alterations in order to minimise complications and maximise benefit. OBJECTIVE OF THE STUDY Sepsis is an important clinical problem causing morbidity and mortality in surgical patients (Cerra, 1982; McGowan & Gorbach, 1983; Wilmore et al , 1983; Meakins, 1985). Survival depends on the adequacy of the immunological reactions to contain and eliminate invading microbes, and on a series of integrated physiological and metabolic responses necessary to maintain energy production and cell function thoughout the body (Clowes et al , 1985). The liver has a central role in intermediary metabolism during sepsis (Wannemacher et al , 1979; Cerra et al , 1979), and yet liver function is frequently disturbed by sepsis (Royle & Kettlewell, 1980; Duff, 1985). It is well recognized that human clinical studies of sepsis are not easy to perform, not only because of restrictions on their scope for ethical reasons, but also because of the difficulty in establishing comparable groups of patients. On the other hand, there are many experimental animal models of sepsis, each with inherent characteristics, which are sometimes of questionable relevance to clinical practice. It is therefore important to study the fundamental changes in liver metabolism, induced by sepsis, in an experimental model which mimics as closely as possible the clinical situation. It is clear that the effect of infection varies with time and yet, surprisingly, there is no information on the time-course of changes within a single experimental model of sepsis. We have therefore studied the temporal changes of the effects of peritonitis on the hepatic metabolism of the rat. The rat liver behaves remarkably like the liver.\\\ccv v00v.bv.b of the human, and the particular septic model used is very reproducible and mimics abdominal sepsis in humans (Wichterman et al , 1980; McGowan & Gorbach, 1983 ). The elucidation of the metabolic changes within the liver and their relationship to hormonal levels could have important clinical consequences. A comparison between the changes in hepatic metabolism during sepsis, and those found in parenterally fed patients was made in the last Chapter. CHAPTER 2 MATERIALS AND METHODS Biochemicals and Enzymes Animals Sepsis induced by caecal-ligation and puncture Sham-operated rats Bile duct-ligated rats Preparation of blood, plasma and tissue samples Determination of metabolites Insulin determination Measurements of lipogenesis and cholesterolegenesis Preparation of isolated liver cells Incubation procedure Calculation of rates Preparation of microsomes Determination of enzyme activities in liver: PEPCK Glucose-6-phosphatase HMG CoA Reductase Measurements of radioactivity Statistical Analysis Biochemicals and Enzymes [1-14C]Oleate, [14C]HMGCoA, [3H]mevalonolactone and 3H2O were purchased from the Amersham International, Amersham, Bucks, U.K. Others biochemicals and enzymes were obtained from Boehringer Corp. (London)Ltd., Lewes, Sussex, U.K. Animals Male rats of the Wistar strain weighing about 250g were fed ad libitum on a breeding diet for rats and mice (Special Diet Services, Witham, Essex, U.K.) containing 4% w/w lipid, 21% crude protein and 52% carbohydrate; the residue was non-digestible material. The animals were maintained at an ambient temperature of 22±2° under a 12h light/12h dark cycle (lights on 07.30h). Sepsis induced by caecal-ligation and puncture Operations were carried out under light ether anaesthesia. A midline laparotomy was performed. The caecum was mobilized by incising the mesocaecum and the faeces were milked into the caecum, which was then ligated with a single 3-0 silk ligature in such a manner that bowel continuity was maintained. The antimesenteric surface of the caecum was punctured once with a 21-gauge needle, and the bowel returned to the abdominal cavity. The abdominal wall was closed in two layers and all rats received 0.9% (v/v) NaCl (2.5ml/100g body wt.) subcutaneously. With this procedure an experimental peritonitis is created (Wichterman et al, 1980) with a microbial flora that closely approximates that of human peritonitis (McGowan & Gorbach, 1983) and therefore has proven to be a suitable model of the human disease. Water was offered ad libitum. The rectal temperatures of the septic rats were not different to those of the sham-operated group. It has been reported that blood pressure of rats made septic by this procedure is unchanged (Wichterman et al, 1980). The single puncture with a small neddle gauge (21 G) was chosen so that the peritonitis could develop at a slower rate allowing animals to starve and survive up to 48 h. The mortality rate was 3.7% (one out of 27) at 12 h, 5.5% (two out of 36) at 24 h and 18.9% (15 out of 79) at 48 h , which was lower than that observed by Wichterman et al (1980) using a single puncture operation. Sham-operated rats In the sham-operated animals the caecum was also mobilized by incising the mesocaecum and then immediately returned to the abdominal cavity. All the other steps of the operation and post-operative care were similar to those described for the caecum-ligated animals. Sham-operated rats recovered more promptly from the anaesthesia and did not show any of the signs of sepsis, as described by Wichterman et al (1980), at the various times they were studied. Bile duct-ligated rats A group of animals was subjected to total interruption of their bile acid hepato-enteric circulation. In these animals, after the midline laparotomy was performed, all the small bowel was mobilized up to the duodenal-jejunal junction so that the bile duct entry into the duodenum could be visualized. The bile duct was isolated at approximately one centimeter from the entry point and ligated with a single 3-0 silk ligature . The caecum was then mobilized and all the other steps of the operation and the post-operative care were the same as to those in either experimental (caecum-ligated and punctured) or control (sham-operated) animals. Preparation of blood, plasma and tissue samples Rats (starved 12, 24 or 48h post-operation) were anaesthetized with pentobarbital (50mg/kg body wt.), injected intraperitoneally, and a laparotomy performed. Arterial blood from the abdominal aorta was collected using heparinized syringes (2 ml) and the liver removed. Part of the latter was immediately freeze- clamped (Wollenberger et al, 1960). This was achieved by freezing the tissue between aluminium tongs cooled in liquid nitrogen (i.e. at a temperature of about -190°C) which lowered the temperature of the tissue to about -80°C in less than 0.1 sec. If not used for metabolite determination on the same day, freeze-clamped livers were kept stored in aluminium foil in a liquid nitrogen-refrigerator tank. To measure hepatic metabolites freeze-clamped livers were ground to a fine powder with liquid nitrogen-cooled porcelain mortar and pestel (Williamson et al, 1967) and extracted in ice-cooled 0.75M HClO4. This rapidly inactivated and precipitated protein which was removed by centrifugation at 15000 rpm for 20 min. This HClO4 extract was then neutralised with a 20% solution of potassium hydroxide (KOH) (Analar, BDH) and the metabolic intermediates measured by spectrophotometric methods. The remaining part of the liver was used for measurements of lipid and cholesterol synthesis or determination of enzyme activity. Metabolites were extracted from total blood with 0.75M HCl04 as previously described by Williamson et al (1967). The tubes containing blood and HClO4 were centrifuged at 3000 rpm for 10 min. The supernatant was decanted into pre-weighed labelled 10ml plastic tubes which were then reweighed. One drop of universal indicator (BDH) was added to the supernatant followed by a 20% solution of potassium hydroxide (KOH) (Analar, BDH), which was added drop-wise until the pH was between 7 and 8. After allowing to stand in ice for 10 min, the tubes were reweighed and centrifuged at 3000 rpm for 10 min, and the supernatant, now a neutral extract, was used for metabolite analysis. Pyruvate and acetoacetate were measured immediately after neutralisation and the other metabolites were measured on the same day of sampling or after storage at -20°C within two weeks. Part of the total blood sample was centrifuged at 3000 rpm for 10 min and the plasma was separated and used for determination of free fatty acids, triacylglycerol and insulin concentrations and measurement of the specific radioactivity of plasma water. Determination of metabolites Before giving details about the various enzymatic methods used to determine metabolite concentrations, the principles behind enzymatic analysis are introduced. The enzymatic assay of metabolic substrates is based on the principle that a specific enzymatic reaction in which the substrate participates is coupled with the reduction of NAD/NADP or oxidation of NADH/NADPH. The pyridine nucleotides (NAD, NADP) absorb light at 260 nm, and in the reduced state (NADH, NADPH) they have an additional absorption band with a maximun at 340 nm. By measuring the optical density at 340 nm, the enzymatic conversion of the substrate can be followed directly in a spectrophotometer cuvette. Regardless of whether NAD accepts H+ or whether NADH donates H+, at this wavelength the optical density increases or decreases by 6.22 units (light path 1 cm) with the production or consumption of 1 µmole of NADH/NADPH. Since in a specific enzymatic reaction, 1 µmole of substrate usually co-reacts with 1 µmole of NAD/NADP (or NADH/NADPH), the change in optical density will reflect accurately the amount of substrate consumed by the reaction. Provided the assay conditions are optimum, conversion of the substrate is practically complete and the optical density difference (ODD) can be used to calculate the concentration of the substrate in the blood or tissue sample by multiplying with an appropriate dilution factor. Since many enzymatic reactions are equilibrium reactions, in order to make an end-point measurement the equilibrium of the reaction has to be displaced such that it favours the complete consumption of the substrate. The reaction equilibrium can be influenced by several factors such as increase in substrate or cofactor concentration, variation of pH, presence of trapping agents, or the use of regenerating reactions in which one of the co-substrates may be regenerated by a secondary reaction. If none of the reactants or products of an enzymic reactions is measurable spectrophotometrically, it is often possible to transform one of the products by another enzymatic reaction which in turn can be easily measured (e.g., measurement of glucose, non-esterified fatty acids and triacylglycerols). The former reaction, in which the substrate to be determined is transformed, is known as the auxiliary reaction, whereas the reaction used for the actual measurement is known as the indicator reaction. Both reactions can usually be carried out in the same assay mixture (Bergmeyer H.-U.,1963). Specificity of an enzymatic assay depends on the purity of the enzyme preparation whereas precision depends on the provision of optimum assay conditions. The sensitivity of enzymatic assays is limited by the fact that sufficient conversion of NAD/NADP (or NADH/NADPH) must occur to the extent of producing a measurable change in the optical density. The following metabolites were measured by standard enzymatic methods as described below: Determination of D-Glucose Blood and liver glucose were measured according to the method described by Slein (1963). Reaction sequence: a. Auxiliary reaction Mg++ GLUCOSE + ATP ——————————————> GLUCOSE-6- PHOSPHATE + ADP b. Indicator reaction: Mg++ glucose-6-phosphate GLUCOSE-6-PHOSPHATE dehydrogenase ——————————————————————> 6- PHOSPHOGLUCONATE + NADP+ + NADPH + H+ At pH 7.5, the equilibrium for the indicator reaction is far to the right which ensures the completion of both reactions (since glucose-6-phosphate formed in the former is rapidly used up in the latter reaction). Although hexokinase catalyses the phosphorylation of several other monosaccharides, specificity is provided by glucose-6-phosphate dehydrogenase (G6PD) with which hexose or pentose esters other than glucose-6-phosphate do not react. Buffer solution for assay: 20ml 0.1M tris buffer pH 8.0 2ml 0.1M magnesium chloride 2ml 0.01 M ATP 2ml 1% NADP 0.13ml of G6PD (1mg/ml) This was prepared freshly for each assay. The total volume in each cuvette was 2.0ml. In the sample cuvettes, this consisted of 0.1ml of neutralised HClO4 extract, 0.9ml of distilled water and 1ml of assay buffer; in the control cuvette 1ml of water and 1ml of assay buffer was added. The cuvettes were read at 340nm before , at 10min and 15min after addition of 0.010ml of hexokinase. Measurement of L-(+)-Lactate Concentrations of blood and liver lactate were measured according to the method described by Hohorst (1963). Reaction sequence: Lactate dehydrogenase LACTATE + NAD+ ——————————————> PYRUVATE + NADH + H+ The equilibrium of this reaction lies well on the side of lactate and NAD. Therefore, in order to ensure the complete conversion of lactate, the reaction products have to be removed from the equilibrium. Protons are trapped by an alkaline reaction medium; the pyruvate reacts with hydrazine hydrate in the buffer solution to form pyruvate hydrazone and, in addition, a large excess of NAD and enzyme is used to obtain a sufficiently rapid end point. Lactate dehydrogenase reacts only with L-(+)-lactate and thus provides specificity for the assay. Buffer solution for the assay: 40ml 0.2 M Tris 5ml hydrazine hydrate 100% 25mg EDTA Made up to 100ml of distilled water. The pH of the buffer solution was adjusted to pH 9.5 with 5M hydrochloric acid and it was stored for up to two weeks at 4°C. Before use, 1 ml of 1% (w/v) NAD was added to every 10 ml of assay buffer. The total volume in each cuvette was 2.0ml. In the sample cuvettes this consisted of 0.2ml of neutrilised HClO4 extract, 0.8ml of water and 1ml of assay buffer; the control cuvette contained 1ml of water and 1ml of assay buffer. All cuvettes were read at 340 nm before and at 35 and 45 min after the addition of 0.02ml of lactate dehydrogenase. Measurement of Pyruvate and Acetoacetate Pyruvate and acetoacetate share very similar assay conditions and therefore were measured sequentially in the same sample (same cuvette) according to combination of methods respectively described by Hohorst et al (1959) and a Williamson et al (1962). Reaction sequence: lactate dehydrogenase PYRUVATE + NADH + H+ ————————————————————> LACTATE + NAD ACETOACETATE + 3-OH-butyrate dehydrogenase NADH + H+ —————————————> 3- HYDROXYBUTYRATE + NAD+ The equilibrium of the first reaction at pH 7.0 is sufficiently far to the right to ensure a quantitative measurement of pyruvate levels provided that the NADH concentration is not less than 0.01 mM. At the same pH and with a suitable excess of NADH, at least 98% of the acetoacetate is reduced to D-(-)-3- hydroxybutyrate. However, due to the low activity of 3-hydroxybutyrate dehydrogenase preparations the second reaction proceeds at a much slower rate than the former. Buffer solution for the assay: 10ml of 0.1 Potassium phosphate buffer pH 6.9 1 ml of 0.5% (w/v) NADH cuvette A fresh solution was prepared for each assay. The total volume in each was 2 ml. In the sample cuvettes, this consisted of 1ml of neutralised HClO4 extract and 1ml of assay buffer. The control cuvette contained 1ml of water and 1ml of assay buffer solution. The cuvettes were read at 340 nm before and 10min after the addition of of 0.01ml of lactate dehydrogenase. Hydroxybutyrate dehydrogenase (0.01ml) was then added to they were read again at 35 and 45 min thereafter. 3- each cuvette and Measurement of D-(-)-3-hydroxybutyrate Blood and hepatic concentrations of 3-hydroxybutyrate were measured according to the method described by Williamson et al (1962). Reaction sequence: 3-hydroxybutyrate dehydrogenase 3-OH-BUTYRATE + NAD+ ————————————> NADH + H+ ACETOACETATE + The equilibrium of the reaction at pH 8.0 is reached when approximately 40% of the 3-hydroxybutyrate is oxidised to acetoacetate. However, the presence of hydrazine in the buffer solution traps the acetoacetate formed as a hydrazone and the reaction proceeds quantitatively from the left to the right. Buffer solution for the assay: 70 ml 0.1M tris buffer pH 8.5 0.25 ml of hydrazine hydrate 100% 25mg of EDTA Made up to 100 ml with distilled water. The pH of the assay buffer solution was adjusted to pH 8.5 with 5M hydrochloric acid and it was stored for up to two weeks at 4°C. Before use, 1 ml of 1% (w/v) NAD was added to 10ml of assay buffer for each assay. The total volume in each cuvette was 2 ml. In the sample cuvettes, this consisted of 0.5 ml of neutralised HClO4 extract, 0.5ml of water and 1 ml of assay buffer; the control cuvette contained 1 ml of water and 1 ml of assay buffer. The cuvettes were read at 340 nm before and 50 and 60 min after the addition of 0.01 ml of 3-hydroxybutyrate dehydrogenase. Measurement of L-Alanine Blood and hepatic concentrations were measured according to the method described by Williamson et al (1967). Reaction sequence: alanine dehydrogenase ALANINE + H2O + NAD+ ——————————————> PYRUVATE + NH4+ + NADH In the presence of low H+ ion concentration, at pH 9.0, this reaction proceeds quantitatively from the left to the right. Hydrazine hydrate is included in the buffer solution in order to trap the pyruvate formed by conversion to pyruvate hydrazone. Buffer solution for the assay: 40ml 0.2M tris buffer 04ml hydrazine hydrate 100% 25mg EDTA Made up to 80 ml with distilled water. The pH of the buffer solution was adjusted to pH 9.0 with 5 M hydrochloric acid and it was stored for up to 14 days at 4°C. Before use, 1ml of 1% (w/v) NAD was added to 10ml of assay buffer for each assay. The total volume per cuvette was 2 ml. In the sample cuvettes, this consisted of 0.5 ml of neutralised HClO4 extract,0.5 ml of water and 1 ml of assay buffer; the control cuvette contained 1 ml of water and 1 ml of asssay buffer.All cuvettes were read at 340 nm before and at 50 and 60 min after addition of 0.01ml of alanine dehydrogenase. Determination of D-3-Phosphoglycerate, D-2-Phosphoglycerate and Phosphoenolpyruvate and Pyruvate Hepatic concentrations of pyruvate, PEP, 2-phosphglycerate and phosphoglycerate were measured according to the method described by Czok 3& Eckert (1963). Reaction sequence: lactate dehydrogenase a. LACTATE + NAD+ ——————————————> PYRUVATE + NADH + H+ pyruvate kinase b. PYRUVATE + PHOSPHOENOLPYRUVATE + ADP Mg2+, K+ ATP ———————————> enolase c. PHOSPHENOLPYRUVATE ———————————> 2- PHOSPHOGLYCERATE Mg2+ phosphoglycerate mutase d. 2-PHOSPHOGLYCERATE —————————————> 3- PHOSPHOGLYCERATE At pH 7.4 quantitative conversion of the over-all reaction is assured because of the positions of the equilibrium state of the reactions catalysed by pyruvate kinase and lactate dehydrogenase being far to the right. 3-Phosphglycerate can only be determined under the conditions described here if the assay mixture contains less than 10-3 M inorganic phosphate. The determination of 2-phosphoglycerate, phosphoenolpyruvate and pyruvate is not affected by phosphate. Specificity is secured by the high specificity of the reactions catalysed by enolase and pyruvate kinase. Buffer solution for assay: 10 ml Tris HCL buffer ph 7.4 0.1M 1 ml Mg Cl2 0.1 M 3 ml KCl 1.0 M 1 ml ADP 0.02 M 0.5 ml NADH 0.5 % This was prepared freshly for each assay. The total volume in each cuvette was 2.0 ml. In the sample cuvettes, this consisted of 0.5 ml of neutralised liver HClO4 extract, 0.5 ml of distilled water and 1 ml of assay buffer; in the control cuvette 1 ml of water and 1 ml of assay buffer were added. The cuvettes were read at 340 nm before and for 5-10 min after the addition of lactate dehydrogenase (0.01ml); pyruvate kinase (0.01 ml) was added and the changes in the optical density followed for 5-10min; enolase (0.01ml) was added and the cuvettes were read continuously for 5-10 min; finally phosphoglycerate mutase (0.01 ml) was added to the cuvettes and the change in the optical density followed for 15-20 min. All cuvettes were read using a Gilford recording spectrophotometer. Measurement of Dihydroxyacetone Phosphate, Glyceraldehyde-3-phosphate and Fructose-1,6-bisphosphate D- Hepatic concentrations of dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP) and fructose-1,6-bisphosphate (FDP) were determined according to the method described by Bücher & Hohorst (1963). Reaction sequence: GDH a. L-(-)GLYCEROL-1-PHOSPHATE + NAD+ —————> DHAP +NADH + H+ TIM b. DIHYDROXYACETONE PHOSPHATE ——> GLYCERALDEHYDE -3- PHOSPHATE (GAP) aldolase c. GAP + DHAP ———————> FRUCTOSE-1,6-BISPHOSPHATE (FDP) The equilibrium state of the reactions ensures that the overall reaction proceeds quantitatively. For each mole of FDP formed 2 moles of NADH are oxidised, therefore, the inclusion of the triosephosphate isomerase (TIM) reaction increases the sensitivity of the assay. Buffer solution for the assay: 10 ml Tris HCl ph 7.4 1 ml NADH 0.5% The total volume in each cuvette was 2 ml. In the sample cuvettes, this consisted of 1 ml of neutralised liver HClO4 extract and 1 ml of assay buffer solution; the control cuvette contained 1 ml of distilled water and 1 ml of assay buffer. All cuvettes were read continuously using a Gilford spectrophotometer as follows: before and for 5-10 min after addition of glycerol-3-phosphate dehydrogenase (GDH); before and for 5-10 min after addition of triophosphate isomerase (TIM) and before and for 10-15 min after addition of aldolase. Measurement of Adenosine-5'-Triphosphate and D-glucose-6- phosphate Adenosine-5'-triphosphate (ATP) and glucose -6-phosphate were measured with a combination of the methods described by Lamprecht & (1963) and Hohorst (1963); using the same cuvette. Trautschold Hexokinase phosphorylates glucose with ATP in the presence of Mg++ to give glucose-6phosphate. Glucose-6-phosphate dehydrogenase catalyses the oxidation of glucose-6-phosphate with NADP. Each mole of ATP forms 1 mole of NADPH. Reaction sequence: hexokinase GLUCOSE + ATP ——————————> GLUCOSE-6-PHOSPHATE (G-6-P) + ADP G6P-DH G-6-P + NADP+ ———————> 6-PHOSPHOGLUCONO-LACTONE + NADPH + H+ In the presence of equivalent concentrations of glucose and Mg2+, ATP is virtually quantitatively converted to ADP by hexokinase. The equilibrium of the glucose-6-phosphate reaction lies to the right. Buffer solution for the assay: 10 ml Tris 0.1M pH 7.4 1 mg/ml NADP 2 ml Mg Cl2 0.1 M 0.5 ml 2 Glucose 0.1 M This was prepared freshly for each assay. The final volume per cuvette was ml. In the sample cuvettes this consisted of 0.5 ml of liver HClO4 extract, 0.5 ml of distilled water and 1 ml of buffer solution. The control cuvette contained 1 ml of distilled water and 1 ml of buffer solution.Cuvettes were read at 340 nm before and 5-10 min after 0.005 ml of glucose-6-phosphate dehydrogenase was added and subsequently 10-15 min after hexokinase (0.005 ml) was added. Determination of Acetyl-Coenzyme A Hepatic Acetyl-CoA was measured by the method described by Decker (1963). Reaction sequence: citrate synthase ACETYL-SCoA + OXALOACETATE + H2O ———————> CITRATE + CoASH + H+ malate dehydrogenase L-MALATE + NAD+ ————————————————> OXALOACETATE + NADH + H+ At pH 8.0 the equilibrium of the over-all reaction lies to the right and acetyl-CoA is used quantitatively. Buffer solution used for assay: 15 ml Tris 0.1 M 1 ml L-malate 0.1 M 0.05 ml NADH 1 ml NAD 1 ml ml. pH 8.0 0.5 % 1% malate dehydrogenase This was made freshly for each assay. The total volume per cuvette was 2 In the sample cuvettes this consisted of 1 ml of liver HClO4 extract and 1 ml of buffer solution. The control cuvette contained 1 ml of distilled water and 1 ml of buffer solution. Cuvettes were read before and at 15 min after addition of citrate synthase (0.01 ml). Determination of L-Glutamate and Glutamine Hepatic glutamate was measured according to the method described by Bernt & Bergmeyer (1963). The determination of glutamine was also based on this enzymatic technique. Reaction sequence: glutamate dehydrogenase L-GLUTAMATE + NAD+————————————————> OXOGLUTARATE + NADH + NH4+ glutaminase L-GLUTAMINE –––––––––––––––––––––––––> L-GLUTAMINE + NH4+ The equilibrium lies far to the left. However, under alkaline conditions 9.0) and the presence of excess NAD, oxoglutarate can be trapped with (pH hydrazine and glutamate quantitatively oxidized to oxoglutarate. Buffer solution for assay: 2 ml Hydrazine hydrate 100 % 20 ml Tris 0.2 M 25 mg EDTA Made up to 40 ml with distilled water and the pH (9.0) adjusted using 5 N HCl. This could be kept at 4°C for up to 14 days. To be used for assay (cocktail), NAD (1mg/ml) and ADP (0.5mg/ml) were added to the buffer solution. The total volume per cuvette was 2 ml. In the sample cuvettes this consisted of 1 ml of liver HClO4 extract and 1 ml of cocktail. The control cuvette contained 1 ml of distilled water and 1 ml of cocktail. Cuvettes were read before and 30-40 min after the addition of glutamate dehydrogenase (in glycerol). Glutamine In order to measure glutamine liver HClO4 extracts were pre-incubated in the presence of glutaminase under the contitions described below: 0.5 ml Liver extract 0.5 M Acetate buffer 0.5 M 0.01 ml NH2OH 1M pH 4.8 0.02 ml Glutaminase [5 mg/ml] The total volume per tube was made up to 1 ml with distilled water. An internal standard was added, consisting of 1 mM glutamine (0.2ml) and distilled water (0.8 ml). Tubes were mixed and incubated at 37°C for 45 min. These were then cooled, 0.5 ml of the mixture taken and the concentration of glutamate measured by the enzymatic method described above (Bernt & Bergmeyer, 1963). As glutamine is converted to glutamate by glutaminase, the total concentration of glutamine+glutamate is measured spectrophotometrically. Therefore, the actual concentration of glutamine is calculated by subtracting the concentration of glutamate measured in the liver extract from the total concentration (glutamine + glutamate) found in the glutaminase pre-incubated extract. Measurement of Non-esterified fatty acids The method of Shimizu et al (1979) was used for the measurement of non-esterified fatty acids (NEFA), which is based on the activation of NEFA by acyl-CoA synthetase. Reaction sequence: acyl-Co A synthetase NEFA + COENZYME A + ATP —————————> ACYL COENZYME A + AMP+ PPi myokinase AMP + ATP ———————> 2 ADP pyruvate kinase 2 ADP + PHOSPHOENOLPYRUVATE ————————-> 2 ATP + 2 PYRUVATE lactate dehydrogenase 2 PYRUVATE + 2 NADH LACTATE + 2 NAD+ ————————————————> 2 The reaction calalysed by acyl-CoA synthetase favours the production of acyl coenzyme A provided the pH is 8.0 and there are sufficient amounts of ATP and coenzyme A. Therefore, the production of AMP is used as a measure of NEFA activation through an indicator system provided by successive reactions catalysed by myokinase, pyruvate kinase and lactate dehydrogenase. The equilibrium state of all three reactions in this indicator system is sufficiently far to the right to ensure that the overall rate of the reaction is limited only by the concentration of NEFA. Specificity for the method is enhanced fact that Acyl CoA synthetase only activates monocarboxylic acids with 6 to 18 carbon atoms and does not affect other carboxylic acids and lipids. Buffer solution for the assay: 200 ml 0.1 M tris pH 8.0 35.1 mg EDTA (o.6 mM) by the 406.6 mg magnesium chloride (10mM) 2 ml Triton X-100 This was stored at 4°C. Immediately before each assay, the following reagents were added to 36 ml of buffer solution: 1.6 ml NADH 0.5% (w/v) 1 ml ATP 0.1 M 1 ml phosphoenolpyruvate 0.2 M 0.2 ml myokinase 2mg/ml 0.4 ml pyruvate kinase 1mg/ml 0.2 ml lactate dehydrogenase 10mg/ml The total volume per cuvette was 2.005 ml. The sample cuvettes contained 0.04ml of plasma, 1.95ml of buffer mixture and 0.015ml of acyl-CoA synthetase. The plasma volume was replaced by 0.04ml of water in the control cuvette. All cuvettes were read at 340 nm before and 20 min after the addition of coenzyme A. Further readings were taken at 5 min intervals until the readings of the optical density were constant. Measurement of Triacylglycerol Plasma triacylglycerol concentration was measured according to the method described by Eggstein & Kreutz (1966). Plasma triacylglycerol were initially hydrolysed to glycerol and non-esterified fatty acids. The glycerol was measured by the enzymatic techique described below. The concentration of measured in neutralised HClO4 extract was subtracted from the free total glycerol, glycerol measured after alkaline hydrolysis of the plasma sample to obtain glyceride-glycerol concentration which, in turn, was taken to be an estimate of the the triacylglycerol content of the plasma. Reaction sequence: a. Hydrolysis: at 70°C TRIACYLGLYCEROL + 3 H2O ————————> GLYCEROL + 3 FATTY ACIDS alchoholic KOH b. Indicator reaction: glycerokinase pyruvate kinase lactate dehydrogenase GLYCEROL + PHOSPHOENOLPYRUVATE ——————> GLYCEROL-3-PO4 + LACTATE ATP ADP ATP NADH NAD+ Hydrolysis: To 0.2 ml of plasma 0.5ml of alcoholic KOH (0.5N KOH in 98% ethanol) was added. The mixture was incubated at 70°C for 30 min and then cooled. Thereafter, 1.5ml of 0.1 M MgSO4 was added to precipitate protein, and the samples were centrifuged at 3000 rpm for 10 min. The triacylglycerol present in plasma produced equimolar quantities of glycerol. This glycerol produced by hydrolysis was contained in the supernatant solution, 0.5ml of which was used for the enzymatic measurement of total glycerol. Measurement of glycerol Reaction sequence: glycerokinase GLYCEROL + ATP ———————————> GLYCEROL-3-PO4 + ADP pyruvate kinase ADP + PHOSPHOENOLPYRUVATE —————————> ATP + PYRUVATE lactate dehydrogenase PYRUVATE + NADH + H+ ————————————> LACTATE + NAD+ The equilibrium state for all three reactions can be acheived at pH 7.4 and in the presence of Mg++ ions. It is far to the right to ensure that there is a quantitative comsumption of glycerol in the first reaction and that the indicator reactions can rapidly proceed to completion. Buffer solution for the assay: 30 ml 0.1 M tris buffer pH 7.4 3 ml 0.1 M magnesium chloride 35 mg phosphenolpyruvate 50 mg ATP 12.5 mg NADH 0.2 ml lactate dehydrogenase 0.2 ml pyruvate kinase This was made up immediately before use and the pH adjusted to pH 7.4 with 0.2 M tris buffer. Triacylglycerol The total final volume per cuvette was 2 ml. In the sample cuvettes, 0.5ml of the hydrolysed plasma sample was added to 1 ml of assay buffer and 0.5 ml of distilled water. Glycerol The sample cuvettes contained 1 ml of assay buffer and 1 ml of neutralised HClO4 extract. Blank cuvettes contained 0.5 ml of buffer mixture and 1.5 ml of distilled water. The control cuvette contained 1 ml of distilled water and 1 ml of assay buffer. All cuvettes were read at 340 nm before and 40 min after addition of 0.01 ml of glycerokinase. They were then re-read every 10 min until there was no further change in the optical density. Calculations for blood and liver metabolites: All metabolite calculations were based on the change in optical density measured at 340 nm in the sample cuvettes following addition of enzyme, and after subtraction of the non-specific change which occured in the control cuvettes. Therefore: Optical density difference (ODD) = Change in absorbance for sample cuvette - Change in absorbance for control cuvettes Since the molar extinction coefficient of NADH is 6.22 cm2/µmol, the amount of substrate in the cuvette = ODD / 6.22 X total volume in cuvette. This is multipied by the dilution factor for each sample to give the concentration of substrate; In the blood: In the liver: 1 g frozen liver = 0.8 ml ; 1 g liver + 4 ml PCA = 4.8 Insulin Determination Insulin was measured by radioimmunoassay with a rat insulin standard (Albano et al, 1972) using activated charcoal for separation of the free and bound hormone. A disequilibrium assay was set up with a 3-day incubation period and all plasma samples were assayed in duplicate with 20 µl of plasma in each tube. The essential principle of the insulin asay, as for any other radioimmunoassay technique, is the reaction of a fixed amount of specific insulin antibody with a mixture of the plasma sample to be assayed and a constant amount of radioactively labbelled pure insulin. The reaction is initiated by incubating the plasma sample with insulin antibody for approximately 48 hours. The radioactively labelled insulin is then added to compete for the remaining binding sites on the antibody complex. The reaction is allowed to approach completion over the subsequent 24 hours, and the antibody-bound insulin is separated from the free hormone by differential adsorption on to albumin-coated charcoal. The distribution of the radioactivity between the free and bound forms of insulin is then determined. It is expected that the amount of radioactively labelled insulin bound to antibody to decrease as the concentration of the unlabelled insulin in the plasma sample increases due to competition for the limited number of binding sites available. Therefore, results obtained with samples of unknown insulin concentration can be compared with standard curves obtained by the measurement of samples to which known amounts of pure insulin standard have been added. Measurements of lipogenesis and cholesterolegenesis Lipogenesis and cholesterolegenesis were measured in vivo with 3H2O as previously described ( Jungas et al, 1968; Robinson et al, 1978). The rats were injected intraperitoneally with 5 mCi (0.25ml) of 3H2O, and tissues removed one hour later; 5 min before removal of tissue the rats were anaesthetized with pentobarbital (50mg/kg body wt.). Heparinised blood was collected for measurement of the specific activity of plasma water. The tissues (in duplicate into 50 ml tubes) were hydrolysed under alkaline conditions by addition of 3 ml of 30% KOH and heated in a water bath at 70°C for 15 min. Absolute alcohol was then added and the samples returned to the 70°C water bath for a further 175 min (Stansbie et al, 1976). The non-saponifiable lipids were extracted three times with light petroleum (b.p. 40-60°C; total vol. 20 ml) and the organic extract was washed twice with distilled water and transfered to scintillation vials. After evaporation of the solvent, the 3H radioactivity of the residue was measured by scintillation counting in half of the samples to determine the total incorporation of 3H2O into the non-saponifiable fraction. Cholesterol and squalene were isolated from the non-saponifiable residue, from the other half of the samples, by chromatography on thin-layer plates of silica gel H developed with chloroform/methanol (99:1, v/v). The plate was dried and the cholesterol-containing band was located by spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v). This area of the plate was scraped into a scintillation vial, and the 3H content of the sterol fractions were determined by scintillation counting (Gibbons et al, 1983). The aqueous phase after extraction of the non-saponifiable lipid was acidified to pH 1.0 with 6N H2SO4, the labelled fatty acid fraction was extracted three times with light petroleum and the organic extract washed twice with distilled water. The extract was transferred into vials to dry (Stansbie et al, 1976) . After evaporation of the solvent, the 3H radioactivity of the residue was determined by scintillation counting. Preparation of isolated liver cells Hepatocytes were prepared essentially as described by Berry & Friend (1969) with the modifications described by Krebs et al (1974). Routine tests indicated that about 90% of the hepatocytes excluded Trypan Blue. Operative technique, liver perfusion and isolation of hepatocytes The rat was anaesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg body wt.). Heparin (150 units; 0.15 ml) was then administered via a saphenous vein. A laparotomy through a vertical mid-line incision was performed. Two midtransverse incisions were made to the left and the right of the mid-line to allow an extended exposure of the abdominal cavity. The portal vein and the descending vena cava were exposed by moving the intestines to the left side of the abdominal cavity. One loose silk ligature (3-0) was placed around the descending vena cava immediately above the right renal vein, and two ligatures, about 1 cm apart, were placed around the portal vein. The portal vein was then cannulated using a 17 gauge metalic intravenous cannula. After withdrawal of the inner needle, the cannula was secured by tying the ligature that was closest to the liver. The other tie was tightened to prevent blood from flowing out of the lower part of the portal vein and to tie off the common bile duct. The cannula was therefore firmly in place and the appearance of blood back-flow at the cannula end from the penetrated portal vein confirmed the success of the cannulation. The thorax was immediately opened, through a transverse incision. A loose ligature was placed around the inferior vena cava just below the heart. A 16 gauge plastic cannula was used to cannulate this vessel through the right atrium and secured with one 3-0 silk tie. Liver perfusion and treatment with collagenase: The method of liver perfusion is that described by Hems et al (1966). A water-jacketed oxygenator was used and the temperature kept approximately at 38°C. The perfusion medium used was the saline of Krebs & Henseleit (1932) from which calcium is omitted because it decreases the activity of the enzyme. The total volume of the perfusion fluid was initially 150 ml. The first 40 ml of the medium passing through the liver were discarded in order to remove the bulk of the blood cells. At this point 30 mg of the crude collagenase (Boehringer Corporation, London), suspended in a small volume of perfusion medium were added. Pure collagenase is not suitable because the contaminating proteinases in the crude preparation play a very important role. The flow rate was approximately 25 ml /min. The perfusion medium was kept in equilibrium with O2/CO2 (95:5 ; v/v). The perfusion was continued for 20-30 min and any leaked perfusion fluid appearing in the abdominal cavity was collected and returned to the reservoir. The action of the enzyme was reflected by the degree of leaking; when this had reached a rate of about 10 ml/min, usually after 25 min, the perfusion was discontinued and the liver was transferred to a beaker. The medium used in the perfusion was added to the broken-up liver tissue as this was put into a nylon cloth (mesh size 0.44 X 0.44 mm) which was on the top of a 100 ml disposable plastic beaker. The liver tissue was then gently broken apart with a pair of scisors and the cells teased out with a plastic spatula, always keeping the perfusion medium (which already contained cells washed out from the liver during the perfusion) flowing through the nylon cloth. The filtrate was centrifuged at 50 g for 2 min and the cells were re-suspended in about 30 ml of the Ca++-containing Krebs-Henseleit medium. The suspension was centrifuged again at 50 g for 2 min and the pellet weighed and resuspended in 16 volumes of the KrebsHenseleit medium containing Ca++, when it was ready to be incubated. Isolated hepatocytes were used immediately after preparation. Incubation procedure The isolated hepatocytes were incubated with constant shaking, at 65 oscillations per min, for 20, 40 or 60 minutes in Krebs-Henseleit saline (Krebs & Henseleit, 1932) containing essentially fatty acid-free albumin (final concentration 2.5% w/v), the gas phase was O2/CO2 (95:5) and the temperature was 37.5° C. The total incubation volume containing 65-95mg wet wt. of cells and added substrates (5mM lactate, 5mM pyruvate, 5mM alanine, 5mM glutamine, 5mM dihydroxyacetone, 5mM butyrate and 2mM [1-14C]oleate; 40µci/ml) was 4 ml. Incubations were stopped by addition of 0.4ml of 20% (w/v) HClO4 , the mixture was centrifuged to remove denatured protein and the supernatant was neutralized with KOH. The precipitate of KClO4 was removed by centrifugation. All incubations with [1-14C]Oleate were performed in duplicate in KrebsHenseleit saline (Krebs & Henseleit, 1932) containing dialysed albumin (fatty acid poor, final concentration 2.5%, w/v). One of each pair was incubated in a 25ml Erlenmeyer flask with a centre well and rubber seal. The incubation was stopped by the injection of 0.4 ml of 20 % (w/v) HClO4 with a long-needled syringe (Whitelaw & Williamson, 1977). Sodium hydroxide (NaOH; 0.3 ml) was injected into the centre well and metabolic CO2 collected by shaking the flasks for a further 2 h at room temperature. The NaOH was then removed for measurements of radioactivity and the deproteinised incubation medium was treated as described above. The reaction in the duplicate flask was stopped by centrifugation for 1 min at 5000 g to separate cells from the medium. Chloroform/methanol [2:1 (v/v); 20 vol.] was added to the cell pellet and the lipids were extracted as described by Folch et al (1957). The radioactivity in the chloroform phase was used as a measure of the [114C]Oleate converted into esterified fats. The disappearance of radioactivity which was extracted by chloroform from the incubation medium (minus pellet) was used to calculate the oleate uptake. This was determined as follows: Potassium phosphate buffer (0.1 M) at pH 7.0 was added to the medium (1.5 ml/0.5 ml). The mixture was extracted with chloroform (6 ml) by shaking for 90 sec. The chloroform and aqueous phases were separated by centrifugation, the upper phase was removed and the organic phase washed with 1 ml of potassium phosphate buffer, pH 7.0. The radioactivity in a sample of the chloroform phase was measured. This method relies on the fact that esterified lipids are not released in appreciable amounts to the incubation medium, as shown by Whitelaw & Williamson (1976). The radioactivity in ketone bodies was estimated by measuring the radioactivity in acid-soluble compounds in the supernatant. Calculation of rates: The time-courses of glucose synthesis from the gluconeogenic precursors and of ketone body formation from long- and short-chain fatty acids, in isolated liver cells, were linear between 20 and 60 minutes, and were calculated as follows: Preparation of microsomes The animals (starved 48h post-operation) were anaesthetized with an intraperitonial injection of pentobarbital (50mg/kg body wt). Each liver was removed, minced and put into a pre-weighed ice-cooled 50 ml beaker containing 10ml of NaF sucrose (0.25 M sucrose; 50 mM NaF) buffer. More NaF sucrose buffer was added to the beaker to a final volume/liver wt. of (10:1). Livers were then homogenized using a glass homogenizer kept in ice. After homogenization samples were centrifuged a 800 g (15 min), 16,000 g (20 min) and again at 16,000 g (20 min) to separate the microsomes from the cell wall, red cells and cytosol during the first spin; from the mitochondria and the lysosomes during the second spin; and from the remaining mitochondria and lysosomes during the third spin. The supernatant of the third spin was carefully transferred to 12.5ml tubes and ultracentrifuged for 1 h at 110,000 g to precipitate the microsomes. The supernatant was then discarded and the microsomes were re-suspended in 20 mM imidazole/chloride - 5 mM dithiothreitol assayed for HMGCoA reductase activity. Determination of hepatic enzyme activity HMGCoA Reductase Buffer solutions required for the assay: Buffer A 20mM Imidazole /Chloride (pH 7.4) 5mM Dithiothreitol Buffer B 350mM Potassium phosphate buffer 40mM EDTA 20mM Dithiothreitol buffer and Cofactors NADP+ 10.5mg in 0.5ml H2O Glucose-6-phosphate 33.5mg in 0.5ml H2O Glucose-6-phosphate dehydrogenase 19U (50µl)+0.45ml H2O These were mixed together. Buffer B (1.38ml) and H2O (0.38ml) were added - The cofactor mixture Microsomes, prepared as described previously, were suspended in 20mM imidazole/chloride - 5 mM dithiothreitol (Buffer A) and incubated in duplicate in the presence or absence of alkaline phosphatase at 37°C for 30 min , so that the total and the expressed amount of enzyme activity could be measured. Before incubation portions (10µl) of the homogenate were taken in duplicate to measure the protein content. Three control incubations each containing buffer A instead of microsomal homogenate. To the pre-incubated microsomes the cofactor mixture was added and a solution of (R,S) [3-14C]HMG-CoA (80,000 dpm; 6.4 nmol; 8µl) in KH2PO4 (0.1 M) was added to each tube giving a final (R,S)-HMG-CoA concentration of 145 µM. Each tube was capped and placed in a water bath at 37°C. The rack in which the tubes were placed was shaken at 70 oscillations per min. Afer 20 min each tube was removed from the water bath and the reaction stopped by the immediate addition of HCl (12 M, 5µl). An internal standard , consisting in a solution in water of (R,S)[2-3H ]mevalonolactone (5µl - 10,986 dpm), was added to each tube. The mevalonic acid was lactonised by incubationg the tubes at 37°C for 30 min. At this stage , if necessary , the samples could be stored for 1-3 days at -20°C. Thin Layer Chromatography of Mevalonolactone To precipitate protein , the tubes were centrifuged at 1000 g for 10 min. Portions (40µl) of the supernatant of each sample were loaded into preadsorbent strips of a multiple channel, glass-backed, Whatman LK5D silica gel TLC plate (20 X 20 cm; 0.8 cm wide channels). The plates were thoroughly dried for 4 h in a TLC tank containing activated silica gel (coarse mesh). They were then developed with ethylacetate/acetone (4:1,v/v) until the solvent, had reached 1 cm from the top of the plate. The TLC tank contained 75 ml of the solvent, was lined with chromatography paper and had been pre-equilibrated for at least 30 min. The plates were dried and the mevalonolactone-containing band was located by spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v) and visualisation under ultra-violet light. This area of the plate was scraped from the plates with a razor blade and transferred into scintillation glass vials. Ethanol (1 ml) was added, followed by 10 ml of a toluene-based scintillation cocktail (PPO/POPOP/Toluene ; w/w/v ; 0.3 : 0.01 : 100). The 3H and 14C contents of the sterol fraction were determined by scintillation counting for 30 min per sample. Microsomal Protein Determination The method of Lowry et al (1951) was used, as modified to assay protein in the presence of sulphydryl reagents, by Geyger and Bessman (1972). The standard curve was constructed using 0-150 µg of bovine serum albumin. After the microsomes were re-suspended in 20mM imidazole/chloride - 5 mM thiothreitol (Buffer A) and were ready for the incubation, a portion of the suspension from each sample (10µl) was taken in duplicate into 10 ml tubes to measure protein. The dithiothreitol was oxidised by heating in the presence of H2O2. Reagents: A. 1 % CuSO4 5 H2O B. 5.4% NaK Tartratel C. 10% Na2CO3 ; 0.5 M NaOH Copper Reagent: 1 ml of reagent A was mixed with 1 ml of reagent B 1 ml of this new mix (A+B) was added to 10 ml of reagent C Phenol Reagent: The phenol reagent (F&C) was diluted with distilled water (v/v; 1:10) Distilled water was added to the standards and the protein-containing tubes up to a final volume per sample of 0.75 ml. The tubes were mixed and 0.75 ml of the copper reagent added; 15 µl of 3% H2O2 were also added to each tube and they were mixed again. The tubes were transferred to a water bath at 60°C for 10 min. Phenol reagent (1.5 ml) was added to each sample and standard , mixed and each tube transferred again to the water bath at 60°C for another 10 min. Tubes were cooled and each standard and each sample was transferred to the same glass cuvette to be read in the spectrophotometer at 750 nM. The standard curve was then constructed and the concentration of protein in the samples calculated based on this standard curve. Calculation of enzyme activity The 14C dpm content of each sample was corrected for the recovery of [23H]mevalonolactone and by subtracting the average 14C dpm value obtained from the control incubations (usually about 25 dpm). The corrected amount of 14C dpm was then converted to pmol of mevalonolactone (the specific radioactivity of the substrate used was 12.5 dpm/pmol). Therefore, the following equation was used to calculate enzyme activity: HMG-CoA reductase activity was expressed as pmol of mevalonolactone produced per min per mg of microsomal protein and was the average of duplicate assays. Measurements of radioactivity Measurements of 3H and 14C radioactivity by liquid scintillation counting were performed using an LS 9800 Liquid Scintillation System (Beckman Instruments Inc., Irvine, CA, USA). The sample to be measured was dissolved in a toluene-based scintillation fluid (10 ml). Depending on the specific scintillation cocktail or radionuclide use, distilled water and or ethanol was added to the vials. The vials were fitted with a screw cap and when all the contents (sample, scintillation fluid and additions ; e.g. water, ethanol) and were counted as described by Williamson et at (1975). The counting procedure is based on the fact that ionising ß-particles produced by the decay of 3H or 14C atoms lose some of their kinetic energy to the scintillant. This energy is converted into photons. Initially the kinetic energy is absorbed by the toluene and is subsequently transferred to the scintillant present in the scintillation fluid. The photon emissions are detected as voltage pulses, proportional in magnitude to the energy of the ß-particles, by two photomultiplier tubes. Only coincident pulses are counted by the machine to eliminate random events. The overall yield for a particular radionuclide will be reduced by several factors, which are termed quenching. The level of quenching was monitored and the counting energy channels optimised for each sample by an automatic quench compensation method. When in the dual-label counting mode (3H-14C), the machine was calibrated with a series of differently quenched samples containing either 3H or 14C of known dpm. The energy channels were set such that there was no spillover from 3H into 14C. The quenching data and shannel setting during either single or dual-lable counting, were stored in the instrument as a 'user counting program'. A microprocessor built into the machine automatically calculated the 3H and/or 14C dpm using this program. Statistical Analysis Experimental results were expressed as the mean ± the standard error of the mean (S.E.M.) accompanied by the number of observations (n). The S.E.M. is mathematically defined as: Results were analysed using the Student t-test. CHAPTER 2 MATERIALS AND METHODS Biochemicals and Enzymes [1-14C]Oleate, [14C]HMGCoA, [3H]mevalonolactone and 3H2O were purchased from the Amersham International, Amersham, Bucks, U.K. Others biochemicals and enzymes were obtained from Boehringer Corp. (London)Ltd., Lewes, Sussex, U.K. Animals Male rats of the Wistar strain weighing about 250g were fed ad libitum on a breeding diet for rats and mice (Special Diet Services, Witham, Essex, U.K.) containing 4% w/w lipid, 21% crude protein and 52% carbohydrate; the residue was non-digestible material. The animals were maintained at an ambient temperature of 22±2°C under a 12h light/12h dark cycle (lights on 07.30h). Sepsis induced by caecal-ligation and puncture Operations were carried out under light ether anaesthesia. A midline laparotomy was performed. The caecum was mobilized by incising the mesocaecum and the faeces were milked into the caecum, which was then ligated with a single 3-0 silk ligature in such a manner that bowel continuity was maintained (Fig. 2.1. & 2.2.). Fig. 2.1. The anti-mesenteric surface of the caecum was punctured once with a 21-gauge needle, and the bowel returned to the abdominal cavity (Fig. 2.2.). Fig. 2.2. The abdominal wall was closed in two layers and all rats received 0.9% (v/v) NaCl (2.5ml/100g body wt.) subcutaneously. With this procedure an experimental peritonitis is created (Wichterman et al , 1980) with a microbial flora that closely approximates that of human peritonitis (McGowan & Gorbach, 1983) and therefore has proven to be a suitable model of the human disease. Water was offered ad libitum . The rectal temperatures of the septic rats were not different to those of the sham-operated group. It has been reported that blood pressure of rats made septic by this procedure is unchanged (Wichterman et al , 1980). The single puncture with a small needle gauge (21 G) was chosen so that the peritonitis could develop at a slower rate allowing animals to starve and survive up to 48 h. The mortality rate was 3.7% (one out of 27) at 12 h, 5.5% (two out of 36) at 24 h and 18.9% (15 out of 79) at 48 h , which was lower than that observed by Wichterman et al (1980) using a single puncture operation. Sham-operated rats In the sham-operated animals the caecum was also mobilized by incising the mesocaecum and then immediately returned to the abdominal cavity (Fig. 2.3.). All the other steps of the operation and post-operative care were similar to those described for the caecum-ligated animals. Sham-operated rats recovered more promptly from the anaesthesia and did not show any of the signs of sepsis, as described by Wichterman et al (1980), at the various times they were studied. Fig. 2.3. Bile duct-ligated rats A group of animals was subjected to total interruption of their bile acid hepato-enteric circulation. In these animals, after the midline laparotomy was performed, all the small bowel was mobilized up to the duodenal-jejunal junction so that the bile duct entry into the duodenum could be visualized. The bile duct was isolated at approximately one centimeter from the entry point and ligated with a single 3-0 silk ligature . The caecum was then mobilized and all the other steps of the operation and the post-operative care were the same as to those in either experimental (caecum-ligated and punctured) or control (shamoperated) animals. Preparation of blood, plasma and tissue samples Rats (starved 12, 24 or 48h post-operation) were anaesthetized with pentobarbital (50mg/kg body wt.), injected intraperitoneally, and a laparotomy performed. Arterial blood from the abdominal aorta was collected using heparinized syringes (2 ml) and the liver removed. Part of the latter was immediately freeze-clamped (Wollenberger et al , 1960). This was achieved by freezing the tissue between aluminium tongs cooled in liquid nitrogen (i.e. at a temperature of about -190°C) which lowered the temperature of the tissue to about -80°C in less than 0.1 sec. If not extracted for metabolite determination on the same day, freeze-clamped livers were kept stored in aluminium foil in a liquid nitrogen-refrigerator tank. To measure hepatic metabolites freezeclamped livers were ground to a fine powder with liquid nitrogen-cooled porcelain mortar and pestle (Williamson et al , 1967) and extracted in icecooled 0.75M HClO4. This rapidly inactivated and precipitated protein which was removed by centrifugation at 15000 rpm for 20 min. This HClO4 extract was then neutralised with a 20% solution of potassium hydroxide (KOH) (Analar, BDH) and the metabolic intermediates measured by spectrophotometric methods. The remaining part of the liver was used for measurements of lipid and cholesterol synthesis or determination of enzyme activity. Metabolites were extracted from total blood with 0.75M HCl04 as previously described by Williamson et al (1967). The tubes containing blood and HClO4 were centrifuged at 3000 rpm for 10 min. The supernatant was decanted into pre-weighed labelled 10ml plastic tubes which were then reweighed. One drop of universal indicator (BDH) was added to the supernatant followed by a 20% solution of potassium hydroxide (KOH) (Analar, BDH), which was added drop-wise until the pH was between 7 and 8. After allowing to stand in ice for 10 min, the tubes were reweighed and centrifuged at 3000 rpm for 10 min, and the supernatant, now a neutral extract, was used for metabolite analysis. Pyruvate and acetoacetate were measured immediately after neutralisation and the other metabolites were measured on the same day of sampling or after storage at -20°C within two weeks. Part of the total blood sample was centrifuged at 3000 rpm for 10 min and the plasma was separated and used for determination of free fatty acids, triacylglycerol and insulin concentrations and measurement of the specific radioactivity of plasma water. Determination of metabolites Before giving details about the various enzymatic methods used to determine metabolite concentrations, the principles behind enzymatic analysis are introduced. The enzymatic assay of metabolic substrates is based on the principle that a specific enzymatic reaction in which the substrate participates is coupled with the reduction of NAD/NADP or oxidation of NADH/NADPH. The pyridine nucleotides (NAD, NADP) absorb light at 260 nm, and in the reduced state (NADH, NADPH) they have an additional absorption band with a maximun at 340 nm. By measuring the optical density at 340 nm, the enzymatic conversion of the substrate can be followed directly in a spectrophotometer cuvette. Regardless of whether NAD accepts H+ or whether NADH donates H+, at this wavelength the optical density increases or decreases by 6.22 units (light path 1 cm) with the production or consumption of 1 µmole of NADH/NADPH. Since in a specific enzymatic reaction, 1 µmole of substrate usually co-reacts with 1 µmole of NAD/NADP (or NADH/NADPH), the change in optical density will reflect accurately the amount of substrate consumed by the reaction. Provided the assay conditions are optimum, conversion of the substrate is practically complete and the optical density difference (ODD) can be used to calculate the concentration of the substrate in the blood or tissue sample by multiplying with an appropriate dilution factor. Since many enzymatic reactions are equilibrium reactions, in order to make an end-point measurement the equilibrium of the reaction has to be displaced such that it favours the complete consumption of the substrate. The reaction equilibrium can be influenced by several factors such as increase in substrate or cofactor concentration, variation of pH, presence of trapping agents, or the use of regenerating reactions in which one of the co-substrates may be regenerated by a secondary reaction. If none of the reactants or products of an enzymic reactions is measurable spectrophotometrically, it is often possible to transform one of the products by another enzymatic reaction which in turn can be easily measured (e.g., measurement of glucose, non-esterified fatty acids and triacylglycerols). The former reaction, in which the substrate to be determined is transformed, is known as the auxiliary reaction, whereas the reaction used for the actual measurement is known as the indicator reaction. Both reactions can usually be carried out in the same assay mixture (Bergmeyer,1963). Specificity of an enzymatic assay depends on the purity of the enzyme preparation whereas precision depends on the provision of optimum assay conditions. The sensitivity of enzymatic assays is limited by the fact that sufficient conversion of NAD/NADP (or NADH/NADPH) must occur to produce a measurable change in the optical density. The following metabolites were measured by standard enzymatic methods as described below: Determination of D-Glucose Blood and liver glucose were measured according to the method described by Slein (1963). Reaction sequence: a. Auxiliary reaction GLUCOSE + Mg++ ATP ——————————————> GLUCOSE-6- PHOSPHATE + ADP b. Indicator reaction: Mg++ glucose-6-phosphate GLUCOSE-6-PHOSPHATE dehydrogenase 6- ——————————————————————> PHOSPHOGLUCONATE + NADP+ + NADPH + H+ At pH 7.5, the equilibrium for the indicator reaction is far to the right which ensures the completion of both reactions (since glucose-6-phosphate formed in the former is rapidly used up in the latter Although hexokinase catalyses the phosphorylation of monosaccharides, specificity is provided by glucose-6-phosphate dehydrogenase (G6PD) with which hexose or pentose esters other than glucose-6-phosphate do not react. reaction). several other Buffer solution for assay: 20ml 0.1M tris buffer pH 8.0 2ml 0.1M magnesium chloride 2ml 0.01 M ATP 2ml 1% NADP 0.13ml of G6PD (1mg/ml) This was prepared freshly for each assay. The total volume in each cuvette was 2.0ml. In the sample cuvettes, this consisted of 0.1ml of neutralised HClO4 extract, 0.9ml of distilled water and 1ml of assay buffer; in the control cuvette 1ml of water and 1ml of assay buffer was cuvettes were read at 340nm before , at 10min and 15min after addition added. The of 0.010ml of hexokinase. Measurement of L-(+)-Lactate Concentrations of blood and liver lactate were measured according to the method described by Hohorst (1963). Reaction sequence: Lactate dehydrogenase LACTATE + NAD+ ——————————————> PYRUVATE + NADH + H+ The equilibrium of this reaction lies well on the side of lactate and NAD. Therefore, in order to ensure the complete conversion of lactate, the reaction products have to be removed from the equilibrium. are trapped by an alkaline reaction medium; the pyruvate reacts with hydrazine hydrate in the buffer form pyruvate hydrazone and, in addition, a large excess of solution to NAD and enzyme is used to obtain a sufficiently rapid end point. Lactate dehydrogenase reacts only with L-(+)-lactate and thus provides for the assay. Protons specificity Buffer solution for the assay: 40ml 0.2 M Tris 5ml hydrazine hydrate 100% 25mg EDTA Made up to 100ml of distilled water. The pH of the buffer solution was adjusted to pH 9.5 with 5M hydrochloric acid and it was stored for up to two weeks at 4°C. Before use, 1 ml of 1% (w/v) NAD was added to every 10 ml of assay buffer. The total volume in each cuvette was 2.0ml. In the sample cuvettes 0.2ml of neutralised HClO4 extract, 0.8ml of water and this consisted of 1ml of assay buffer; the control cuvette contained 1ml of water and 1ml of assay buffer. All cuvettes were read at 340 nm before and at 35 and 45 min after the addition of 0.02ml of lactate dehydrogenase. Measurement of Pyruvate and Acetoacetate Pyruvate and acetoacetate share very similar assay conditions and therefore were measured sequentially in the same sample (same cuvette) according to a combination of methods respectively described by Hohorst et al (1959) and Williamson et al (1962). Reaction sequence: lactate dehydrogenase PYRUVATE + NADH + H+ ————————————————————> LACTATE + NAD 3-OH-butyrate dehydrogenase ACETOACETATE + NADH HYDROXYBUTYRATE + NAD+ + H+ —————————————> The equilibrium of the first reaction at pH 7.0 is sufficiently far to 3- the right to ensure a quantitative measurement of pyruvate levels provided that the NADH concentration is not less than 0.01 mM. At the same pH and with a suitable excess of NADH, at least 98% of the acetoacetate reduced to D-(-)-3-hydroxybutyrate. However, due to the low activity of 3- is hydroxybutyrate dehydrogenase preparations the second reaction proceeds at a much slower rate than the former. Buffer solution for the assay: 10ml of 0.1 Potassium phosphate buffer pH 6.9 1 ml of 0.5% (w/v) NADH A fresh solution was prepared for each assay. The total volume in each cuvette was 2 ml. In the sample cuvettes, this consisted of 1ml of neutralised HClO4 extract and 1ml of assay buffer. The control cuvette contained 1ml of water and 1ml of assay buffer solution. The cuvettes were read at 340 nm before and 10min after the addition of of 0.01ml of lactate dehydrogenase. 3-Hydroxybutyrate dehydrogenase (0.01ml) was then added to each cuvette and they were read again at 35 and 45 min thereafter. Measurement of D-(-)-3-hydroxybutyrate Blood and hepatic concentrations of 3-hydroxybutyrate were measured to the method described by Williamson et according al (1962). Reaction sequence: 3-hydroxybutyrate dehydrogenase 3-OH-BUTYRATE + NAD+ ————————————> NADH + H+ The equilibrium of the reaction at pH 8.0 is reached when ACETOACETATE + approximately 40% of the 3-hydroxybutyrate is oxidised to acetoacetate. However, the presence of hydrazine in the buffer solution acetoacetate formed as a hydrazone and the reaction quantitatively from the left to the right. traps the proceeds Buffer solution for the assay: 70 ml 0.1M tris buffer pH 8.5 0.25 ml of hydrazine hydrate 100% 25mg of EDTA Made up to 100 ml with distilled water. The pH of the assay buffer solution was adjusted to pH 8.5 with 5M hydrochloric acid and it was stored for up to two weeks at 4°C. Before use, 1 ml of 1% (w/v) NAD was added to 10ml of assay buffer for each assay. volume in each cuvette was 2 ml. In the sample consisted of 0.5 ml of neutralised HClO4 extract, 0.5ml of cuvettes, this water and 1 ml of assay buffer; the control cuvette contained 1 ml of water and 1 ml of assay buffer. The cuvettes were read at 340 nm before and 50 and 60 min after the addition of 0.01 ml of 3-hydroxybutyrate The total dehydrogenase. Measurement of L-Alanine Blood and hepatic concentrations were measured according to the method described by Williamson et al (1967). Reaction sequence: alanine dehydrogenase ALANINE + H2O + NAD+ ——————————————> PYRUVATE + NH4+ + NADH In the presence of low H+ ion concentration, at pH 9.0, this reaction proceeds quantitatively from the left to the right. Hydrazine hydrate is included in the buffer solution in order to trap the pyruvate formed by conversion to pyruvate hydrazone. Buffer solution for the assay: 40ml 0.2M tris buffer 04ml hydrazine hydrate 100% 25mg EDTA Made up to 80 ml with distilled water. The pH of the buffer solution was adjusted to pH 9.0 with 5 M hydrochloric acid and it was stored for up to 14 days at 4°C. Before use, 1ml of 1% (w/v) NAD was added to 10ml of assay buffer for each assay. The total volume per cuvette was 2 ml. In the sample cuvettes, ml of neutralised HClO4 extract,0.5 ml of water this consisted of 0.5 and 1 ml of assay buffer; the control cuvette contained 1 ml of water and 1 ml of asssay buffer.All cuvettes were read at 340 nm before and at 50 and 60 min after addition of 0.01ml of alanine dehydrogenase. Determination of D-3-Phosphoglycerate, D-2-Phosphoglycerate and Phosphoenolpyruvate and Pyruvate Hepatic concentrations of pyruvate, PEP, 2-phosphglycerate and phosphoglycerate were measured according to the method 3- described by Czok & Eckert (1963). Reaction sequence: lactate dehydrogenase a. PYRUVATE LACTATE + NAD+ + NADH + H+ -——————————————> pyruvate kinase b. PHOSPHOENOLPYRUVATE + ADP ———————————> PYRUVATE + ATP Mg2+, K+ enolase c. 2-PHOSPHOGLYCERATE ———————————> PHOSPHOENOLPYRUVATE Mg2+ phosphoglycerate mutase d. 3-PHOSPHOGLYCERATE PHOSPHOGLYCERATE —————————————> 2- At pH 7.4 quantitative conversion of the substitute is assured because of positions of the equilibrium state of the reactions catalysed by pyruvate kinase and lactate dehydrogenase being far to the Phosphglycerate can only be determined under the here if the assay mixture contains less than 10-3 conditions described M inorganic phosphate. The determination of 2-phosphoglycerate, phosphoenolpyruvate and pyruvate is not affected by phosphate. Specificity is secured by the high specificity of the reactions catalysed by enolase right. the 3- and pyruvate kinase. Buffer solution for assay: 10 ml Tris HCL buffer ph 7.4 0.1M 1 ml Mg Cl2 3 ml KCl 1.0 M 1 ml ADP 0.02 M 0.5 ml 0.1 M NADH 0.5 % This was prepared freshly for each assay. The total volume in each cuvette was 2.0 ml. In the sample cuvettes, this consisted of 0.5 ml of HClO4 extract, 0.5 ml of distilled water and 1 ml of neutralised liver assay buffer; in the control cuvette 1 ml of water and 1 ml of assay buffer The cuvettes were read at 340 nm before and for 5-10 min addition of lactate dehydrogenase (0.01ml); pyruvate kinase (0.01 ml) was added and the changes in the optical density followed for 5-10min; enolase (0.01ml) was added and the cuvettes continuously for 5-10 min; finally phosphoglycerate mutase (0.01 ml) was added to the cuvettes and the change in the optical density followed for 15-20 min. All cuvettes were read using a Gilford recording spectrophotometer. were added. after the were read Measurement of Dihydroxyacetone Phosphate, D-Glyceraldehyde-3- phosphate and Fructose- 1,6-bisphosphate Hepatic concentrations of dihydroxyacetone phosphate glyceraldehyde 3-phosphate (GAP) and fructose 1,6-bisphosphate determined acording to the method described by Bücher & (DHAP) , were Hohorst (1963). Reaction sequence: GDH DHAP +NADH + H+ —————> L-(-)GLYCEROL-1-PHOSPHATE + a. NAD+ TIM b. GLYCERALDEHYDE -3-PHOSPHATE (GAP) ——> DIHYDROXYACETONE PHOSPHATE aldolase c. FRUCTOSE-1,6-BISPHOSPHATE (FDP) ———————> GAP + DHAP The equilibrium state of the reactions ensures that the overall reaction proceeds quantitatively. For each mole of FDP formed 2 moles of NADH are oxidised, therefore, the inclusion of the triosephosphate isomerase (TIM) reaction increases the sensitivity of the assay. Buffer solution for the assay: 10 ml Tris HCl ph 7.4 1 ml NADH 0.5% The total volume in each cuvette was 2 ml. In the sample cuvettes, this consisted of 1 ml of neutralised liver HClO4 extract and 1 ml of assay buffer solution; the control cuvette contained 1 ml of distilled water and 1 ml of assay buffer. All cuvettes were read continuously using spectrophotometer as follows: before and for 5-10 min after glycerol-3-phosphate dehydrogenase (GDH); before and for 5-10 min after addition of triophosphate isomerase (TIM) and before and for 10-15 min after addition of aldolase. a Gilford addition of Measurement of Adenosine-5'-Triphosphate and D-glucose-6- phosphate Adenosine-5'-triphosphate (ATP) and glucose -6-phosphate were measured with a combination of the methods described by Lamprecht Trautschold (1963) and Hohorst (1963); using the same cuvette. & phosphorylates glucose with ATP in the Hexokinase presence of Mg++ to give glucose-6-phosphate. Glucose-6-phosphate dehydrogenase catalyses the oxidation of glucose-6-phosphate with NADP. Each mole of ATP forms 1 mole of NADPH. Reaction sequence: hexokinase GLUCOSE + ATP ——————————> GLUCOSE-6-PHOSPHATE (G-6-P) + ADP G6P-DH G-6-P + NADP+ ———————> 6-PHOSPHOGLUCONO-LACTONE + NADPH + H+ In the presence of equivalent concentrations of glucose and Mg2+, virtually quantitatively converted to ADP by hexokinase. The ATP equilibrium is of the glucose-6-phosphate reaction lies to the right. Buffer solution for the assay: 10 ml Tris 0.1M pH 7.4 1 mg/ml NADP 2 ml Mg Cl2 0.1 M 0.5 ml Glucose 0.1 M This was prepared freshly for each assay. The final volume per cuvette was 2 ml. In the sample cuvettes this consisted of 0.5 ml of liver HClO4 extract, 0.5 ml of distilled water and 1 ml of buffer solution. The control cuvette contained 1 ml of distilled water and 1 ml solution.Cuvettes were read at 340 nm before and 5-10 min after 0.005 ml of glucose-6-phosphate dehydrogenase was added and subsequently 10- 15 min after hexokinase (0.005 ml) was added. of buffer Determination of Acetyl-Coenzyme A Hepatic acetyl-CoA was measured by the method described by Decker (1963). Reaction sequence: citrate synthase ACETYL-SCoA + OXALOACETATE + H2O ———————> CITRATE + CoASH + H+ malate dehydrogenase L-MALATE + NAD+ ————————————————> OXALOACETATE + NADH + H+ At pH 8.0 the equilibrium of the over-all reaction lies to the right and acetyl-CoA is used quantitatively. Buffer solution used for assay: 15 ml Tris 0.1 M 1 ml L-malate 0.05 ml NADH 1 ml NAD 1 ml pH 8.0 0.1 M 0.5 % 1% malate dehydrogenase This was made freshly for each assay. The total volume per cuvette was 2 ml. In the sample cuvettes this consisted of 1 ml of liver HClO4 extract and 1 ml of buffer solution. The control cuvette contained 1 ml of distilled water and 1 ml of buffer solution. Cuvettes were read before and at 15 min after addition of citrate synthase (0.01 ml). Determination of L-Glutamate and Glutamine Hepatic glutamate was measured according to the method described Bergmeyer (1963). The determination of glutamine was enzymatic technique. Reaction sequence: glutamate dehydrogenase by Bernt & also based on this L-GLUTAMATE + NAD+————————————————> OXOGLUTARATE + NADH + NH4+ glutaminase L-GLUTAMINE –––––––––––––––––––––––––> L-GLUTAMINE + NH4+ The equilibrium lies far to the left. However, under alkaline conditions and the presence of excess NAD, oxoglutarate can be hydrazine and glutamate quantitatively oxidized to oxoglutarate. (pH 9.0) trapped with Buffer solution for assay: 2 ml Hydrazine hydrate 100 % 20 ml Tris 0.2 M 25 mg EDTA Made up to 40 ml with distilled water and the pH (9.0) adjusted using 5 N HCl. This could be kept at 4°C for up to 14 days. To be used for assay (cocktail), NAD (1mg/ml) and ADP (0.5mg/ml) were added to the buffer solution. The total volume per cuvette was 2 ml. In the sample of 1 ml of liver HClO4 extract and 1 ml of cuvettes this consisted cocktail. The control cuvette contained 1 ml of distilled water and 1 ml Cuvettes were read before and 30-40 min after the addition of glutamate of cocktail. dehydrogenase (in glycerol). Glutamine In order to measure glutamine liver HClO4 extracts were presence of glutaminase under the contitions 0.5 ml pre-incubated in the described below: Liver extract 0.5 M Acetate buffer 0.5 M pH 4.8 0.01 ml NH2OH 1M 0.02 ml Glutaminase [5 mg/ml] The total volume per tube was made up to 1 ml with distilled water. An internal standard was added, consisting of 1 mM glutamine (0.2ml) and distilled water (0.8 ml). Tubes were mixed and incubated at 37°C for 45 min. These were then cooled, 0.5 ml of the mixture taken and the concentration of glutamate measured by the enzymatic method described above (Bernt & Bergmeyer, 1963). As glutamine is converted to glutamate by glutaminase, the total concentration of glutamine+glutamate is measured spectrophotometrically. Therefore, the actual concentration of glutamine is calculated by subtracting the concentration of glutamate measured in the liver extract from the total concentration (glutamine + glutamate) found in the glutaminase pre- incubated extract. Measurement of Non-esterified fatty acids The method of Shimizu et al (1979) was used for the measurement of nonesterified fatty acids (NEFA), which is based on the activation of NEFA by acyl-CoA synthetase. Reaction sequence: acyl-Co A synthetase NEFA + COENZYME A + ATP —————————> ACYL COENZYME A + AMP+ PPi myokinase AMP + ATP ———————> 2 ADP pyruvate kinase 2 ADP + PHOSPHOENOLPYRUVATE ————————-> 2 ATP + 2 PYRUVATE lactate dehydrogenase 2 PYRUVATE + 2 NADH ————————————————> 2 LACTATE + 2 NAD+ The reaction calalysed by acyl-CoA synthetase favours the production of acyl coenzyme A provided the pH is 8.0 and there are sufficient amounts of ATP and coenzyme A. Therefore, the production of AMP is used as a measure of NEFA activation through an indicator system provided by successive reactions catalysed by myokinase, pyruvate kinase and lactate dehydrogenase. The equilibrium state of all three reactions in this indicator system is sufficiently far to the right to ensure that the overall rate of the reaction is limited only concentration of NEFA. Specificity for the method is enhanced by the fact that Acyl CoA synthetase only activates monocarboxylic acids by the with 6 to 18 carbon atoms and does not affect other carboxylic acids and lipids. Buffer solution for the assay: 200 ml 0.1 M tris pH 8.0 35.1 mg EDTA (0.6 mM) 406.6 mg magnesium chloride (10mM) 2 ml Triton X-100 This was stored at 4°C. Immediately before each assay, the following reagents were added to 36 ml of buffer solution: 1.6 ml NADH 0.5% (w/v) 1 ml ATP 0.1 M 1 ml phosphoenolpyruvate 0.2 M 0.2 ml myokinase 2mg/ml 0.4 ml pyruvate kinase 1mg/ml 0.2 ml lactate dehydrogenase 10mg/ml The total volume per cuvette was 2.005 ml. The sample cuvettes contained 0.04ml of plasma, 1.95ml of buffer mixture and 0.015ml of acyl-CoA synthetase. The plasma volume was replaced by 0.04ml of water in the control cuvette. All cuvettes were read at 340 nm before and 20 min after the addition of coenzyme A. Further readings were taken intervals until the readings of the optical density were constant. at 5 min Measurement of Triacylglycerol Plasma triacylglycerol concentration was measured according to the method described by Eggstein & Kreutz (1966). Plasma triacylglycerol were hydrolysed to glycerol and non-esterified fatty acids. The glycerol measured by the enzymatic techique described concentration of free glycerol, measured in the below. neutralised extract was subtracted from the total glycerol measured alkaline hydrolysis of the plasma sample to obtain the glyceride-glycerol concentration which, in turn, was taken to be an estimate triacylglycerol content of the plasma. Reaction sequence: a. Hydrolysis: at 70°C initially was The HClO4 after of the TRIACYLGLYCEROL + 3 H2O ————————> GLYCEROL + 3 FATTY ACIDS alchoholic KOH b. Indicator reaction: glycerokinase pyruvate kinase lactate dehydrogenase GLYCEROL + PHOSPHOENOLPYRUVATE ——————> GLYCEROL-3-PO4 + LACTATE ATP ADP NADH ATP NAD+ Hydrolysis: To 0.2 ml of plasma 0.5ml of alcoholic KOH (0.5N KOH in 98% ethanol) was added. The mixture was incubated at 70°C for 30 min and then 1.5ml of 0.1 M MgSO4 was added to precipitate cooled. protein, samples were centrifuged at 3000 rpm for 10 min. The present in plasma produced equimolar quantities of glycerol produced by hydrolysis was contained in the solution, 0.5ml of which was used for the enzymatic measurement of total glycerol. Thereafter, and the triacylglycerol glycerol. This supernatant Measurement of glycerol Reaction sequence: glycerokinase GLYCEROL + ATP ———————————> GLYCEROL-3-PO4 + ADP pyruvate kinase ADP + PHOSPHOENOLPYRUVATE —————————> ATP + PYRUVATE lactate dehydrogenase PYRUVATE + NADH + H+ ————————————> LACTATE + NAD+ The equilibrium state for all three reactions can be acheived at pH the presence of Mg++ ions. It is far to the right to ensure that 7.4 and in there is a quantitative consumption of glycerol in the first reaction and that the indicator reactions can rapidly proceed to completion. Buffer solution for the assay: 30 ml 0.1 M tris buffer pH 7.4 3 ml 0.1 M magnesium chloride 35 mg phosphoenolpyruvate 50 mg ATP 12.5 mg NADH 0.2 ml lactate dehydrogenase 0.2 ml pyruvate kinase This was made up immediately before use and the pH adjusted to pH 7.4 with 0.2 M tris buffer. Triacylglycerol The total final volume per cuvette was 2 ml. In the sample cuvettes, hydrolysed plasma sample was added to 1 ml of assay 0.5ml of the buffer and 0.5 ml of distilled water. Glycerol The sample cuvettes contained 1 ml of assay buffer and 1 ml of neutralised HClO4 extract. Blank cuvettes contained 0.5 ml of buffer mixture and 1.5 ml of distilled water. The control cuvette contained 1 ml of distilled water and 1 ml of assay buffer. All cuvettes were read at 340 nm before and 40 min after addition of 0.01 ml of glycerokinase. were then re-read every 10 min until there was no further change in the optical density. They Calculations for blood and liver metabolites All metabolite calculations were based on the change in optical density measured at 340 nm in the sample cuvettes following addition of enzyme, and after subtraction of the non-specific change which occurred in the control cuvettes. Therefore: Optical density difference (ODD) = Change in absorbance for sample cuvette - Change in absorbance for control cuvettes Since the molar extinction coefficient of NADH is 6.22 cm2/µmol, the amount of substrate in the cuvette = ODD / 6.22 X total volume in cuvette. This is multipied by the dilution factor for each sample to give the concentration of substrate; In the blood: In the liver: 1 g frozen liver = 0.8 ml ; 1 g liver + 4 ml PCA = 4.8 Insulin Determination Insulin was measured by radioimmunoassay with a rat insulin standard (Albano et al , 1972) using activated charcoal for separation of the free and bound hormone. A disequilibrium assay was set up with a 3-day incubation period and all plasma samples were assayed in duplicate with 20 µl of plasma in each tube. The essential principle of the insulin asay, as for any other radioimmunoassay technique, is the reaction of a fixed amount of specific insulin antibody with a mixture of the plasma sample to be assayed and a constant amount of radioactively labbelled pure insulin. The reaction is initiated by incubating the plasma sample with insulin antibody for approximately 48 hours. The radioactively labelled insulin is then added to compete for the remaining binding sites on the antibody complex. The reaction is allowed to approach completion over the subsequent 24 hours, and the antibody-bound insulin is separated from the free hormone by differential adsorption on to albumin-coated charcoal. The distribution of the radioactivity between the free and bound forms of insulin is then determined. It is expected that the amount of radioactively labelled insulin bound to antibody to decrease as the concentration of the unlabelled insulin in the plasma sample increases due to competition for the limited number of binding sites available. Therefore, results obtained with samples of unknown insulin concentration can be compared with standard curves obtained by the measurement of samples to which known amounts of pure insulin standard have been added. Measurements of lipogenesis and cholesterolegenesis Lipogenesis and cholesterolegenesis were measured in vivo with 3H2O as previously described ( Jungas et al , 1968; Robinson et al , 1978). The rats were injected intraperitoneally with 5 mCi (0.25ml) of 3H2O, and tissues removed one hour later; 5 min before removal of tissue the rats were anaesthetized with pentobarbital (50mg/kg body wt.). Heparinised blood was collected for measurement of the specific activity of plasma water. The tissues (in duplicate into 50 ml tubes) were hydrolysed under alkaline conditions by addition of 3 ml of 30% KOH and heated in a water bath at 70°C for 15 min. Absolute alcohol was then added and the samples returned to the 70°C water bath for a further 175 min (Stansbie et al , 1976). The non-saponifiable lipids were extracted three times with light petroleum (b.p. 40-60°C; total vol. 20 ml) and the organic extract was washed twice with distilled water and transfered to scintillation vials. After evaporation of the solvent, the 3H radioactivity of the residue was measured by scintillation counting in half of the samples to determine the total incorporation of 3H O into the non-saponifiable fraction. Cholesterol and squalene were isolated 2 from the non-saponifiable residue, from the other half of the samples, by chromatography on thin-layer plates of silica gel H developed with chloroform/methanol (99:1, v/v). The plate was dried and the cholesterolcontaining band was located by spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v). This area of the plate was scraped into a scintillation vial, and the 3H content of the sterol fractions were determined by scintillation counting (Gibbons et al , 1983). The aqueous phase after extraction of the non-saponifiable lipid was acidified to pH 1.0 with 6N H2SO4, the labelled fatty acid fraction was extracted three times with light petroleum and the organic extract washed twice with distilled water. The extract was transferred into vials to dry (Stansbie et al , 1976) . After evaporation of the solvent, the 3H radioactivity of the residue was determined by scintillation counting. Preparation of isolated hepatocytes Hepatocytes were prepared essentially as described by Berry & Friend (1969) with the modifications described by Krebs et al (1974). Routine tests indicated that about 90% of the hepatocytes excluded Trypan Blue. Operative technique, liver perfusion and isolation of hepatocytes The rat was anaesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg body wt.). Heparin (150 units; 0.15 ml) was then administered via a saphenous vein. A laparotomy through a vertical mid-line incision was performed. Two mid-transverse incisions were made to the left and the right of the mid-line to allow an extended exposure of the abdominal cavity. The portal vein and the descending vena cava were exposed by moving the intestines to the left side of the abdominal cavity. One loose silk ligature (3-0) was placed around the descending vena cava immediately above the right renal vein, and two ligatures, about 1 cm apart, were placed around the portal vein. The portal vein was then cannulated using a 17 gauge metallic intravenous cannula. After withdrawal of the inner needle, the cannula was secured by tying the ligature that was closest to the liver. The other tie was tightened to prevent blood from flowing out of the lower part of the portal vein and to tie off the common bile duct. The cannula was therefore firmly in place and the appearance of blood back-flow at the cannula end from the penetrated portal vein confirmed the success of the cannulation. The thorax was immediately opened, through a transverse incision. A loose ligature was placed around the inferior vena cava just below the heart. A 16 gauge plastic cannula was used to cannulate this vessel through the right atrium and secured with one 3-0 silk tie. Liver perfusion and treatment with collagenase: The method of liver perfusion was that described by Hems et al (1966). A water-jacketed oxygenator (Fig. 2.4.) was used and the temperature kept approximately at 38°C. The perfusion medium used was the saline of Krebs & Henseleit (1932) from which calcium was omitted because it decreases the activity of the enzyme. The total volume of the perfusion fluid was initially 150 ml. The first 40 ml of the medium passing through the liver were discarded in order to remove the bulk of the blood cells. At this point 30 mg of the crude collagenase (Boehringer Corporation, London), suspended in a small volume of perfusion medium were added. Pure collagenase is not suitable because the contaminating proteinases in the crude preparation play a very important role. The flow rate was approximately 25 ml /min. The perfusion medium was kept in equilibrium with O2/CO2 (95:5 ; v/v). The perfusion was continued for 20-30 min and any leaked perfusion fluid appearing in the abdominal cavity was collected and returned to the reservoir (Fig. 2.4.). The action of the enzyme was reflected by the degree of leaking; when this had reached a rate of about 10 ml/min, usually after 25 min, the perfusion was discontinued and the liver was transferred to a beaker. The medium used in the perfusion was added to the broken-up liver tissue as this was put into a nylon cloth (mesh size 0.44 X 0.44 mm) which was on the top of a 100 ml disposable plastic beaker. Fig. 2.4. The liver tissue was then gently broken apart with a pair of scissors and the cells teased out with a plastic spatula, always keeping the perfusion medium (which already contained cells washed out from the liver during the perfusion) flowing through the nylon cloth. The filtrate was centrifuged at 50 g for 2 min and the cells were re-suspended in about 30 ml of the Ca++-containing Krebs-Henseleit medium. The suspension was centrifuged again at 50 g for 2 min and the pellet weighed and resuspended in 16 volumes of the Krebs-Henseleit medium containing Ca++, when it was ready to be incubated. Isolated hepatocytes were used immediately after preparation. Incubation procedure The isolated hepatocytes were incubated with constant shaking, at 65 oscillations per min, for 20, 40 or 60 minutes in Krebs-Henseleit saline (Krebs & Henseleit, 1932) containing essentially fatty acid-free albumin (final concentration 2.5% w/v), the gas phase was O2/CO2 (95:5) and the temperature was 37.5° C. The total incubation volume containing 65-95mg wet wt. of cells and added substrates (5mM lactate, 5mM pyruvate, 5mM alanine, 5mM glutamine, 5mM dihydroxyacetone, 5mM butyrate and 2mM [1-14C]oleate; 40µci/ml) was 4 ml. Incubations were stopped by addition of 0.4ml of 20% (w/v) HClO4 , the mixture was centrifuged to remove denatured protein and the supernatant was neutralized with KOH. The precipitate of KClO4 was removed by centrifugation. All incubations with [1-14C]Oleate were performed in duplicate in KrebsHenseleit saline (Krebs & Henseleit, 1932) containing dialysed albumin (fatty acid poor, final concentration 2.5%, w/v). One of each pair was incubated in a 25ml Erlenmeyer flask with a centre well and rubber seal. The incubation was stopped by the injection of 0.4 ml of 20 % (w/v) HClO4 with a long-needled syringe (Whitelaw & Williamson, 1977). Sodium hydroxide (NaOH; 0.3 ml) was injected into the centre well and metabolic CO2 collected by shaking the flasks for a further 2 h at room temperature. The NaOH was then removed for measurements of radioactivity and the deproteinised incubation medium was treated as described above. The reaction in the duplicate flask was stopped by centrifugation for 1 min at 5000 g to separate cells from the medium. Chloroform/methanol [2:1 (v/v); 20 vol.] was added to the cell pellet and the lipids were extracted as described by Folch et al (1957). The radioactivity in the chloroform phase was used as a measure of the [1-14C]oleate converted into esterified fats. The disappearance of radioactivity which was extracted by chloroform from the incubation medium (minus pellet) was used to calculate the oleate uptake. This was determined as follows: potassium phosphate buffer (0.1 M) at pH 7.0 was added to the medium (1.5 ml/0.5 ml). The mixture was extracted with chloroform (6 ml) by shaking for 90 sec. The chloroform and aqueous phases were separated by centrifugation, the upper phase was removed and the organic phase washed with 1 ml of potassium phosphate buffer, pH 7.0. The radioactivity in a sample of the chloroform phase was measured. This method relies on the fact that esterified lipids are not released in appreciable amounts to the incubation medium, as shown by Whitelaw & Williamson (1976). The radioactivity in ketone bodies was estimated by measuring the radioactivity in acid-soluble compounds in the supernatant. Calculation of rates: The time-courses of glucose synthesis from the gluconeogenic precursors and of ketone body formation from long- and short-chain fatty acids, in isolated liver cells, were linear between 20 and 60 minutes, and were calculated as follows: Preparation of microsomes The animals (starved 48h post-operation) were anaesthetized with an intraperitoneal injection of pentobarbital (50mg/kg body wt). Each liver was removed, minced and put into a pre-weighed ice-cooled 50 ml beaker containing 10ml of NaF sucrose (0.25 M sucrose; 50 mM NaF) buffer. More NaF sucrose buffer was added to the beaker to a final volume/liver wt. of (10:1). Livers were then homogenized using a glass homogenizer kept in ice. After homogenization samples were centrifuged at 800 g (15 min), 16,000 g (20 min) and again at 16,000 g (20 min) to separate the microsomes from the cell wall, red cells and cytosol during the first spin; from the mitochondria and the lysosomes during the second spin; and from the remaining mitochondria and lysosomes during the third spin. The supernatant of the third spin was carefully transferred to 12.5ml tubes and ultracentrifuged for 1 h at 110,000 g to precipitate the microsomes. The supernatant was then discarded and the microsomes were re-suspended in 20 mM imidazole/chloride - 5 mM dithiothreitol buffer and assayed for HMGCoA reductase activity. Determination of hepatic enzyme activity HMGCoA Reductase Buffer solutions required for the assay: Buffer A 20mM Imidazole /Chloride (pH 7.4) 5mM Dithiothreitol Buffer B 350mM Potassium phosphate buffer 40mM EDTA 20mM Dithiothreitol Cofactors NADP+ 10.5mg in 0.5ml H2O Glucose-6-phosphate Glucose-6-phosphate dehydrogenase 33.5mg in 0.5ml H2O 19U (50µl)+0.45ml H2O These were mixed together. Buffer B (1.38ml) and H2O (0.38ml) were added - The cofactor mixture Hydroxymethylglutaryl-CoA reductase activity was measured as described by Brown et al (1979). Microsomes, prepared as described previously, were suspended in 20mM imidazole/chloride - 5 mM dithiothreitol (Buffer A) and incubated in duplicate in the presence or absence of alkaline phosphatase at 37°C for 30 min , so that the total and the expressed amount of enzyme activity could be measured. Before incubation portions (10 µl) of the homogenate were taken in duplicate to measure the protein content. Three control incubations each containing buffer A instead of microsomal homogenate. To the pre-incubated microsomes the cofactor mixture was added and a solution of (R,S) [314C]HMG-CoA (80,000 dpm; 6.4 nmol; 8 µl) in KH2PO4 (0.1 M) was added to each tube giving a final (R,S)-HMG-CoA concentration of 145 µM. Each tube was capped and placed in a water bath at 37°C. The rack in which the tubes were placed was shaken at 70 oscillations per min. Afer 20 min each tube was removed from the water bath and the reaction stopped by the immediate addition of HCl (12 M, 5 µl). An internal standard, consisting of a solution in water of (R,S)[23H ]mevalonolactone (5 µl - 10,986 dpm), was added to each tube. The mevalonic acid was lactonised by incubating the tubes at 37°C for 30 min. At this stage , if necessary , the samples could be stored for 1-3 days at -20°C. Thin Layer Chromatography of Mevalonolactone To precipitate protein , the tubes were centrifuged at 1000 g for 10 min. Portions (40µl) of the supernatant of each sample were loaded into preadsorbent strips of a multiple channel, glass-backed, Whatman LK5D silica gel TLC plate (20 X 20 cm; 0.8 cm wide channels). The plates were thoroughly dried for 4 h in a TLC tank containing activated silica gel (coarse mesh). They were then developed with ethylacetate/acetone (4:1,v/v) until the solvent, had reached 1 cm from the top of the plate. The TLC tank contained 75 ml of the solvent, was lined with chromatography paper and had been pre-equilibrated for at least 30 min. The plates were dried and the mevalonolactone-containing band was located by spraying with a solution of Rhodamine 6G in acetone (0.1%,w/v) and visualisation under ultra-violet light. This area of the plate was scraped from the plates with a razor blade and transferred into scintillation glass vials. Ethanol (1 ml) was added, followed by 10 ml of a toluene-based scintillation cocktail (PPO/POPOP/Toluene ; w/w/v ; 0.3 : 0.01 : 100). The 3H and 14C contents of the sterol fraction were determined by scintillation counting for 30 min per sample. Microsomal Protein Determination The method of Lowry et al (1951) was used, as modified to assay protein in the presence of sulphydryl reagents, by Geiger and Bessman (1972). The standard curve was constructed using 0-150 µg of bovine serum albumin. After the microsomes were re-suspended in 20mM imidazole/chloride - 5 mM thiothreitol (Buffer A) and were ready for the incubation, a portion of the suspension from each sample (10µl) was taken in duplicate into 10 ml tubes to measure protein. The dithiothreitol was oxidised by heating in the presence of H2O2. Reagents: A. 1 % CuSO4 5 H2O B. C. 5.4% NaK Tartrate 10% Na2CO3 ; 0.5 M NaOH Copper Reagent: 1 ml of reagent A was mixed with 1 ml of reagent B 1 ml of this new mix (A+B) was added to 10 ml of reagent C Phenol Reagent: The phenol reagent was diluted with distilled water (v/v; 1:10) Distilled water was added to the standards and the protein-containing tubes up to a final volume per sample of 0.75 ml. The tubes were mixed and 0.75 ml of the copper reagent added; 15 µl of 3% H2O2 were also added to each tube and they were mixed again. The tubes were transferred to a water bath at 60°C for 10 min. Phenol reagent (1.5 ml) was added to each sample and standard , mixed and each tube transferred again to the water bath at 60°C for another 10 min. Tubes were cooled and each standard and each sample was transferred to the same glass cuvette to be read in the spectrophotometer at 750 nM. The standard curve was then constructed and the concentration of protein in the samples calculated based on this standard curve. Calculation of enzyme activity The 14C dpm content of each sample was corrected for the recovery of [23H]mevalonolactone and by subtracting the average 14C dpm value obtained from the control incubations (usually about 25 dpm). The corrected amount of 14C dpm was then converted to pmol of mevalonolactone (the specific radioactivity of the substrate used was 12.5 dpm/pmol). Therefore, the following equation was used to calculate enzyme activity: HMG-CoA reductase activity was expressed as pmol of mevalonolactone produced per min per mg of microsomal protein and was the average of duplicate assays. Determination of lipoprotein lipase activity Lipoprotein lipase is an extracellular enzyme that is physiologically active at luminal surface of the capillary endothelia of most extrahepatic tissues (for review see; Cryer, 1981). It is responsible for the hydrolysis of plasma lipoprotein triacylglycerol and, therefore, controls the rate limiting step in the removal of triacylglycerol fatty acids from the circulation. The method used in this work to measure lipoprotein lipase acitivity was essentially that described by Oller do Nascimento & Williamson (1986). This involved the use of radioactive triacylglycerol ([3H]-triolein), labelled in the fatty acid moiety, which is then incorporated into a triacylglycerol emulsion from which the release of radioactive fatty acid is measured, indicating the activity of lipoprotein lipase. The results were expressed as nmol of free fatty acid released per min per mg of adipose tissue. Measurements of radioactivity Measurements of 3H and 14C radioactivity by liquid scintillation counting were performed using an LS 9800 Liquid Scintillation System (Beckman Instruments Inc., Irvine, CA, USA). The sample to be measured was dissolved in a toluene-based scintillation fluid (10 ml). Depending on the specific scintillation cocktail or radionuclide use, distilled water and or ethanol was added to the vials. The vials were fitted with a screw cap and when all the contents (sample, scintillation fluid and additions ; e.g. water, ethanol) and were counted as described by Williamson et al (1975). The counting procedure is based on the fact that ionising ß-particles produced by the decay of 3H or 14C atoms lose some of their kinetic energy to the scintillant. This energy is converted into photons. Initially the kinetic energy is absorbed by the toluene and is subsequently transferred to the scintillant present in the scintillation fluid. The photon emissions are detected as voltage pulses, proportional in magnitude to the energy of the ß-particles, by two photomultiplier tubes. Only coincident pulses are counted by the machine to eliminate random events. The overall yield for a particular radionuclide will be reduced by several factors, which are termed quenching. The level of quenching was monitored and the counting energy channels optimised for each sample by an automatic quench compensation method. When in the dual-label counting mode (3H-14C), the machine was calibrated with a series of differently quenched samples containing either 3H or 14C of known dpm. The energy channels were set such that there was no spillover from 3H into 14C. The quenching data and channel setting during either single or dual-lable counting, were stored in the instrument as a 'user counting program'. A microprocessor built into the machine automatically calculated the 3H and/or 14C dpm using this program. Statistical Analysis Experimental results were expressed as the mean ± the standard error of the mean (S.E.M.) accompanied by the number of observations (n). The representation used for mean values, and the mathematical definition of S.E.M. are shown5 below: Results were analysed using the Student t-test. CHAPTER 4 Time course of changes in rates of gluconeogenesis in isolated hepatocytes during induction of sepsis Introduction Carbohydrate metabolism is substantially affected by sepsis. Glycogen reserves are depleted in early sepsis, during the fuel mobilization characteristic of the ebb phase. During the flow phase, when sepsis is usually accompanied by diminished or virtually no carbohydrate intake, there is increased demand for glucose, especially by glucose-dependent tissues including the brain, nerve cells, red blood cells, kidney medulla, and the cells involved in the infectious-inflamatory reaction and the repair process (Frayn KN, 1986). This requirement for glucose is met by the synthesis of glucose de novo - gluconeogenesis (Fig. 4.1.). The catabolism of glucose in these glycolytic tissues forms a large amount of lactate (Kelleher et al , 1982) which is taken up by the liver as a prime precursor for glucose synthesis. During sepsis, the liver uses other substrates to produce glucose such as pyruvate, glycerol, alanine and other gluconeogenic amino acids. The contribution of these precursors reported in the literature varies, e.g. alanine utilization for hepatic gluconeogenesis, in the presence of sepsis, has been reported to range from 30 to 70% (Forse & Kinney, 1985). The increased muscle proteolysis induced by sepsis (Long et al , 1977; Hasselgren et al , 1986) and the reported accelerated metabolism of branched chain amino acids with formation of alanine during sepsis (Biesel & Wannemacher, 1980) provide a continuous supply of precursors for glucose synthesis from muscle. Glucagon and catecholamines stimulate gluconeogenesis by their ability to activate adenylate cyclase, increasing the concentration of cyclic AMP in the hepatocytes (Newsholme & Leech, 1983). Both hormones are reported to be increased during sepsis (Cerra et al , 1980; Watters et al , 1986). Insulin, however, is known to antagonize adrenaline or glucagon action, by lowering the concentration of cyclic AMP within the liver cells, inhibiting gluconeogenesis (Newsholme & Leech, 1983). Higher concentrations of plasma insulin were found in caecal-ligated and punctured rats (Fig. 3.5. - Chapter 3). This has also been reported in other forms of sepsis (Biesel & Wannemacher, 1980; Neufeld et al , 1980 and 1982; Clemens et al , 1984; Shaw et al , 1985). Thus during sepsis some conditions favour increased glucose synthesis, namely, increased availability of substrates (Figs. 3.2.; 3.3.; 3.4 - Chapter 3) and elevated concentrations of gluconeogenic hormones. On the other hand, the presence of increased concentrations of plasma insulin may inhibit gluconeogenesis. It is therefore perhaps not surprising that there is controversy regarding the effects of sepsis on hepatic glucose metabolism. In some studies gluconeogenesis has been reported to be increased (Wannemacher et al , 1980; Lang et al , 1984; Kelleher et al , 1982; Shaw et al , 1985) while in others it is decreased (Wannemacher et al , 1976; Wilmore DW, 1977;Guillem et al , 1982; Clemens et al , 1984). Such widely differing results are presumably due to several factors such as the source and type of sepsis, the nutritional and hormonal status of the animal and the stage of the infection. To investigate changes with time in the rate of gluconeogenesis, hepatocytes were isolated from caecal-ligated and punctured and sham-operated rats, and glucose synthesis from various gluconeogenic precursors measured in vitro. Design of the study Rats, after being made septic by caecal-ligation and puncture or subjected to shamoperation, were starved for 12, 24 or 48 h before isolation of hepatocytes (Fig 4.2.). Fig 4.2. OP 12 h 24 h 48 h Hepatocyte isolation Rats Incubation 1. no substrate 2. lactate 3. pyruvate 20' 40' 4. alanine 5. glutamine 6. dihydroxyacetone 60' For details of the technique of hepatocyte isolation and incubation procedures see Chapter 2. Cells from caecal-ligated and punctured rats and from sham-operated rats were incubated with various gluconeogenic precursors at 12, 24 and 48 h after the operation (Fig. 4.2.) and rates of glucose synthesis were measured. Results The basal rates of glucose formation by hepatocytes from septic and shamoperated rats were low and similar at 12, 24 and 48 h after the operation (Fig. 4.3) Fig. 4.3. Rate of gluconeogenesis - no substrate (5) µmol/g wet wt. cells/min 0.20 (5) (5) (8) (5) Sham Sham-op Septic (6) 0.10 X + S.E.M. ( ) nº of observations 0.00 12 1 24 2 48 3 Time after the operation (h) The addition of various gluconeogenic precursors, namely lactate, alanine, pyruvate, glutamine and dihydroxyacetone, at the final concentration of 5 mM, resulted in increases of 3- to 4-fold in the rates of glucose synthesis as compared to basal rates. However, hepatocytes from caecal-ligated and punctured rats produced glucose at significantly lower rates when compared to those of sham-operated rats. When lactate was the substrate the rate of glucose synthesis, at 24 and 48 h after the operation, was significantly lower when compared to those of sham-operated rats (Fig. 4.4.). Fig. 4.4. Rate of gluconeogenesis from lactate [5mM] (5) µmol/g wet wt. cells/min 0.8 ** (5) 0.6 (5) 0.4 (8) * (6) (5) Sham-op Sham Septic * P < 0.05 ** P < 0.01 0.2 X + S.E.M. ( ) nº of observations 0.0 12 1 24 2 48 3 Time after the operation (h) Clemens et al (1984), using a caecal-ligation and double puncture model, also found decreased production of glucose from lactate [5mM], in perfused livers from septic rats stimulated with phenylephrine, as early as at 6 h after the operation. When hepatocytes were incubated with lactate, at various concentrations, rates of glucose synthesis were progressively lower in isolated liver cells from caecalligated rats (n=4) as the concentration of lactate in the incubation medium increased, when compared to those of hepatocytes from sham-operated rats (n=3) at 48 h after the operation (Fig. 4.5). This depression in glucose synthesis in isolated liver cells from septic rats could be overcome when hepatocytes were incubated with lactate at similar concentration to those found in the blood in vivo (septic [2.5mM]; sham-operated [1.5mM] - see Fig. 3.2. - Chapter 3). Under these conditions the rate of glucose production was approximately the same (0.51 µmol/g wet wt. cells/min - Fig. 4.5.). This suggests that provided the availability of gluconeogenic precursors is increased by sepsis, as indicated by the increased concentrations of lactate, pyruvate and alanine in the blood and in the liver of caecal-ligated and punctured rats (see Chapter 3), gluconeogenesis in vivo may be maintained during sepsis. Fig. 4.5. Rates of gluconeogenesis from lactate 0.8 µmol/g wet wt. cells/min 0.7 0.6 Sham-op Septic 0.5 0.4 0.3 X ± S.E.M. 48 h after the operation 0.2 0.1 [0] [0.625] [[1.25] [2.5] [5] [10] Incubation concentration [mM] Gluconeogenic rates in hepatocytes from septic rats were significantly lower than those of liver cells isolated from sham-operated rats (at 24 h and 48 h) when alanine [5mM] was the substrate (Fig. 4.6.). Fig. 4.6. Rate of glucose synthesis from alanine [5mM] µmol/g wet wt. cells/min 0.50 0.40 (5) * (5) (5) ** (6) (5) 0.30 Sham-op Septic (8) * P < 0.05 0.20 ** P < 0.01 0.10 X + S.E.M. ( ) nº of observations 0.00 12 1 24 2 Time after the operation (h) 48 3 Perfused livers from septic rats, subjected to caecal-ligation and double puncture, produced glucose from alanine [10mM] at significantly lower rates than perfused livers from sham-operated rats at 18 h after the operations (Guillem et al , 1982). However, rates of gluconeogenesis in vivo from [1-14C]alanine increased in rats with pneumoccocal infection as compared to fasted control rats (Wannemacher et al , 1980). When pyruvate was the substrate, isolated liver cells from caecal-ligated and punctured rats formed glucose at significantly lower rates at all time points studied (12 h, 24 h and 48 h - Fig. 4.7.). Fig. 4.7. Rate of gluconeogenesis from pyruvate [5mM] 0.70 (5) 0.60 µmol/g wet wt. cells/min (8) 0.50 (5) * (5) ** (5) 0.40 Sham-op Septic ** (6) * P < 0.05 0.30 ** P < 0.01 0.20 X + S.E.M. ( ) nº of observations 0.10 0.00 12 1 24 2 48 3 Time after the operation (h) Hepatocytes from rats with pneumoccocal infection synthesized glucose from pyruvate [10mM] at lower rates than hepatocytes from fasted control rats 40 h after the injection of bacteria; at 24 h after bacterial injection rates of gluconeogenesis were similar to those of controls (Wannemacher et al , 1980). Hepatocytes from septic rats produced glucose from glutamine at significantly lower rates as compared to those of isolated liver cells (24 h and 48 h) from sham-operated rats (Fig. 4.8.). Fig. 4.8. Rate of gluconeogenesis from glutamine [5mM] (8) 0.50 (5) µmol/g wet wt. cells/min 0.40 (5) 0.30 * * (5) (5) Sham-op Septic (6) 0.20 * P < 0.01 0.10 X + S.E.M. ( ) nº of observations 0.00 12 1 24 2 48 3 Time after the operation (h) The reason for this difference in the rate of glucose formation from glutamine, between the two groups of cells, appeared to be due to the fact that the rates of glucose synthesis in hepatocytes from sham-operated rats increased with time while those of liver cells from septic rats remained unchanged with the progression of sepsis. Sepsis had no effect on the production of glucose from dihydroxyacetone at 12 and 24 h after the operations. However, rates of glucose synthesis in isolated liver cells from septic rats were significantly lower at 48 h after the operation as compared to those of hepatocytes from sham-operated animals (Fig. 4.9.). Fig. 4.9. Rate of gluconeogenesis from dihydroxyacetone [5mM] 1.2 µmol/g wet wt. cells/min 1.0 (8) (5) (5) (5) (5) * (6) 0.8 Sham-op Septic 0.6 * P < 0.01 0.4 X + S.E.M. ( ) nº of observations 0.2 0.0 12 1 24 2 48 3 Time after the operation (h) Discussion Despite causing no appreciable change in the basal rate of production of glucose, at the time points studied, gluconeogenesis was progressively and significantly impaired in hepatocytes isolated from septic rats in the presence of added substrates. This inhibition of glucose synthesis in vitro appears to affect all substrates tested at 48 h after the operation, when rates of glucose formation were approximately 35% lower in hepatocytes from septic rats, despite the fact the gluconeogenic precursors used enter into the gluconeogenic pathway at different levels (Fig. 4.1.). However, if hepatocytes were incubated with lactate at similar concentrations to those found in the blood in vivo (Fig. 3.2. - Chapter 3; Fig. 4.5.), the rate of glucose production was about the same suggesting gluconeogenesis in vivo may be maintained in sepsis by increasing the availability of hepatic glucose precursors (Figs. 3.2.; 3.3.; 3.4. - Chapter 3). A similar conclusion has been reached by Spitzer et al (1985) studying hepatocytes isolated from rats treated with continuous infusion of endotoxin. Injection of endotoxin into mice decreases activities of the gluconeogenic enzymes (Fig. 4.1.), glucose 6-phosphatase, fructose 1-6-biphosphatase and phosphoenolpyruvate carboxykinase (McCallum & Berry, 1972; Ripe & Berry, 1972). The endotoxin treatment appears to inhibit the glucocorticoid induction of phosphoenolpyruvate carboxykinase at a late post-receptor induction step (Shackleford et al , 1986). Very recent evidence suggests stimulation of the glycolytic enzyme, 6phosphofructokinase, as the primary site of inhibition in vitro of gluconeogenesis by endotoxin injection into rats (Knowles et al , 1987); it would therefore be predictable that the concentrations of intermediates preceding the fructose bisphosphatase/PKF-1 cycle should be increased. Against this view is the fact that, in the present studies, hepatic concentrations of glucose-6-phosphate were significantly elevated in septic rats at 24 and 48 h after the operation (Figs. 3.15 & 3.16 - Chapter 3). It is known that addition of endotoxin does not inhibit hepatic gluconeogenesis in vitro (Filkins & Cornell, 1974), which suggests that its effects in vivo are indirect. Recent evidence supports the view that endotoxin exerts most of its metabolic effects via the action of endogenous mediators produced by cells of haemotopoietic origin (Tracey et al , 1986; Beutler & Cerami, 1987). There is also evidence that pneumoccocal infection decreases the hepatic activity of glucose 6-phosphatase by 50% (Cannonico et al , 1977). In the present studies, accumulation of hepatic gluconeogenic intermediates up to glucose-6-phosphate, with no change in glucose concentration in the livers of septic rats (Table 3.1.; Fig. 3.15. - Chapter 3), suggests an inhibition of gluconeogenic flux at the level of glucose 6-phosphatase. The unchanged concentration of hepatic or blood glucose presumably reflecting a balance between decreased peripheral utilization and hepatic production. If sepsis causes similar changes in enzyme concentrations in the rat this would explain the decreased capacity for hepatic gluconeogenesis in vitro . The decrease in hepatic acetyl-CoA concentration in vivo (Fig. 3.17. - Chapter 3), which is an obligatory activator of pyruvate carboxylase (Fig. 4.1.; Scrutton & Griffiths, 1981), may play a role in diminishing flux through this enzyme, therefore, also limiting gluconeogenesis during sepsis. The absence of hypoglycaemia in the septic rats (Fig. 3.1.- Chapter 3) suggests that this impairment of gluconeogenesis may be overcome in vivo by increased concentration of precursors, mainly lactate and alanine, which are supplied in increased amount from muscle in sepsis (Biesel & Wannemacher, 1980; Hasselgren et al , 1984). CHAPTER 5 Time course of changes in the metabolism of [1-14C]oleate and the rate of ketogenesis in isolated liver cells after induction of sepsis Introduction Sepsis in man and experimental animals causes a number of changes in lipid metabolism (Blackburn, 1977; Beisel & Wannemacher, 1980; Neufeld et al , 1982). These include hypertriacylglycerolaemia (Blackburn, 1977; Beisel & Wannemacher, 1980), decreased blood ketone bodies (Neufeld et al , 1976) and changes in plasma free fatty acid concentrations and lipoprotein lipase activity (Robin et al , 1981; Lanza-Jacoby et al , 1982). It is therefore, important to obtain information on the effects of sepsis on the hepatic metabolism of fatty acids during starvation. Metabolism of long- and short-chain fatty acids During starvation plasma non-esterified fatty acids are derived exclusively from the hydrolysis of triacylglycerol within the adipose tissue and the rate of mobilization of fatty acids from this tissue controls their concentration in the plasma. The liver has the capacity to remove a large proportion of non-esterified fatty acids from the circulation (Newsholme & Start, 1973). The rate of hepatic uptake appears to be regulated by the concentration of non-esterified fatty acids in plasma. Once in the hepatocyte, these non-esterified fatty acids can be esterified to form triacylglycerol or be partially oxidized to form ketone bodies, or be totally oxidized into the Krebs cycle to form metabolic CO2 (Fig. 5.1.). Long-chain fatty acids have to be activated and transported into the mitochondria for oxidation via the carnitine acyl-CoA transferase system, whereas short-chain fatty acids are not be esterified and do not require the acylCoA transport system to enter the mitochondria for ß-oxidation (Fig. 5.1.; for review see McGarry & Foster, 1980). Fig. 5.1. Changes in plasma non-esterified fatty acids and blood and hepatic ketone bodies concentrations during sepsis While plasma non-esterified fatty acid concentrations have been reported to be variable, esterification by the liver has been reported to be increased by sepsis (Beisel & Wannemacher, 1980). The increased production of triacylglycerol by the liver together with diminished activity of lipoprotein lipase in peripheral tissues have been proposed as the mechanism responsible for hypertriacylglycerolaemia during sepsis (Robin et al , 1981; Lanza-Jacoby et al , 1982). During the fed state, the concentration of ketone bodies (acetoacetate and 3hydroxybutyrate) in the liver and blood is very low (Williamson et al , 1967). During the starved state, large quantities of ketone bodies are produced by the liver and are transported via the blood to peripheral tissues where they are used as important alternative substrates. The acetyl-CoA formed in the metabolism of ketone bodies can either be oxidized in the Krebs cycle to provide energy or in particular tissues, such as the developing brain or lactating mammary gland, it can be used as precursor for lipid synthesis (Williamson & Whitelaw, 1978; Williamson, 1981). However, if starvation is accompanied by sepsis, blood and hepatic concentrations of ketone bodies decrease to values similar to those of the fed state (Neufeld et al, 1976 and 1977). Possible reasons for this failure to develop hyperketonaemia, in the starved septic state, are increased utilization of ketone bodies or decreased ketone body formation. The latter may be the result of poor perfusion of adipose tissue leading to decreased mobilization of non-esterified fatty acids, and/or decreased hepatic blood flow (Williamson, 1981). Increased utilization in peripheral tissues does not appear to be the cause because infused 3-hydroxybutyrate reaches similar blood concentrations, in infected as well as in fasted control sheep (Radcliffe et al , 1981). Wannemacher et al (1979), studying pneumococcal sepsis in rats, proposed that the failure of septic rats to become ketonaemic during starvation was the result of reduced ketogenic capacity of the liver and , possibly, a decrease in the hepatic supply of fatty acids. In order to obtain more information and understand the mechanisms reponsible for the failure to develop during sepsis the hyperketonaemia associated with starvation , rates of ketogenesis from long- and short-chain fatty acids in isolated hepatocytes, from caecalligated and punctured rats as compared to sham-operated animals, were measured. The metabolism of [1-14C]oleate, namely the rate of uptake and distribution into the pathways of esterification, ketogenesis and 14CO2 formation, by isolated liver cells from septic and sham-operated rats, was also studied. Design of the studies Rats were subjected to caecal-ligation and puncture or sham-operation and starved for 12, 24 or 48 h when they were studied (Fig 5.2.). Fig 5.2. OP 12 h 24 h 48 h Hepatocyte isolation Rats Incubation 1. no substrate 2. butyrate 3. oleate 20' 40' 60' For details of the technique of liver cell isolation and incubation procedures see Chapter 2. Isolated liver cells from a group of rats, studied at 48 h after the operation, were incubated with [1-14C]oleate. The uptake and the conversion of [1-14C]oleate into its metabolic products were studied. Results Time course of rates of ketogenesis from long- and short-chain fatty acids in isolated hepatocytes There were no appreciable changes in the rate of ketone body formation at any of the time points studied in the absence of added substrate (Fig. 5.3.). Fig. 5.3. When hepatocytes from caecal-ligated and punctured rats and sham-operated rats were incubated with [5mM]butyrate, a short-chain fatty acid, there were no significant differences in the rate of ketogenesis (Fig. 5.4.). Fig. 5.4. However, isolated liver cells from septic rats produced ketone bodies at a significantly lower rate, at all time points studied, when the long-chain fatty acid [2mM]oleate was the substrate (Fig. 5.5.). Fig. 5.5. Similar results were found in isolated perfused livers from rats with pneumoccocal infection (Wannemacher et al , 1979). The metabolism of [1-14C]oleate during sepsis Table 5.1. shows the uptake and conversion of [1-14C]oleate to its metabolic products. The rate of ketone body formation from [1-14C]oleate was significantly lower in septic rats as compared with sham-operated rats, due to a decreased rate of D-3-hydroxybutyrate formation (Table 5.1.). This change in the [3hydroxybutyrate]/ [acetoacetate] ratio would be expected if ß-oxidation is depressed (Williamson et al , 1967). The rate of removal of [1-14C]oleate from the medium (uptake) by isolated liver cells from septic rats was 23% lower as compared with sham-operated animals (Table 5.1. ; Fig. 5.6.). Although there was no significant difference in the absolute amounts of [114C]oleate esterified by hepatocytes from septic rats (Table 5.1.), the percentage was significantly higher (Fig. 5.6.) than that of sham-operated rats (45±2.8% versus 36±0.73% ; P < 0.05, n=6). The percentage of [1-14C]oleate converted to metabolic 14CO2 in hepatocytes from septic rats was twice (Fig. 5.6.) that of sham-operated rats (3.5±0.5% versus 1.8±0.02% ; P < 0.05, n=6) and may be a contributory factor to the decreased ketogenesis as it reflects more acetyl-CoA entering the Krebs cycle in the hepatocytes from septic rats. The rate of formation of acid-soluble (A-S) products, mainly ketone bodies, was significantly lower in hepatocytes from septic rats (Table 5.1.; Fig. 5.6.) as was the percentage of [1-14C]oleate converted to these products ( 40±4.5% versus 60±4.8% ; P < 0.05, n=6). Table 5.1. Effects of sepsis on metabolism of [1-14C]oleate in isolated hepatocytes from 48 h starved rats _________________________________________________________________________ Results are mean values ± SEM with number of observations shown in parentheses. Significance of difference by Student's t-test from the sham-operated rats: * P < 0.01 ; ** P < 0.001. __________________________________________________________________ _______ Rate (µmol/g wet wt. cells/min) _________________________________________________________ Sham-operated Septic Change (%) _______________________________________________________________________ Acetoacetate 0.52±0.027 (6) 0.55±0.055 (6) + 6 3-Hydroxybutyrate 1.15±0.062 (6) 0.66±0.056 (6) ** - 57 Total ketone bodies 1.66±0.058 (6) 1.23±0.077 (6) ** - 26 Removal of [1-14]oleate 0.55±0.044 (6) 0.42±0.053 (6) - 23 Esterification of [1-14C]oleate [1-14C]oleate - 14CO2 0.20±0.004 (6) 0.19±0.012 (6) - 5 0.010±0.001 (6) 0.015±0.002 (6) + 50 [1-14C]oleate - 14C acid 0.33±0.028 (6) 0.17±0.020 (6) * - 48 soluble products _______________________________________________________________________ Fig. 5.6. Metabolism of [1 - C]oleate14 % [1-14C]oleate distribution 100 80 Esterification A-S products Metabolic CO 2 60 40 20 0 Sham-op 48 h after the operation Discussion Septic The concentration of plasma non-esterified fatty acids has been reported to be increased (Blackburn, 1977) or decreased (Neufeld et al , 1982) by sepsis. In the present study the slight decrease (13% ; Fig. 3.7. - Chapter 3) was not significant. Nevertheless, in vitro ketogenesis from the added long-chain fatty acid, oleate, was markedly depressed (Fig. 5.5.) indicating that the septic liver has a decreased capacity to produce ketone bodies. Others have shown that perfused livers from rats with pneumococcal infection produce significantly less ketone bodies from oleate as compared to perfused livers from fasted controls (Wannemacher et al , 1979). When medium chain fatty acids, such as octanoic acid, are the substrate, isolated perfused livers from infected rats produce as much ketone bodies as livers from starved controls (Wannemacher et al , 1979). Similar results were found when butyrate was the substrate in isolated liver cells from caecal-ligated and punctured rats, as compared to hepatocytes from sham-operated rats (Fig. 5.4.) The fact that ketogenesis remained unchanged, in isolated hepatocytes from septic rats, when the short-chain fatty acid butyrate was the substrate, whereas it was significantly lower when the long-chain fatty acid oleate was the substrate, suggests that a site of inhibition of ketogenesis in the septic liver may be the entry of long-chain fatty acid into the mitochondria via the carnitine acyl-CoA transferase system (McGarry & Foster , 1980). Long-chain fatty acyl-CoA would, alternatively, go into the esterification pathway leading to formation of triacylglycerols and phospholipids. One physiologically important regulatory mechanism of the entry of long-chain acyl-CoA into the mitochondria is the modulation of the flux through carnitine palmitoyltransferase I by malonyl-CoA, an intermediate of the lipogenic pathway. Although the precise mechanism of malonyl-CoA inhibition on carnitine acyltransferase I in the rat liver mitochondria has not yet been fully established, available evidence indicates a competitive type of inhibition against long-chain acyl-CoA substrates (McGarry & Foster, 1980). During starvation, at low rates of lipogenesis, decreased malonyl-CoA concentrations permit the diversion of long-chain fatty acid into oxidative pathways in the mitochondria. However, despite the fact that donor rats are starved, hepatocytes from septic rats divert more long-chain fatty acid to esterification (Fig. 5.6.) than to partial oxidation in the mitochondria to yield ketone bodies (Fig 5.5.). The rate of hepatic lipogenesis in vivo is significantly increased (Fig. 6.2. Chapter 6) in starved septic rats. Although lipogenesis is considered to be positively correlated with cytosolic malonyl-CoA concentrations it must be emphasized that hepatic carnitine acyl-CoA transferase I (CAT I) is less sensitive to inhibition by malonyl-CoA in livers from starved rats (Cook et al , 1980). Sepsis induced by injection of bacteria has been reported not to increase hepatic malonyl-CoA (Wannemacher et al , 1979) or alternatively to increase this metabolite (Vary et al , 1986). There is also evidence that the sensitivity of carnitine acyl transferase I to malonyl-CoA can be rapidly increased by insulin (Gamble & Cook, 1985) and, therefore, it may be pertinent that our septic rats had higher plasma insulin concentration (Fig. 3.5. Chapter 3). It must however be emphasised that lipogenesis has not been measured in the present hepatocyte experiments. A decreased [D-3-hydroxybutyrate]/[acetoacetate] ratio (Table 5.1.) together with the lower concentrations of acetyl-CoA ( Fig. 3.17. - Chapter 3) in livers from septic rats support the view of depressed ß-oxidation in sepsis, as a result of the diminished flux of long-chain acyl-CoA into the mitochondria. An alternative reason for the decreased entry of long-chain acyl-CoA into the mitochondria is that the high insulin concentrations may suppress the putative role of glucagon to phosphorylate and increase the activity of carnitine acyl transferase I. Recent experiments have suggested that glucagon can increase the affinity of the enzyme complex (CPT1+CPT2) for palmitoyl-CoA and increase the phosphorylation of the protein (Harano et al , 1985). Further work has suggested that CPT activity might be regulated by both protein kinase A and by Ca++/calmodulin-dependent protein kinase activities (Kojima et al , 1986). The reason for the impairment of ketogenesis in livers from septic rats, therefore, appears to be multifactorial and involves a possible decrease in hepatic uptake of nonesterified fatty acids, depression of the entry of long-chain acyl-CoA into the mitochondria for ß-oxidation and lower hepatic concentration of acetyl-CoA. The increase in the percentage of metabolic CO2 formation, by hepatocytes from septic rats, may be another contributory factor for it indicates more acetyl-CoA entering the Krebs cycle for oxidation, thus decreasing the availability of acetyl-CoA as the immediate cause for the decreased ketogenesis. This depression of the entry of long-chain acyl-CoA may be of clinical importance in deciding what lipid emulsion to infuse, as part of nutrients required, in intravenous nutrition. The use of recently available lipid emulsions, which are enriched with mediumchain fatty acids, might be theoretically beneficial for septic patients (Eckhart et al , 1980). However reports on the use of these long-chain triacylglycerol (LCT)/mediumchain triacylglycerol (MCT) emulsions are controversial. Stein et al (1986) studied caecalligated rats receiving intravenous nutrition with either MCT/LCT or LCT emulsions as part of or as the only non-protein calorie source. Rats fed with MCT/LCT mixture alone, or in association with glucose, showed significantly lower survival rates as compared to rats fed with other nutrient mixtures. In contrast, Dennison et al (1986), in a randomized crossover trial, showed a significant increase in blood ketone body concentration accompanied by a decrease in plasma tricylglycerol concentration, in patients parenterally fed with half of their non-nitrogen calories as an MCT/LCT (1:1) 10% lipid emulsion, as compared to the concentrations measured when the same patients were receiving a 10% lipid emulsion containing only LCT. The increase in ketone body concentration suggests increased hepatic utilization, whereas the decrease in plasma triacylglycerol suggests enhanced clearance with MCT. While receiving MCT, patients also had a better nitrogen retention. Therefore, if the depression in the entry of long-chain acyl-CoA in the liver of the septic rat is also present in the liver of septic patients, MCT containing lipid emulsions may be theoretically regarded as a better caloric source during sepsis. Nevertheless further investigation, in the form of randomized control studies involving septic patients requiring intravenous feeding is needed. CHAPTER 5 Time course of changes in the metabolism of [1-14C]oleate and ketogenesis in isolated liver cells after induction of sepsis Introduction Sepsis in man and experimental animals causes a number of changes in lipid metabolism (Blackburn, 1977; Beisel & Wannemacher, 1980; Neufeld et al , 1982). These include hypertriacylglycerolaemia (Blackburn, 1977; Beisel & Wannemacher, 1980), decreased blood ketone bodies (Neufeld et al , 1976) and changes in plasma free fatty acids concentration and lipoprotein lipase activity (Robin et al , 1981; LanzaJacoby et al , 1982). Therefore, a better understanding of the hepatic metabolism of long- and short-chain fatty acids during starvation in the presence or absence of sepsis is necessary. Metabolism of long- and short-chain fatty acids During starvation plasma non-esterified fatty acids are derived exclusively from the hydrolysis of triacylglycerol within the adipose tissue and the rate of mobilization of fatty acids from this tissue controls their concentration in the plasma. The liver has the capacity to remove a large proportion of non-esterified fatty acids from the circulation (Newsholme & Start, 1973). The rate of hepatic uptake appears to be regulated by the concentration of non-esterified fatty acids in plasma. Once in the hepatocyte, these non-esterified fatty acids can be esterified to form triacylglycerol or be partially oxidized to form ketone bodies, or be totally oxidized into the Krebs cycle to form metabolic CO2 (Fig. 5.1.). Long-chain fatty acids have to be activated and transported into the mitochondria for oxidation via the carnitine acyl-CoA transferase system, whereas short-chain fatty acids can not be esterified and do not require the acyl-CoA transport system to enter the mitochondria and suffer ∫-oxidation (Fig. 5.1.; for review see McGarry & Foster, 1980). Fig. 5.1. Simplified scheme of the hepatic metabolism of long- and short-chain fatty acids Long-chain fatty acids Short-chain fatty acids Long-chain fatty acyl-CoA carnitine Esterified CAT I products Lactate CAT II Pyruvate VLDL carnitine Fatty acyl-CoA Oxaloacetate Short-chain fatty acyl-CoA Acetyl-CoA Acetoacetyl-CoA HMG-CoA CO 2 CO 2 Acetoacetate ß-Hydroxybutyrate Changes in plasma non-esterified fatty acids and blood and hepatic ketone bodies concentrations during sepsis While plasma non- esterified fatty acid concentrations have been reported to be variable, esterification by the liver has been reported to be increased by sepsis (Beisel & Wannemacher, 1980). The increased production of triacylglycerol by the liver together with diminished activity of lipoprotein lipase in peripheral tissues have been proposed as the mechanism responsible for hypertriacyglycerolaemia during sepsis (Robin et al, 1981; LanzaJacoby et al , 1982). During the fed state, the concentration of ketone bodies (acetoacetate and 3-hydroxybutyrate) in the liver and blood is very low (Williamson et al , 1967). During the starved state, when the supply of carbohydrate is limited, large quantities of ketone bodies are produced by the liver and are transported via the blood to peripheral tissues where they are used as important alternative substrates. The acetyl-CoA formed in the metabolism of ketone bodies can either be oxidized in the Krebs cycle to provide energy or in particular tissues, such as, the developing brain or lactating mammary gland, it can be used as precursor for lipid synthesis (Williamson & Whitelaw, 1978; Williamson, 1981). However, if starvation is accompanied by sepsis, blood and hepatic concentrations of ketone bodies decrease to values not different from those of the fed state (Neufeld et al, 1976 and 1977). Possible reasons for this failure to develop hyperketonaemia, in the starved septic state, are increased utilization of ketone bodies or decreased ketone body formation. The latter may be the result of poor perfusion of adipose tissue leading to decreased mobilization of non-esterified fatty acids, and/or decreased hepatic blood flow (Williamson DH, 1981). Increased utilization in peripheral tissues does not appear to be the cause because infused 3-hydroxybutyrate was oxidised to similar amounts of acetoacetate, and both ketone bodies were equally cleared, in infected as well as in fasted control sheep (Radcliffe et al , 1981). Wannemacher et al (1979), studying pneumococcal sepsis in rats, proposed that the failure of septic rats to become ketonemic during starvation was the result of reduced ketogenic capacity of the liver and , possibly, a decrease in the hepatic supply of fatty acids. In order to obtain more information and understand the mechanisms reponsible for the failure to develop during sepsis the hyperketonaemia associated with starvation , rates of ketogenesis from long- and short-chain fatty acids in isolated hepatocytes, from caecal-ligated and punctured rats as compared to sham-operated animals, were measured. The metabolism of [1-14C]oleate, namely the rate of uptake and distribution into esterification, ketogenesis and 14CO2 formation, by isolated liver cells from septic and sham-operated rats, was also studied. Design of the studies Rats were subjected to caecal-ligation and puncture or sham-operation and starved for 12, 24 or 48 h when they were studied (Fig 5.2.). Fig 5.2. OP 12 h 24 h 48 h Hepatocyte isolation Rats Incubation 1. no substrate 2. butyrate 3. oleate 20' 40' 60' For details of the technique of liver cell isolation and incubation procedures see Chapter 2. Isolated liver cells from a group of rats, studied at 48 h after the operation, were incubated with [1-14C]oleate. The uptake and the conversion of [1-14C]oleate into its metabolic products were studied. Results Time course of rates of ketogenesis from long- and short-chain fatty acids in isolated hepatocytes There were no appreciable changes in the rate of ketone body formation at any of the time points studied in the absence of added substrate (Fig. 5.3.). Fig. 5.3. Rate of ketone body formation - no added substrate 0.5 (5) µmol/g wet wt. cells/min 0.4 (5) (3) (3) (5) (5) 0.3 Sham-op Septic 0.2 X + S.E.M. ( ) nº of observations 0.1 0.0 12 1 24 2 48 3 Time after the operation (h) When hepatocytes from caecal-ligated and punctured rats and shamoperated rats were offered [5mM]butyrate, a short-chain fatty acid, as the substrate there were no significant differences in the rate of ketogenesis (Fig. 5.4.). Fig. 5.4. Rate of ketogenesis from butyrate [5mM] 2 (5) (5) µmol/g wet wt. cells/min (5) (3) (3) (5) Sham-op Septic 1 X + S.E.M. ( ) nº of observations 0 12 1 24 2 48 3 Time after the operation (h) However, isolated liver cells from septic rats produced ketone bodies at a significantly lower rate, at all time points studied, when the long-chain fatty acid [2mM]oleate was the substrate (Fig. 5.5.). Fig. 5.5. Rate of ketogenesis from oleate [2mM] (7) µmol/g wet wt. cells/min 1.50 * (8) (3) (4) 1.00 Sham-op Septic ** (4) * (5) * P < 0.05 ** P < 0.01 0.50 X + S.E.M. ( ) nº of observations 0.00 12 1 24 2 48 3 Time after the operation (h) Similar results were found in isolated perfused livers from rats with pneumoccocal infection (Wannemacher et al , 1979). The metabolism of [1-14C]oleate during sepsis Table 5.1. shows the uptake and conversion of [1-14C]oleate to its metabolic products. The rate of ketone body formation from [1-14C]oleate was significantly lower in septic rats as compared with sham-operated rats, due to a decreased rate of D-3-hydroxybutyrate formation (Table 5.1.). This change in the [3- hydroxybutyrate]/[acetoacetate] ratio would be expected if ∫-oxidation is depressed (Williamson et al, 1967). The rate of removal of [1-14C]oleate from the medium (uptake) by isolated liver cells from septic rats was 23% lower as compared with shamoperated animals (Table 5.1. ; Fig. 5.6.). Although there was no significant difference in the absolute amounts of [114C]oleate esterified by hepatocytes from septic rats (Table 5.1.), the percentage was significantly higher (Fig. 5.6.) than that of sham-operated rats (45±2.8% versus 36±0.73% ; P < 0.05, n=6). The percentage of [1-14C]oleate converted to metabolic 14CO in hepatocytes from septic rats was twice (Fig. 5.6.) that of sham-operated rats 2 (3.5±0.5% versus 1.8±0.02% ; P < 0.05, n=6) and may be a contributory factor to the decreased ketogenesis as it reflects more acetyl-CoA entering the Krebs cycle in the hepatocytes from septic rats. Table 5.1. Effects of sepsis on metabolism of [1-14C]oleate in isolated hepatocytes from 48 h starved rats _______________________________________________________________________ Results are mean values ± SEM with number of observations shown in parentheses. Significance of difference by Student's t-test from the sham-operated rats: * P < 0.01 ; ** P < 0.001. _________________________________________________________ Rate (µmol/g wet wt. cells/min) ______________________________________________________ Sham-operated Septic Change (%) _______________________________________________________________________ Acetoacetate 3-Hydroxybutyrate Total ketone bodies Removal of [1-14]oleate 0.52±0.027(6) 1.15±0.062(6) 1.66±0.058(6) 0.55±0.055(6) 0.66±0.056(6) ** 1.23±0.077(6) ** + 6 - 57 - 26 0.55±0.044(6) 0.42±0.053(6) Esterification of [1-14C]oleate 0.20±0.004(6) [1-14C]oleate - 14CO2 0.010±0.001(6) 0.19±0.012(6) - 5 0.015±0.002(6) + 50 [1-14C]oleate - 14C acid 0.33±0.028(6) 0.17±0.020(6) * - 23 - 48 soluble products _______________________________________________________________________ The rate of formation of acid-soluble (A-S) products, mainly ketone bodies, was significantly lower in hepatocytes from septic rats (Table 5.1.; Fig. 5.6.) as was the percentage of [1-14C]oleate converted to these products ( 40±4.5% versus 60±4.8% ; P < 0.05, n=6). Fig. 5.6. Metabolism of [1 - C]oleate14 % [1-14C]oleate distribution 100 80 Esterification A-S products Metabolic CO 2 60 40 20 0 Sham-op 48 h after the operation Septic Discussion The concentration of plasma non-esterified fatty acids has been reported to be increased (Blackburn, 1977) or decreased (Neufeld et al , 1982) by sepsis. In the present study the slight decrease (13% ; Fig. 3.7. - Chapter 3) was not significant. Nevertheless, ketogenesis from the added long-chain fatty acid, oleate, in vitro was markedly depressed (Fig. 5.5.) indicating that the septic liver has a decresed capacity to produce ketone bodies. Others have shown that perfused livers from rats with pneumococcal infection produce significantly less ketone bodies from oleate as compared to perfused livers from fasted controls (Wannemacher et al , 1979). When octanoic acid was the substrate, isolated perfused livers from infected rats produced as much ketone bodies as livers from starved controls (Wannemacher et al , 1979). The same results were found, when butyrate was used as substrate, in isolated liver cells from caecal-ligated and punctured rats as compared to hepatocytes from sham-operated rats (Fig. 5.4.) The fact that ketone body formation remained unchanged when the shortchain fatty acid, butyrate, was the substrate, whereas it was significantly lower in hepatocytes from septic rats, when the long-chain fatty acid oleate was the substrate, suggests that a site of inhibition of ketogenesis in the septic liver may be the entry of long-chain fatty acid into the mitochondria via the carnitine acyl-CoA transferase system (McGarry & Foster , 1980). Long-chain fatty acyl-CoA would, alternatively, go into the esterification pathway leading to formation of triacylglycerols and phospholipids. One physiologically important regulatory mechanism of the entry of longchain acylCoA into the mitochondria is the modulation of the flux through carnitine palmitoyltransferase I by malonyl-CoA, an intermediate of the lipogenic pathway. Although the precise mechanism of malonylCoA inhibition on carnitine acyltransferase I in the rat liver mitochondria has not yet been fully established, available evidence indicates a competitive type of inhibition against long-chain acyl-CoA substrates (McGarry & Foster, 1980). During starvation, at the low rates of lipogenesis, decreased malonyl-CoA concentrations permit the diversion of long-chain fatty acid into oxidative pathways in the mitochondria. However, despite being starved, hepatocytes from septic rats divert more long-chain fatty acid to esterification (Fig. 5.6.) than to partial oxidation in the mitochondria to yield ketone bodies (Fig 5.5.). The rate of hepatic lipogenesis in vivo is significantly increased (Fig. 6.2. Chapter 6) in septic rats. Although lipogenesis is considered to be positively correlated with cytosolic malonyl-CoA concentrations it must be emphasized that hepatic carnitine acyl-CoA transferase I (CAT I) is less sensitive to inhibition by malonyl-CoA in livers from starved rats (Cook et al , 1980). In addition, lipogenesis has not been measured in the present hepatocyte experiments. Sepsis induced by injection of bacteria has been reported not to increase hepatic malonyl-CoA (Wannemacher et al , 1979) or alternatively to increase this metabolite (Vary et al , 1986). There is also evidence that the sensitivity of carnitine acyl transferase I to malonyl-CoA can be rapidly increased by insulin (Gamble & Cook, 1985) and, therefore, it may be pertinent that our septic rats had higher plasma insulin concentration ( Fig. 3.5. - Chapter 3). Decreased [D-3-hydroxybutyrate]/[acetoacetate] ratio (Table 5.1.) together with the lower concentrations of acetyl-CoA ( Fig. 3.17. - Chapter 3) in livers from septic rats support the view of depressed ∫-oxidation in sepsis, as a result of the diminished flux of long-acylCoA into the mitochondria. The reason for the impairment of ketogenesis in livers from septic rats, therefore, appears to be multifactorial and involves a possible decrease in hepatic uptake of non-esterified fatty acids, depression of the entry of long-chain acyl-CoA into the mitochondria for ∫-oxidation and lower hepatic concentration of acetyl-CoA. The increase in the percentage of metabolic CO2 formation, by hepatocytes from septic rats, may be another contributory factor for it indicates more acetyl-CoA entering the Krebs cycle for oxidation, thus decreasing the availability of acetyl-CoA as the immediate cause for the decreased ketogenesis. CHAPTER 6 Time course of changes in rates of lipogenesis and hepatic cholesterogenesis in vivo after the induction of sepsis Introduction Despite the fact lipids constitute one of the principal sources of body energy and sepsis produces various alterations in fat metabolism, many of the metabolic changes induced by sepsis still need clarification. More than six decades have passed since the changes in concentrations of cholesterol in serum, during bacterial infection, were first described (Kipp, 1920). However, little is known of the regulation of cholesterol metabolism during sepsis. Some information has recently been collected on the metabolism of non-esterified fatty acids during infection (Blackburn, 1977; Wannemacher et al , 1979; Beisel & Wannemacher, 1980). The availability of non-esterified fatty acids may be decreased in sepsis as a result of the anti-lipolytic effect of the hyperinsulinaemia (Fig. 3.5. - Chapter 3), to suppress hormone sensitive lipase activity (Steinberg & Khoo, 1977) resulting in lower concentrations of non-esterified fatty acids, as reported in various models of sepsis (Beisel & Wannemacher, 1980). The plasma concentration of non-esterified fatty acids, however, was not significantly changed in the present studies (Fig. 3.7. - Chapter 3). A decrease in the percentage of non-esterified fatty acid uptake (23%) was found in isolated hepatocytes from caecal-ligated and punctured rats (Table 5.1. - Chapter 5). Within hepatocytes, non-esterified fatty acids appear to be preferentially esterified in the livers of septic rats, rather than partially or totally oxidized with formation of ketone bodies or metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al , 1979). The percentage of [1-14C]oleate esterified was significantly higher in isolated hepatocytes from caecal- ligated rat as compared to that of hepatocytes from sham-operated rats (Fig. 5.6. Chapter 5). As a result of decreased oxidation of long-chain fatty acids the rate of ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the hyperketonaemia, associated with starvation, decreased in sepsis (Fig. 3.8. - Chapter 3). Non-esterified fatty acid utilization by the liver, therefore, appears to be decreased in experimental or clinical sepsis with the development of an elevation in plasma concentrations of triacylglycerol (Levin et al , 1972; Kaufmann et al , 1976; Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al , 1981 Alvares & Ramos, 1986), and fatty infiltration of the liver (Mukherjee et al , 1973; Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of increased triacylglycerol synthesis accompanied by a decreased or insufficient production of the proteins needed to export very low density lipoprotein (VLDL) as suggested by Forse & Kinney (1985). However, general protein synthesis in the liver has been reported to be stimulated by sepsis (Long et al , 1977; Hasselgen et al, 1984). Decreased plasma clearance of triacylglycerol as a result of diminished activity of lipoprotein lipase has been reported as contributory to the hypertriacylglycerolaemia induced by sepsis (Kaufmann et al , 1976; Robin et al , 1981; Lanza-Jacoby et al , 1982). Hepatic lipogenesis in vitro , measured by the incorporation of [1-14C]acetate into lipids in hepatocytes isolated from rats with pneumococcal infection, is increased in response to sepsis (Canonico et al , 1977). There appears to be no information, however, on rates of lipid synthesis in vivo in the liver or adipose tissue after induction of sepsis. Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated rats (Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats (Paceet al ,1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from rats with pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the flow of acetylCoA toward lipogenesis and cholesterolegenesis in vivo rather than to ketogenesis or oxidation in the Krebs tricarboxylic acid cycle (Fig. 6.1.). Fig. 6.1. Total cholesterol concentration in serum has been reported to increase, decrease, or remain unchanged during sepsis (Fiser et al , 1971 and 1972; Royle & Kettlewell, 1980; Alvares & Ramos, 1986). Such variation in cholesterol concentration provides little information on the dynamics involved in cholesterol synthesis, transport and utilization. Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic acid, showed increased incorporation of the labelled mevalonate into free cholesterol in plasma when compared to control monkeys (Fiser et al , 1971). In vitro , [14C]acetate incorporation into cholesterol by isolated hepatocytes from rats with pneumococcal infection was significantly increased as compared to the incorporation by hepatocytes from control rats (Cannonico et al , 1977). However, there appears to be no information on the rates of hepatic cholesterol synthesis in vivo in response to sepsis. The entero-hepatic circulation of bile acids plays an essential role in the absorption of cholesterol from the intestine and in the regulation of cholesterol metabolism in the liver (for review see : Myant, 1981 and Gibbons et al , 1982). The diversion of bile acids, or the complete interruption of the bile duct, leads to the absence of bile acids in the intestine, which in turn, supresses the normal uptake of cholesterol from the intestinal lumen. There is some evidence that it is the transport of intestinal cholesterol to the liver which directly determines the rates of hepatic cholesterol synthesis (Weis & Dietschy, 1975). This view is supported by the fact that restoration of the entero-hepatic circulation, in rats subjected to biliary diversion, failed to prevent the rise in hepatic cholesterogenesis, whereas, the infusion of chylomicron cholesterol led to a prevention in the rise of hepatic sterol synthesis rates (Gibbons et al , 1982). In addition, bile acids inhibit cholesterol 7Ïhydroxylase (the rate-limiting enzyme in the process of bile acids formation from cholesterol) and, therefore, would prevent the catabolism and removal of cholesterol from the liver, resulting in suppression of HMG-CoA reductase activity (Nervi & Dietschy, 1978). The possibility of bile acids having a direct effect on HMG-CoA reductase, in the liver, would suggest diversion rather than obstruction of the bile duct as the resonable approach to study the metabolism of cholesterol in the liver. However, opposing the idea of a direct bile acid effect is the fact that partial or complete biliary obstruction causes an increase, rather than the expected decrease, in hepatic cholesterol synthesis (Fredrickson et al , 1954; Gibbons et al , 1982). To investigate the time course of changes in vivo in lipogenesis in the liver, and in white and brown adipose tissue, and hepatic cholesterogenesis, after the induction of sepsis, caecal-ligated and punctured rats were studied. Sham-operated rats were used as controls. As the absorption of bile acids and cholesterol in the intestinal lumen of caecalligated and punctured rats may be impaired, due to the development of an adynamic ileus in these rats, ligation of the bile duct of a group of septic and sham-operated rats was also performed. This complete interruption of the entero-hepatic circulation of bile acids may help to evaluate the contribution of the impairment in the absorption of cholesterol and bile acids from the gut to hepatic cholesterogenesis in sepsis. Experimental Design Rats, after being subjected to caecal-ligation and puncture or sham-operation, were fasted for 12, 24 or 48 h before livers were removed and studied (Fig.6.2.). Fig. 6.2. For details of the operations and the techniques involved in the measurement of lipogenesis, cholesterogenesis, preparation of microsomes and the measurement of hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and lipoprotein lipase activities see Chapter 2. A group of rats had their bile duct ligated immediately before either caecal-ligation and puncture or sham-operation was performed (Chapter 2). Results Effects of sepsis on hepatic lipogenesis The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo was significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48 h after the operations, as compared to that of livers from sham-operated animals (Fig. 6.3.). Fig. 6.3. Rate of H 3 incorporation O into fatty acids 2 ** (7) (4) µmol/g wet wt. liver/h 4.0 3.0 * (18) Sham-op Septic (3) (13) 2.0 * P < 0.05 ** P < 0.001 (6) 1.0 X + S.E.M. ( ) nº of observations 0.0 12 24 48 Time after the operation (h) Increased rates of formation of total non-saponifiable lipids in vivo were also found to be present in livers from septic rats when compared to those found in livers from sham-operated rats (Fig. 6.4.) as early as at 12 h after the operation. Fig. 6.4. Rate of H3 incorporation into non-saponifiable lipids 2O * (4) 7 6 µmol/g wet wt. liver/h Sham-op Septic ** (7) 5 ** (18) 4 3 * P < 0.05 (3) (6) ** P < 0.001 2 (13) X + S.E.M. ( ) nº of observations 1 0 12 24 48 Time after the operation (h) Therefore, livers from septic rats have significantly increased rates of total lipogenesis as compared to those of sham-operated rats. This increase in lipid synthesis appears to occur both in rates of formation of saponifiable (fatty acids) and nonsaponifiable (squalene, cholesterol, other sterols) fractions (Fig. 6.5.). Fig. 6.5. Rate of H 10 3 incorporation into lipids 2O * * µmol/g wet wt. liver/h 8 * 6 saponified non-saponified 4 2 * P < 0.001 0 sham septic 1 2 12 h sham septic 3 4 24 h sham septic 5 6 48 h Effects of sepsis on squalene and cholesterol synthesis in vivo and hydroxymethylglutarylCoA reductase activity in the liver The non-saponifiable fraction was separated into the various sterol fractions. Sepsis produced a significant increase in the rate of squalene formation in the liver at 24 h after the operation (Fig. 6.6.). No change in rate was observed at the other time points studied. Fig. 6.6. Rate of H 3 incorporation into squalene O 2 * (7) (4) 0.50 (18) µmol/g wet wt. liver/h 0.40 (3) Sham-op Septic (13) 0.30 (6) 0.20 * P < 0.05 0.10 X + S.E.M. ( ) nº of observations 0.00 12 24 48 Time after the operation (h) Starvation caused the incorporation of 3H2O into cholesterol to decrease with time in both sham-operated and caecal-ligated and punctured rats. However, the rates of cholesterol synthesis in the livers of septic rats in vivo were significantly higher than those found in the livers of sham-operated rats. This increase (2.3 fold) occurred as early as 12 h after the operations reaching a four-fold increase at 48 h (Fig. 6.7.). Fig. 6.7. Rate of H 3incorporation 2O into cholesterol * (4) 4.0 sham-op septic µmol/g wet wt. liver/h 3.0 2.0 ** (11) ** (18) (3) * P < 0.05 ** P < 0.001 (11) 1.0 (12) X + S.E.M. ( ) nº of observations 0.0 12 24 48 Time after the operation (h) The total activity of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the livers of septic rats was significantly increased compared to that found in livers of shamoperated rats at 48 h after the operations (Fig. 6.8.). Fig. 6.8. Total HMG-CoA reductase activity pmol mevalonate/mg protein/min * (7) 20 sham-op septic 10 (4) * P < 0.02 X + S.E.M. ( ) nº of observations 0 48 h 48 h after the operation HMG-CoA reductase is phosphorylated by a protein kinase and phosphorylation leads to inactivation of the enzyme (Gibbons et al , 1982; Gibson & Parker, 1987). The activity of both the phosphorylated ('inactive' form) and the non-phosphorylated or expressed form of this enzyme (Fig. 6.9.) was measured in livers from septic and shamoperated rats at 48 h after the operation. Sepsis induced no appreciable change in the expressed activity of HMG-CoA reductase (non-phosphorylated or 'active'). As there was a significant increase in the total enzyme activity (Fig. 6.8.) this was mainly due to an increase in activity of the phosphorylated or 'inactive' form of the enzyme (Fig. 6.9.). Fig. 6.9. HMG-CoA reductase activity * (7) pmol mevalonate/mg protein/min 25 20 Sham-op Septic 15 10 * P < 0.01 (7) (4) (4) 5 X + S.E.M. ( ) Nº of observations 0 'active' 1 phosphorylated 2 48 h after the operation It may be of note to mention that the technique of rapid sampling of the liver for the preparation of liver microsomal fraction, as described by Easom & Zammit (1984; 1986), was not used in the present studies, and, therefore, the measured activity of the dephosphorylated or 'active' form of the enzyme may not correspond to the actual total enzyme activity. Nevertheless, the same sampling technique was used for both septic and sham-operated rats. Effects of sepsis on lipoprotein lipase activity and rates of lipogenesis in vivo in adipose tissue In order to obtain some information on the changes in lipid metabolism in adipose tissue, and to compare to those found in the liver, rates of lipogenesis and lipoprotein lipase activity were measured in white and brown adipose tissue of septic and shamoperated rats. Sepis caused no appreciable change in the rate of total lipid synthesis in white adipose tissue at 12 h after the operation, however, in contrast to the findings in the liver, sepsis decreased the lipogenic rate at 48 h in adipose tissue (Fig. 6.10.) when compared to that of sham-operated rats. Fig. 6.10. The rate of lipogenesis in brown adipose tissue of septic rats was significantly decreased when compared to that of sham-operated rats, at 12 h after the operations. However, at 48 h, brown adipose tissue from septic rats synthesized lipids at similar rates to those of sham-operated rats (Fig.6.11.). Fig. 6.11. Lipoprotein lipase activity was measured in adipose tissue from septic and shamoperated rats at 48 h after the operations. Sepsis did not cause appreciable change in lipoprotein lipase activity in white adipose tissue (Septic : 0.302±0.035 , n=9 ; Shamoperated : 0.253±0.048 , n=8 ; mean values ± S.E.M.; enzyme activity expressed as nmol of FFA released/min/mg of tissue). However, this enzyme was significantly more active in brown adipose tissue from septic rats at 48 h ( Septic : 1.37±0.26 , n=9 ; Sham-operated : 0.61±0.16 , n=8 ; P < 0.05 - Student t-test). The effect of the complete interruption of the entero-hepatic circulation of bile acids accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver in vivo The complete interruption of the entero-hepatic circulation of bile acids obtained by bile duct ligation (BDL) produced a significant increase in the rates of cholesterolegenesis in livers of sham-operated+BDL rats as compared to those of shamoperated rats at 48 h after the operations. There was, however, no significant further increase in the rates of cholesterol synthesis in the livers of septic rats+BDL as compared to caecal-ligated and punctured rats (Fig. 6.12.). Fig. 6.12. There was no appreciable effect of bile duct ligation (BDL) on the rate of fatty acid synthesis in sham-operated animals (Sham-op+BDL : 1.95±0.13, n=4 ; Sham-op : 1.95±0.24, n=13; 3H2O incorporation expressed as µmol of labelled fatty acid/g wet wt. liver/h). The stimulatory effect produced by bile-duct ligation was, therefore, specific for cholesterol synthesis. It is important to add that caecal-ligated and punctured rats which also had bile duct ligation were hyperinsulinaemic, as compared to sham-operated rats which had their bile duct ligated (Septic+BDL : 35.5±6.02 (n-4); Sham-operated : 18.7±1.76 (n=4) - mean values ± S.E.M. expressed as µunit/ml of plasma ; P < 0.05 - Student t test). Discussion Hypertriacylglycerolaemia is a common finding during sepsis (Fig. 3.6. - Chapter 3) and has been thought to be a consequence of both increased triacylglycerol production and decreased triacylglycerol clearance from the circulation (Kaufman et al , 1976; Robin et al , 1981). Hepatic esterification of [1-14C]oleate was increased by this model of sepsis (Fig. 5.6. - Chapter 5) and this was accompanied by unchanged lipoprotein lipase activity in white adipose tissue from septic rats at 48 h after the operation. In contrast, lipoprotein lipase activity has been reported to be decreased by infection (Robin et al , 1981; LanzaJacoby et al , 1982). A dose-dependent suppression of lipoprotein lipase activity when adipocytes were exposed either to purified recombinant interleukin-1 (Price et al , 1986) or to cachectin (Price et al , 1986) has been shown in vitro . The in vivo rates of lipogenesis in white adipose tissue, however, were decreased by sepsis (Fig. 6.10.) at 48 h after the operation. Pekala et al , have shown that crude preparations of cachectin were able to suppress the activity of key lipogenic enzymes such as acetyl-CoA carboxylase and fatty acid synthetase in the white adipocytes, which in turn would probably inhibit lipogenesis. This supports the view that "hormones" in the crude medium from endotoxin-stimulated macrophages are capable of promoting important changes in the metabolism of the adipose cell (Price et al , 1986). Sepsis did not induce appreciable change in the rate of lipid synthesis in brown adipose tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase activity was, however, significantly increased in this tissue in septic rats, at the same time point, suggesting the available triacylglycerol is being oxidised and used for chemical energy or heat production. Although high rates of lipogenesis may be necessary for the production of sufficient amounts of non-esterified fatty acids for the activation of thermogenin (Cannon & Nedergaard, 1985), thermogenesis can also occur without stimulated lipogenesis, the two being regulated independently (Himms-Hagen, 1985). Sepsis caused a significant increase in the rate of hepatic production of lipids in vivo at all time points studied (Fig. 6.5.). There was increased production not only of fatty acids, represented by the saponifiable lipid fraction (Fig. 6.3.) but also in the total non-saponifiable lipid fraction (Fig. 6.4.). During sepsis many factors create an increased demand for cholesterol, including increased hepatic production of lipoproteins (necessary to complement the increased triacylglycerol synthesis) and possibly increased cell membrane synthesis in tissues involved with defence and repair. As this need for cholesterol cannot be met from dietary sources because the gut is not always available to absorb nutrients for example during intra-abdominal sepsis, endogenous synthesis within the liver is expected to increase. In fact, cholesterol synthesis was significantly increased in vivo in the livers of septic rats as early as 12 h after the operation (Fig. 6.7.), whereas squalene synthesis was increased only at 24 h (Fig. 6.6.). Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.5. & 6.7.) accompanied by decreased rates of ketogenesis (see Chapter 5), in the presence of diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest sepsis directs the flow of acetyl-CoA preferentially toward lipid and cholesterol synthesis rather than to partial oxidation, with ketone body production, or complete oxidation in the tricarboxylic acid cycle. As absorption of bile acids and cholesterol may be partially impaired in this model of sepsis, due to the presence of an adynamic ileus, in response to the localized peritonitis, this may be a contributory factor to the raised cholesterol synthesis in the livers of septic rats. Although hepatic fatty acid synthesis was not affected by bile duct ligation (BDL) in sham-operated rats, at 48 h after the operation, cholesterol synthesis rates in the livers of these rats were increased to levels comparable to those of septic rats (Fig. 6.12.). However, rats subjected to subcutaneous bacterial injection (Canonico et al , 1977), and therefore less likely to develop an adynamic ileus, had increased rates of cholesterogenesis in vitro . This finding does not support the view that partial interruption of the bile acid hepatoenteric circulation is the main reason for the increase in cholesterol synthesis in vivo , found in this model of sepsis, and therefore favours an independent effect of sepsis. Septic rats had increased total HMG-CoA reductase activity (Fig. 6.9.) associated with augmented cholesterol synthesis (Fig. 6.8.). There was, however, no change in the activity of the 'active' or expressed form of the enzyme (Fig. 6.10.). This is perhaps surprising in view of the fact that insulin, at least in the fed state, is considered to rapidly increase the proportion of this enzyme in the active form (Easom & Zammit, 1986). Insulin, present in higher concentrations in the plasma of septic rats at all time points studied (Fig. 3.5. - Chapter 3), may have exerted its longer-term effect by increasing the synthesis of this enzyme rather than changing the activation state. The increased rates of hepatic lipogenesis, induced by caecal-ligation and puncture, give support to the view of an inhibition in the entry of long-chain acyl-CoA in the mitochondria of hepatocytes from septic rats by higher concentrations of malonyl-CoA, an intermediate in the lipogenic pathway (see Chapter 5). This stimulation in the rates of hepatic lipogenesis and cholesterogenesis by sepsis may well be due to greater availability of lactate (Figs. 3.2. & 3.9. - Chapter 3) and pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the blood and liver, and the presence of hyperinsulinaemia (Fig. 3.5. - Chapter 3). CHAPTER 6 Time course of changes in lipogenesis and hepatic cholesterolegenesis in vivo after the induction of sepsis Introduction Despite the fact lipids constitute one of the principal sources of body energy and sepsis produce various alterations on fat metabolism, many of these metabolic changes induced by sepsis still need clarification. More than six decades have passed since the changes in concentrations of cholesterol in serum, during bacterial infection, were first described (Kipp, 1920). However, little is known on the regulation of cholesterol metabolism during sepsis. Some information has recently been collected on the metabolism of nonesterified fatty acids during infection.The availability of non-esterified fatty acids may be decreased in sepsis as a result of the inhibitory effect of hyperinsulinaemia (Fig. 3.5. - Chapter 3) on lypolysis , suppressing hormone sensitivity lipase activity (Steinberg & Khoo, 1977) resulting in lower concentrations of non- esterified fatty acids, as reported in various models of sepsis (Beisel & Wannemacher, 1980). Plasma concentration of non esterified fatty acids, however, remained unchanged in the present studies (Fig. 3.7. - Chapter 3). A decrease in the percentage of non-esterified fatty acid uptake (23%) was found in livers from caecal ligated and punctured rats (Table 5.1. - Chapter 5). Once in the hepatocytes, non-esterified fatty acids suffer preferential esterification in the livers of septic rats, rather than partial or total oxidation with formation of ketone bodies or metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al , 1979). The percentage of [1-14C]oleate esterified was significantly higher in isolated hepatocytes from caecal- ligated rat as compared to that of hepatocytes from shamoperated rats (Fig. 5.6. - Chapter 5). As a result of decreased oxidation of long-chain fatty acids the rate of ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the hyperketonaemia, in adaptation to starvation, abolished by sepsis (Fig. 3.8. - Chapter 3). Non-esterified fatty acid utilization by the liver, therefore, appears to be decreased in experimental or clinical sepsis with the development of elevation in plasma concentrations of triacylglycerols (Levin et al , 1972; Kaufmann et al , 1976; Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al , 1981 Alvares & Ramos, 1986), and fatty infiltration of the liver (Mukherjee et al , 1973; Pace et al , 1977; Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of increased triacylglycerol synthesis accompanied by a decreased or insuficient production of the proteins needed to export very low density lipoprotein (VLDL) as suggested by Forse & Kinney (1985). However, protein synthesis in the liver has been reported to be stimulated by sepsis (Long et al , 1979; Hasselgen et al, 1984). Decreased plasma clearence of triacylglycerol as a result of diminished activity of lipoprotein lipase has been reported as contributory to the hypertriacylglycerolaemia induced by sepsis (Kaufmann et al , 1976; Robin et al , 1981; Lanza-Jacoby et al , 1982). Hepatic lipogenesis in vitro , measured by the incorporation of [114C]acetate into lipids in hepatocytes isolated from rats with pneumococcal infection, is increased in response to sepsis (Canonico et al , 1977). There appears to be no information, however, on rates of lipid synthesis in vivo in the liver or adipose tissue after induction of sepsis. Total cholesterol concentration in serum has been reported to increase, decrease, or remain unchanged during sepsis (Lindell et al , 1964; Fiser et al , 1971; Alvares & Ramos, 1986). Such variation in cholesterol concentration provides little information into the dynamics involved in cholesterol synthesis, transport and utilization. Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic acid, showed increased incorporation of the labelled mevalonate into free cholesterol in plasma when compared to control monkeys (Fiser et al , 1971). In vitro , [14C]acetate incorporation into cholesterol by isolated hepatocytes from rats with pneumococcal infection was significantly increased as compared to the incorporation by hepatocytes from control rats (Cannonico et al , 1977). However, there appears to be no information on the rates of hepatic cholesterol synthesis in vivo in response to sepsis. Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated rats (Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats (Pace et al , 1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from rats with pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the flow of acetyl-CoA toward lipogenesis and cholesterolegenesis in vivo rather than to ketogenesis or oxidation in the Krebs tricarboxilic acid cycle. To investigate the time course of changes in vivo in lipogenesis in the liver and white and brown adipose tissue, and hepatic cholesterolegenesis, after the induction of sepsis, caecal-ligated and punctured rats were studied. Sham-operated rats were used as controls. Experimental Design Rats, after being subjected to caecal-ligation and puncture or shamoperation, were fasted for 12, 24 or 48 h before livers were removed and studied (Fig.6.1.). Fig. 6.1. OP 12 h 24 h 48 h Rats 3 H O injected 2 Livers removed Lipid exctraction Lipogenesis Cholesterolegenesis Microsomal preparation HMGCoA reductase activity For details on the operations and the techniques involved in the measurement of lipogenesis, cholesterolegenesis, preparation of microsomes and the measurement of hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and lipoprotein lipase activities see Chapter 2. A group of rats had their bile duct ligated immediately before either caecal-ligation and puncture or sham-operation was performed (Chapter 2). Results Effects of sepsis on hepatic lipogenesis The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo was significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48 h after the operations, as compared to that of livers from sham-operated animals (Fig. 6.2.). Fig. 6.2. Rate of fatty acid synthesis in the liver ** (7) (4) µmol fatty acid/g wet wt. liver/h 4.0 3.0 * (18) Sham-op Septic (3) (13) 2.0 (6) * P < 0.05 1.0 ** P < 0.001 ( nº of observations ) 0.0 12 24 48 Time after the operations (h) Increased rates of formation of total non-saponifiable lipids in vivo were also found to be present in livers from septic rats when compared to those found in livers from sham operated rats (Fig. 6.3.) as early as at 12 h after the operations. Fig. 6.3. Rate of non-saponifiable lipid synthesis * (4) 7 µmol lipid/gwet wt. liver/h 6 ** (7) 5 ** (18) 4 3 (3) (6) Sham-op Septic * P < 0.05 2 ** P < 0.001 (13) 1 ( nº of observations ) 0 12 24 48 Time after the operations (h) Therefore, livers from septic rats exhibit rates of total lipogenesis significantly increased as compared to those of sham-operated rats. This increase in lipid synthesis appears to be at the expense of increases in rates of formation of both saponifiable (fatty acids) and non-saponifiable (squalene, cholesterol, other sterols) fractions (Fig. 6.4.). Fig. 6.4. Rate of total lipogenesis in the liver µmol lipid formed/g wet wt. liver/h 10 * * 8 * 6 saponified non-saponified 4 2 * P < 0.001 0 sham 1 septic 2 12 h sham septic 3 4 24 h sham septic 5 6 48 h Effects of sepsis on lipoprotein lipase activity and rates of total lipogenesis in vivo in adipose tissue Depite inducing no appreciable change in the rate of total lipid synthesis in white adipose tissue at 12 h after the operation, sepsis did decrease the total lipogenic rate at 48 h (Fig. 6.5.) when compared to that of sham-operated rats. Fig. 6.5. Rate of lipogenesis in white adipose tissue 8 (8) 7 µmol/g wet wt. tissue/h 6 5 (10) (7) * (11) 4 sham-op septic 3 2 * P < 0.05 1 ( nº of observations ) 0 12 48 Time after the operations (h) The rate of total lipogenesis in brown adipose tissue of septic rats was significantly decreased when compared to that of sham-operated rats, at 12 h after the operations. However, at 48 h, brown adipose tissue from septic rats produced lipids at similar rates to those of sham-operated rats (Fig.6.6.). Fig. 6.6. Rate of lipogenesis in brown adipose tissue µmol/g wet wt. tissue/h 20 (7) * (8) 10 (10) (12) sham-op septic * P < 0.05 ( nº of observations ) 0 12 48 Time after the operations (h) Lipoprotein lipase activity was measured in adipose tissue from septic and sham-operated rats at 48 h after the operations. Sepsis did not cause appreciable change in lipoprotein lipase activity in white adipose tissue (septic : 0.302±0.035 , n=9 ; sham-op : 0.253±0.048 , n=8 ; mean±SEM with number of observations in parenthesis). However, this enzyme was significantly more active in brown adipose tissue from septic rats at 48 h (septic : 1.37±0.26 , n=9 ; sham-op : 0.61±0.16 , n=8 ; P < 0.05 - Student t-test). Effects of sepsis on squalene and cholesterol synthesis in vivo and hydroxymethylglutaryl-CoA reductase activity in the liver Sepsis produced significant increase in the rate of squalene formation in the liver at 24 h after the operations (Fig. 6.7.). No effect was observed at the other time points studied. Fig. 6.7. Rate of squalene synthesis in the liver µmol squalene/g wet wt. liver/h * (7) (4) 0.50 (18) 0.40 (3) Sham-op Septic (13) 0.30 (6) 0.20 * P < 0.05 0.10 ( nº of observations ) 0.00 12 24 48 Time after the operations (h) The rates of cholesterol synthesis in the livers of septic rats in vivo were significantly higher than those found in the livers of sham-operated rats. This increase (2.3 fold) occured as early as 12 h after the operations reaching a four-fold increase at 48 h (Fig. 6.8.). Fig. 6.8. Rate of cholesterol synthesis * (4) µmol formed/g wet wt. liver/h 4.0 3.0 2.0 ** (11) ** (18) (3) * P < 0.05 (11) 1.0 sham-op septic (12) ** P < 0.001 ( nº of operations ) 0.0 12 24 Time after the operations (h) 48 The total activity of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the livers of septic rats was significantly increased compared to that found in livers of sham-operated rats at 48 h after the operations (Fig. 6.9.). Fig. 6.9. Total HMG-CoA reductase activity pmol mevalonate/mg protein/min * (7) 20 sham-op septic 10 (4) * P < 0.02 ( nº of observations ) 0 48 h HMG-CoA reductase is phosphorylated by a protein kinase. Phosphorylation leads to inactivation of the enzyme. We have measured the activity of both the phosphrylated ('inactive' form) and the non-phosphorylated or expressed form of this enzyme (Fig. 6.10.) in livers from septic and sham-operated rats at 48 h after the operations. Sepsis induced no appreciable change in the expressed activity of HMGCoA reductase (non-phosphorylated or 'active'). As there was a significant increase in the total enzyme activity (Fig. 6.9.) this was mainly due to an increase in activity of the phosphorylated or 'inactive' form of the enzyme (Fig. 6.10.). Fig. 6.10. HMG-CoA reductase activity 18 * (7) pmol mevalonate/mg protein/min 16 14 12 10 6 sham-op septic (7) 8 (4) 4 (4) 2 * P < 0.01 ( nº of observations ) 0 'active' phosphorilated 48 h after the operations The effect of the complete interruption of the entero-hepatic circulation of bile acids accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver in vivo The complete interruption of the entero-hepatic circulation of bile acids obtained by bile duct ligation (BDL) produced a significant increase in the rates of cholesterolegenesis in livers of shamoperated+BDL rats as compared to those of sham-operated rats at 48 h after the operations. There was, however, no significant further increase in the rates of cholesterol synthesis in the livers of septic rats+BDL as compared to caecal-ligated and punctured rats (Fig. 6.11.). Fig. 6.11. Hepatic cholesterol synthesis and BDL 4 † µmol formed/g wet wt. liver/min (4) 3 (8) (18) 2 1 sham-op septic ( nº of observations ) (12) † P < 0.001 ° sham-op/no BDL 0 No 1BDL BDL 2 48 h after the operations There was no appreciable effect of BDL on the rate of fatty acid synthesis on shamoperated animals (sham-op+BDL : 1.95±0.13, n=4 ; sham-op : 1.95±0.24, n=13). This stimulatory effect produced by bile-duct ligation was, therefore, specific for cholesterol synthesis as found by Fredrickson et al , 1953. Discussion Hypertryacylglycerolaemia is a comon finding during sepsis (Fig. 3.6. Chapter 3) and has been thought to be a consequence of both increased tryacylglycerol production and decreased tryacylglycerol clearence from the circulation (Robin et al , 1981). Hepatic esterification of [1-14C]oleate was increased by this model of sepsis (Fig. 5.6. - Chapter 5) and this was accompanied by unchanged lipoprotein lipase activity in white adipose tissue from septic rats at 48 h after the operations (Fig. 6.5.). The rate of lipogenesis in white adipose tissue, however, was decreased by sepsis (Fig. 6.5.) at the same time point studied. Sepsis did not induce appreciable change in the rate of lipid synthesis in brown adipose tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase activity was, however, significantly increased in this tissue in septic rats, at the same time point studied, suggesting the hydrolysed tryacylglycerol is being oxidised and used for energy or heat production rather than for lipogenesis. Although high rates of lipogenesis may be necessary for the production of sufficient amount of nonesterified fatty acids for the activation of thermogenin (Cannon & Nedergaard, 1985), thermogenesis can also occur without stimulated lipogenesis, the two being regulated independently (Himms-Hagen, 1985). Sepsis caused significant increase in the rate of hepatic production of lipids in vivo at all time points studied (Fig. 6.4.). There was increased production not only of fatty acids, represented by the saponifiable lipid fraction (Fig. 6.2.) but also in the total non-saponifiable lipid fraction (Fig. 6.3.). During sepsis many factors create a large demand for cholesterol, including increased hepatic production of lipoproteins (necessary to match with the increased triacylglycerol synthesis) and possibly increased cell membrane synthesis at tissues involved with defence and repair. As this need for cholesterol can not be met from dietary sources because the gut is not always available to absorb nutrients e.g. during intra-abdominal sepsis, endogenous synthesis within the liver is expected to increase. In fact, cholesterol synthesis was significantly increased in vivo in the livers of septic rats as early as at 12 h after the operations (Fig. 6.8.) while squalene synthesis was increased at 24 h (Fig. 6.7.). Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.4 & 6.8.) accompanied by decreased rates of ketogenesis (see Chapter 5), in the presence of diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest sepsis directs the flow of acetyl-CoA preferentially toward lipid and cholesterol synthesis rather than to partial oxidation with ketone body production or complete oxidation in the tricarboxylic acid cycle. As absorption of bile acids may be partially impaired in this model of sepsis due to the presence of an adynamic ileus, in response to the localized peritonitis, this may be a contributory factor to the raised cholesterol synthesis in the livers of septic rats. Although hepatic fatty acid synthesis was not affected by bile duct ligation (BDL) in sham-operated rats at 48 h after the operation, cholesterol synthesis in the livers of these rats was increased to level comparable to those of septic rats (Fig. 6.11.). However, rats subjected to subcutaneous bacterial injection (Canonico et al , 1977), and therefore less likely to develop an adynamic ileus, presented increased rates of cholesterolegenesis in vitro , questioning the idea of the partial interruption of the bile acid hepato-enteric circulation as the main reason for the increase in cholesterol synthesis in vivo , found in this model, and favouring an independent effect of sepsis. Hydroxymethylgluratyl-CoA reductase activity and cholesterolegenesis were measured in the same animals. Septic rats presented with higher total enzyme activity (Fig. 6.9.) along with aumented cholesterol synthesis (Fig. 6.8.). There was , however , no change in the activity of the 'active' or expressed form of the enzyme (Fig. 6.10.). Insulin, present in higher concentrations in the plasma of septic rats at all time points studied (Fig. 3.5. - Chapter 3) may have exerted its longer-term effect by increasing this enzyme synthesis rather than changing the activation state of the enzyme. The increased rates of hepatic lipogenesis induced by caecal-ligation and puncture give support to the view of an inhibition in the entry of long-chain acyl-CoA in the mitochondria of hepatocytes from septic rats by higher concentrations of malonyl-CoA, an intermediate in the lipogenic pathway (see Chapter 5). This stimulation in the rates of hepatic lipogenesis and cholesterolegenesis by sepsis may well be due to greater availability of lactate (Figs. 3.2. & 3.9. - Chapter 3) and pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the blood and liver, and the presence of hyperinsulinaemia (Fig. 3.5. - Chapter 3). CHAPTER 6 Time course of changes in rates of lipogenesis and hepatic cholesterolegenesis in vivo after the induction of sepsis Introduction Despite the fact lipids constitute one of the principal sources of body energy and sepsis produce various alterations on fat metabolism, many of these metabolic changes induced by sepsis still need clarification. More than six decades have passed since the changes in concentrations of cholesterol in serum, during bacterial infection, were first described (Kipp, 1920). However, little is known on the regulation of cholesterol metabolism during sepsis. Some information has recently been collected on the metabolism of nonesterified fatty acids during infection (Blackburn, 1977; Wannemacher et al , 1979; Beisel & Wannemacher, 1980).The availability of non-esterified fatty acids may be decreased in sepsis as a result of the anti-lipolytic effect of the hyperinsulinaemia (Fig. 3.5. - Chapter 3), to suppress hormone sensitive lipase activity (Steinberg & Khoo, 1977) resulting in lower concentrations of non-esterified fatty acids, as reported in various models of sepsis (Beisel & Wannemacher, 1980). Plasma concentration of non-esterified fatty acids, however, was not significantly changed in the present studies (Fig. 3.7. - Chapter 3). A decrease in the percentage of non-esterified fatty acid uptake (23%) was found in isolated hepatocytes from caecal ligated and punctured rats (Table 5.1. Chapter 5). Whithin hepatocytes, non-esterified fatty acids appear to be preferentially esterified in the livers of septic rats, rather than partially or totally oxidized with formation of ketone bodies or metabolic CO2 respectively (see Chapter 5 ; Wannemacher et al , 1979). The percentage of [1-14C]oleate esterified was significantly higher in isolated hepatocytes from caecal- ligated rat as compared to that of hepatocytes from sham- operated rats (Fig. 5.6. - Chapter 5). As a result of decreased oxidation of long-chain fatty acids the rate of ketogenesis was decreased (Fig. 5.5. - Chapter 5) and the hyperketonaemia, associated with starvation, decreased by sepsis (Fig. 3.8. Chapter 3). Non-esterified fatty acid utilization by the liver, therefore, appears to be decreased in experimental or clinical sepsis with the development of elevation in plasma concentrations of triacylglycerols (Levin et al , 1972; Kaufmann et al , 1976; Blackburn, 1977; Beisel & Wannemacher, 1980; Robin et al , 1981 Alvares & Ramos, 1986), and fatty infiltration of the liver (Mukherjee et al , 1973; Pace et al , 1977; Wannemacher et al , 1979). This hepatic accumulation of fat may be a result of increased triacylglycerol synthesis accompanied by a decreased or insuficient production of the proteins needed to export very low density lipoprotein (VLDL) as suggested by Forse & Kinney (1985). However, genearal protein synthesis in the liver has been reported to be stimulated by sepsis (Long et al , 1979; Hasselgen et al, 1984). Decreased plasma clearence of triacylglycerol as a result of diminished activity of lipoprotein lipase has been reported as contributory to the hypertriacylglycerolaemia induced by sepsis (Kaufmann et al , 1976; Robin et al , 1981; Lanza-Jacoby et al , 1982). Hepatic lipogenesis in vitro , measured by the incorporation of [114C]acetate into lipids in hepatocytes isolated from rats with pneumococcal infection, is increased in response to sepsis (Canonico et al , 1977). There appears to be no information, however, on rates of lipid synthesis in vivo in the liver or adipose tissue after induction of sepsis. Total cholesterol concentration in serum has been reported to increase, decrease, or remain unchanged during sepsis (Lindell et al , 1964; Fiser et al , 1971; Alvares & Ramos, 1986). Such variation in cholesterol concentration provides little information on the dynamics involved in cholesterol synthesis, transport and utilization. Rhesus monkeys with pneumococcal infection, injected with 3H-mevalonic acid, showed increased incorporation of the labelled mevalonate into free cholesterol in plasma when compared to control monkeys (Fiser et al , 1971). In vitro , [14C]acetate incorporation into cholesterol by isolated hepatocytes from rats with pneumococcal infection was significantly increased as compared to the incorporation by hepatocytes from control rats (Cannonico et al , 1977). However, there appears to be no information on the rates of hepatic cholesterol synthesis in vivo in response to sepsis. Increased activities of fatty acid synthetase (FAS) in livers of E coli-treated rats (Lanza-Jacoby et al , 1982), acetyl-CoA carboxylase in fasted infected rats (Pace et al , 1981), and hydroxymethylglutaryl-CoA reductase in hepatocytes from rats with pneumococcal infection (Canonico et al , 1977), suggest sepsis directs the flow of acetyl-CoA toward lipogenesis and cholesterolegenesis in vivo rather than to ketogenesis or oxidation in the Krebs tricarboxylic acid cycle (Fig. 6.1.). Fig. 6.1 To investigate the time course of changes in vivo in lipogenesis in the liver and white and brown adipose tissue, and hepatic cholesterolegenesis, after the induction of sepsis, caecal-ligated and punctured rats were studied. Sham-operated rats were used as controls. Experimental Design Rats, after being subjected to caecal-ligation and puncture or shamoperation, were fasted for 12, 24 or 48 h before livers were removed and studied (Fig.6.2.). Fig. 6.2. OP 12 h 24 h 48 h Rats 3 H O injected 2 Livers removed Lipid exctraction Lipogenesis Cholesterolegenesis Microsomal preparation HMGCoA reductase activity For details on the operations and the techniques involved in the measurement of lipogenesis, cholesterolegenesis, preparation of microsomes and the measurement of hydroxymethylglutaryl-CoA reductase (HMGCoA reductase) and lipoprotein lipase activities see Chapter 2. A group of rats had their bile duct ligated immediately before either caecal-ligation and puncture or sham-operation was performed (Chapter 2). Results Effects of sepsis on hepatic lipogenesis The rate of fatty acid synthesis (saponifiable lipid fraction) in vivo was significantly increased in the livers of caecal-ligated and punctured rats, at 24 and 48 h after the operations, as compared to that of livers from sham-operated animals (Fig. 6.3.). Fig. 6.3. Rate of H 3 incorporation O into fatty acids 2 ** (7) (4) µmol/g wet wt. liver/h 4.0 3.0 * (18) Sham-op Septic (3) (13) 2.0 * P < 0.05 ** P < 0.001 (6) 1.0 X + S.E.M. ( ) nº of observations 0.0 12 24 48 Time after the operation (h) Increased rates of formation of total non-saponifiable lipids in vivo were also found to be present in livers from septic rats when compared to those found in livers from sham-operated rats (Fig. 6.4.) as early as at 12 h after the operation. Fig. 6.4. Rate of H3 incorporation into non-saponifiable lipids 2O * (4) 7 6 µmol/g wet wt. liver/h Sham-op Septic ** (7) 5 ** (18) 4 3 (3) * P < 0.05 (6) ** P < 0.001 2 (13) X + S.E.M. ( ) nº of observations 1 0 12 24 48 Time after the operation (h) Therefore, livers from septic rats have significantly increased rates of total lipogenesis as compared to those of sham-operated rats. This increase in lipid synthesis appears to occur both in rates of formation of saponifiable (fatty acids) and non-saponifiable (squalene, cholesterol, other sterols) fractions (Fig. 6.5.). Fig. 6.5. Rate of H 10 3 incorporation into lipids 2O * * µmol/g wet wt. liver/h 8 * 6 saponified non-saponified 4 2 * P < 0.001 0 sham 1 septic 2 12 h sham septic 3 4 24 h sham septic 5 6 48 h Effects of sepsis on squalene and cholesterol synthesis in vivo and hydroxymethylglutaryl-CoA reductase activity in the liver The non-saponifiable fraction was separated into the various sterol fractions. Sepsis produced a significant increase in the rate of squalene formation in the liver at 24 h after the operation (Fig. 6.6.). No change in rate was observed at the other time points studied. Fig. 6.6. Rate of H 3 incorporation into squalene O 2 * (7) (4) 0.50 (18) µmol/g wet wt. liver/h 0.40 (3) Sham-op Septic (13) 0.30 (6) 0.20 * P < 0.05 0.10 X + S.E.M. ( ) nº of observations 0.00 12 24 48 Time after the operation (h) Starvation caused the incorporation of 3H2O into cholesterol to decrease with time in both sham-operated and caecal-ligated and punctured rats. However, the rates of cholesterol synthesis in the livers of septic rats in vivo were significantly higher than those found in the livers of sham-operated rats. This increase (2.3 fold) occured as early as 12 h after the operations reaching a four-fold increase at 48 h (Fig. 6.7.). Fig. 6.7. Rate of H 3incorporation 2O into cholesterol * (4) 4.0 sham-op septic µmol/g wet wt. liver/h 3.0 2.0 ** (11) ** (18) (3) * P < 0.05 ** P < 0.001 (11) 1.0 (12) X + S.E.M. ( ) nº of observations 0.0 12 24 48 Time after the operation (h) The total activity of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the livers of septic rats was significantly increased compared to that found in livers of sham-operated rats at 48 h after the operations (Fig. 6.8.). Fig. 6.8. Total HMG-CoA reductase activity pmol mevalonate/mg protein/min * (7) 20 sham-op septic 10 (4) * P < 0.02 X + S.E.M. ( ) nº of observations 0 48 h 48 h after the operation HMG-CoA reductase is phosphorylated by a protein kinase. Phosphorylation leads to inactivation of the enzyme. The activity of both the phosphorylated ('inactive' form) and the non-phosphorylated or expressed form of this enzyme (Fig. 6.9.) was measured in livers from septic and sham-operated rats at 48 h after the operation. Sepsis induced no appreciable change in the expressed activity of HMG-CoA reductase (non-phosphorylated or 'active'). As there was a significant increase in the total enzyme activity (Fig. 6.8.) this was mainly due to an increase in activity of the phosphorylated or 'inactive' form of the enzyme (Fig. 6.9.). Fig. 6.9. HMG-CoA reductase activity 18 * (7) pmol mevalonate/mg protein/min 16 14 12 sham-op septic 10 (7) 8 6 (4) * P < 0.01 4 (4) 2 X + S.E.M. ( ) nº of observations 0 'active' phosphorilated phosphorylated 48 h after the operation Effects of sepsis on lipoprotein lipase activity and rates of lipogenesis in vivo in adipose tissue In order to obtain some information on the changes in lipid metabolism in adipose tissue, and to compared to those found in the liver, rates of lipogenesis and lipoprotein lipase activity were measured in white and brown adipose tissue of septic and sham-operated rats. Sepis caused no appreciable change in the rate of total lipid synthesis in white adipose tissue at 12 h after the operation, however, in contrast to the findings in the liver, sepsis decreased the lipogenic rate at 48 h in adipose tissue (Fig. 6.10.) when compared to that of sham-operated rats. Fig. 6.10. Rate of lipogenesis in white adipose tissue 8 (8) 6 (µmol/g tissue/h) 3H2O incorporation into lipids 7 5 (10) (7) 4 (11) sham-op septic 3 2 X + S.E.M. ( ) nº of observations 1 0 12 48 Time after the operation (h) The rate of lipogenesis in brown adipose tissue of septic rats was significantly decreased when compared to that of sham-operated rats, at 12 h after the operations. However, at 48 h, brown adipose tissue from septic rats synthesized lipids at similar rates to those of sham-operated rats (Fig.6.11.). Fig. 6.11. Rate of lipogenesis in brown adipose tissue (µmol/g of tissue/h) 3H2O incorporation into lipids 20 (7) (10) (8) 10 (12) sham-op septic * P < 0.05 X + S.E.M. ( ) nº of observations 0 12 48 Time after the operation (h) Lipoprotein lipase activity was measured in adipose tissue from septic and sham-operated rats at 48 h after the operations. Sepsis did not cause appreciable change in lipoprotein lipase activity in white adipose tissue (septic : 0.302±0.035 , n=9 ; sham-operated : 0.253±0.048 , n=8 ; mean±SEM with number of observations in parenthesis). However, this enzyme was significantly more active in brown adipose tissue from septic rats at 48 h (septic : 1.37±0.26 , n=9 ; sham-operated : 0.61±0.16 , n=8 ; P < 0.05 - Student t-test). The effect of the complete interruption of the entero-hepatic circulation of bile acids accompanied by sepsis on the rates of cholesterol and fatty acid synthesis in the liver in vivo The entero-hepatic circulation of bile acids plays an essential role in the absortion of cholesterol from the intestine and in the regulation of cholesterol metabolism in the liver (for review see : Myant, 1981 and Gibbons et al , 1982). The diversion of bile acids, or the complete interruption of the bile duct, lead to the absence of bile acids in the intestine, which in turn, supresses the normal uptake of cholesterol from the intestinal lumen. There is some evidence that it is the transport of intestinal cholesterol to the liver which directly determines the rates of hepatic cholesterol synthesis (Weis & Dietschy, 1975). This view is supported by the fact that restoration of the entero-hepatic circulation, in rats subjected to biliary diversion, failed to prevent the rise in hepatic cholesterolegenesis, whereas, the infusion of chylomicron cholesterol lead to a prevention in the rise of hepatic sterol synthesis rates (Gibbons et al , 1982). In addition, bile acids inhibit cholesterol 7Ï-hydroxylase (the rate-limiting enzyme in the process of bile acids formation from cholesterol) and, therefore, would prevent the catabolism and removal of cholesterol from the liver, resulting in suppression of HMG-CoA reductase activity (Nervi & Dietschy, 1978). The possibility of bile acids having a direct effect on HMG-CoA reductase, in the liver, would suggest diversion rather than obstruction of the bile duct as the resonable approach to study the metabolism of cholesterol in the liver. However, opposing the idea of a direct bile acid effect is the fact that partial or complete biliary obstruction cause an increase, rather than the expected decrease, in hepatic cholesterol synthesis (Fredrickson et al , 1953; Gibbons et al , 1982). The absorption of bile acids and cholesterol in the intestinal lumen of caecal-ligated and punctured rats may be impaired due to the development of an adynamic ileus in these rats. Therefore, we decided to produce a complete interruption of the entero-hepatic circulation of bile acids, by ligating the bile duct of septic and sham-operated rats at 48 h after the operation, to try to evaluate the contribution of the impairment in the absorption of cholesterol and bile acids from the gut to the elevated rates of hepatic cholesterogenesis found in this septic model (Fig. 6.7.). The complete interruption of the entero-hepatic circulation of bile acids obtained by bile duct ligation (BDL) produced a significant increase in the rates of cholesterolegenesis in livers of shamoperated+BDL rats as compared to those of sham-operated rats at 48 h after the operations. There was, however, no significant further increase in the rates of cholesterol synthesis in the livers of septic rats+BDL as compared to caecal-ligated and punctured rats (Fig. 6.12.). Fig. 6.12. Hepatic cholesterol synthesis and BDL 4 ² 3 ( µmol/g wet wt. liver/h) 3H2O incorporation into cholesterol (4) (8) ² P < 0.001 ° sham-op/no BDL (18) 2 sham-op septic 1 (12) X + S.E.M. ( ) nº of observations 0 No 1BDL BDL 2 48 h after the operation There was no appreciable effect of BDL on the rate of fatty acid synthesis in shamoperated animals (sham-op+BDL : 1.95±0.13, n=4 ; sham-op : 1.95±0.24, n=13). This stimulatory effect produced by bile-duct ligation was, therefore, specific for cholesterol synthesis. Discussion Hypertriacylglycerolaemia is a comon finding during sepsis (Fig. 3.6. Chapter 3) and has been thought to be a consequence of both increased triacylglycerol production and decreased tryacylglycerol clearance from the circulation (Robin et al , 1981). Hepatic esterification of [1-14C]oleate was increased by this model of sepsis (Fig. 5.6. - Chapter 5) and this was accompanied by unchanged lipoprotein lipase activity in white adipose tissue from septic rats at 48 h after the operation. The rate of lipogenesis in white adipose tissue, however, was decreased by sepsis (Fig. 6.10.) at the same time point. Sepsis did not induce appreciable change in the rate of lipid synthesis in brown adipose tissue of 48 h caecal-ligated and punctured rats. Lipoprotein lipase activity was, however, significantly increased in this tissue in septic rats, at the same time point, suggesting the hydrolysed triacylglycerol is being oxidised and used for energy or heat production rather than for lipogenesis. Although high rates of lipogenesis may be necessary for the production of sufficient amounts of nonesterified fatty acids for the activation of thermogenin (Cannon & Nedergaard, 1985), thermogenesis can also occur without stimulated lipogenesis, the two being regulated independently (Himms-Hagen, 1985). Sepsis caused a significant increase in the rate of hepatic production of lipids in vivo at all time points studied (Fig. 6.5.). There was increased production not only of fatty acids, represented by the saponifiable lipid fraction (Fig. 6.3.) but also in the total non-saponifiable lipid fraction (Fig. 6.4.). During sepsis many factors create a large demand for cholesterol, including increased hepatic production of lipoproteins (necessary to match with the increased triacylglycerol synthesis) and possibly increased cell membrane synthesis at tissues involved with defence and repair. As this need for cholesterol cannot be met from dietary sources because the gut is not always available to absorb nutrients e.g. during intra-abdominal sepsis, endogenous synthesis within the liver is expected to increase. In fact, cholesterol synthesis was significantly increased in vivo in the livers of septic rats as early as 12 h after the operation (Fig. 6.7.) while squalene synthesis was increased only at 24 h (Fig. 6.6.). Increased rates of lipogenesis and cholesterolegenesis (Figs. 6.5. & 6.7.) accompanied by decreased rates of ketogenesis (see Chapter 5), in the presence of diminished hepatic concentrations of acetyl-CoA (Fig. 3.18. - Chapter 3) suggest sepsis directs the flow of acetyl-CoA preferentially toward lipid and cholesterol synthesis rather than to partial oxidation with ketone body production or complete oxidation in the tricarboxylic acid cycle. As absorption of bile acids and cholesterol may be partially impaired in this model of sepsis due to the presence of an adynamic ileus, in response to the localized peritonitis, this may be a contributory factor to the raised cholesterol synthesis in the livers of septic rats. Although hepatic fatty acid synthesis was not affected by bile duct ligation (BDL) in sham-operated rats, at 48 h after the operation, cholesterol synthesis rates in the livers of these rats were increased to levels comparable to those of septic rats (Fig. 6.12.). However, rats subjected to subcutaneous bacterial injection (Canonico et al , 1977), and therefore less likely to develop an adynamic ileus, had increased rates of cholesterogenesis in vitro . This finding does not support the view that partial interruption of the bile acid hepatoenteric circulation is the main reason for the increase in cholesterol synthesis in vivo ,found in this model of sepsis, and therefore favours an independent effect of sepsis. Hydroxymethylglutaryl-CoA reductase activity and cholesterolegenesis were measured in the same animals. Septic rats presented with higher total enzyme activity (Fig. 6.9.) along with augmented cholesterol synthesis (Fig. 6.8.). There was , however , no change in the activity of the 'active' or expressed form of the enzyme (Fig. 6.10.). Insulin, present in higher concentrations in the plasma of septic rats at all time points studied (Fig. 3.5. - Chapter 3) may have exerted its longer-term effect by increasing this enzyme synthesis rather than changing the activation state of the enzyme. The increased rates of hepatic lipogenesis induced by caecal-ligation and puncture give support to the view of an inhibition in the entry of long-chain acyl-CoA in the mitochondria of hepatocytes from septic rats by higher concentrations of malonyl-CoA, an intermediate in the lipogenic pathway (see Chapter 5). This stimulation in the rates of hepatic lipogenesis and cholesterolegenesis by sepsis may well be due to greater availability of lactate (Figs. 3.2. & 3.9. - Chapter 3) and pyruvate (Figs. 3.3. & 3.10. - Chapter 3) in the blood and liver, and the presence of hyperinsulinaemia (Fig. 3.5. - Chapter 3). CHAPTER 7 The effects of modulation of plasma insulin on hepatic metabolism during the induction of sepsis Introduction The model of sepsis used in the present work caused substantial changes in hepatic and peripheral metabolism. These included increased blood and hepatic concentrations of gluconeogenic precursors, namely, lactate, pyruvate and alanine but no change in glucose concentration (Chapter 3). There was also an impairment of the hepatic capacity for gluconeogenesis and ketogenesis (Chapters 4 & 5) in vitro , and stimulation of lipogenesis and cholesterogenesis in vivo (Chapter 6). Thus despite being starved, the septic rats, in terms of hepatic metabolism, behaved more like fed than fasted rats. Caecal-ligated and punctured rats had higher plasma concentrations of insulin at 12, 24 and 48 h after the operation (Fig. 3.5. - Chapter 3). Hyperinsulinaemia has also been reported to occur in various experimental models of sepsis (Ryan et al , 1974; Wichterman et al , 1979; Beisel & Wannemacher, 1980; Neufeld et al , 1980; Neufeld et al , 1982; Clemens et al , 1984) and in the septic man (Shaw et al , 1985; Long et al , 1985; Frayn, 1986). Higher concentrations of plasma insulin have also been reported during endotoxaemia (Yelich & Filkins, 1980; Neufeld et al , 1982). Insulin is known to stimulate lipogenesis and decrease gluconeogenesis and ketogenesis by its short-term actions on the activation state of key enzymes (e.g. acetyl-CoA carboxylase) and by longer-term regulation of enzyme concentration (Denton et al , 1981). Therefore, the key factor which prevented the septic rats from responding in the same way as the sham-operated rats to starvation may be the raised plasma insulin. Although plasma glucagon is also increased during sepsis (Beisel & Wannemacher, 1980; Shaw et al , 1985) its stimulatory effects on ketogenesis and gluconeogenesis and its inhibitory effects on lipogenesis may be antagonized by the elevated plasma insulin concentration. A possible approach to investigate the postulated regulatory action of insulin, upon the metabolic changes in sepsis, is to make the septic rats insulin-deficient. Streptozotocin, an N-methylnitrosourea derivative, is a potent diabetogenic agent, being extremely toxic to the ß-cell both in vitro and in vivo (Bolaffi et al , 1986). Its toxicity for the ß-cells is linked to its capacity to accumulate rapidly in these cells (Gaulton et al , 1985). Streptozotocin can supress insulin synthesis and secretion as early as 1 h after exposure, this being followed by progressive necrosis and death of the ß-cell, in 1-3 days, resulting in permanent hyperglycaemia (for review see: Cooperstein & Watkins, 1981; Weiss, 1982). Neufeld et al (1980) reported a reversal of the hyperketonaemic state induced by diabetes associated with infection (when total ketone body concentration reached 12 µmol/ml of plasma) by insulin replacement (2 Units), in rats previously (24 h) injected with streptozotocin (100mg/kg). Another possibility to block insulin release in vivo in septic rats is the use of mannoheptulose (Fig. 7.1.). This is a seven-carbon sugar, found in significant amounts in avocado pears (for review see Simon & Kraicer, 1966 and Simon et al , 1972), which has the ability to decrease insulin secretion by competitive inhibition of glucokinase and hexokinase in the ß-cell (Coore & Randle, 1964; Malaisse et al , 1968). Fig. 7.1. Glucokinase is only present in the liver and pancreas and controls glucose phosphorylation, the limiting-step of glycolysis. The metabolism of glucose to lactate and pyruvate appears to be a very important signal for insulin release (Randle et al , 1968), and as the capacity of the glycolytic pathway to handle glucose is decreased, by the competitive action of mannoheptulose, so insulin release is decreased. Therefore, to investigate the role of hyperinsulinaemia in the changes in hepatic metabolism during the induction of sepsis, short-term decrease in plasma insulin, by administration of mannoheptulose or streptozotocin, was induced in caecal-ligated and punctured rats. The effects of mannoheptulose treatment in sham-operated rats was also studied. To investigate whether the changes brought about by mannoheptulose treatment of septic rats was due to the inhibition of insulin secretion the hormone was administered with mannoheptulose to a group of septic rats, at 48 h after the induction of sepsis. Experimental design Rats, after being subjected to caecal-ligation and puncture or sham-operation, were fasted for 24 or 48 h when livers were removed (Fig. 7.2.) and blood collected. Fig. 7.2. Treatment to lower plasma insulin consisted of injection of either mannoheptulose (500mg/200g body wt. - s.c.) or streptozotocin (50mg/kg body wt. - i.v.), 3 h before removal of liver and 2 h before 3H2O injection (i.p.). To a group of septic rats, treated with mannoheptulose, neutral insulin (Actrapid - 2 Units) was injected subcutaneously 15 min after the mannoheptulose injection (at 2 h and 45 min before removal of liver) at 48 h after the induction of sepsis. For details of the operations and the techniques involved in the measurements of blood and hepatic metabolites, lipogenesis, cholesterogenesis, preparation of microsomes and the measurement of hydroxymethylglutaryl-CoA (HMGCoA) reductase activity see Chapter 2. Results For purposes of comparison, the results of septic and sham-operated rats, presented in Chapter 3 and Chapter 6, will also be included in the Figures of this chapter. The effects of mannoheptulose or streptozotocin treatment on changes in concentrations of blood glucose, lactate, pyruvate and alanine during the induction of sepsis As already discussed sepsis caused no appreciable change in blood glucose concentration at 24 and 48 h after the operation, but septic rats treated with either mannoheptulose or streptozotocin became hyperglycaemic (Fig. 7.3.). Fig. 7.3. This could be the result of either increased hepatic glucose formation or decreased glucose utilization in the periphery , or a combination of these factors. Sepsis is known to cause hyperlactataemia. Septic rats had increased blood lactate concentrations at 24 and 48 h after the operation. Mannoheptulose treatment was unable to reverse the hyperlactataemia found in septic rats at either time points (24 h and 48 h). When septic rats were treated with streptozotocin, lactate concentration was not different from either septic or sham-operated rats at 48 h after the operation (Fig. 7.4.). Fig. 7.4. Blood pyruvate concentration was unchanged by sepsis at 24 h after the operation. At 48 h septic rats had significantly increased blood concentrations of pyruvate as compared to those of sham-operated rats. Treatment of septic rats with mannoheptulose reversed this increased blood pyruvate concentration induced by sepsis at both 24 and 48 h after the operation. Septic rats treated with streptozotocin, however, had higher concentrations of blood pyruvate at 48 h after the operation (Fig. 7.5.). Fig. 7.5. Blood alanine concentration was not appreciably changed by sepsis at 24 h after the operation. However it was significantly elevated in caecal-ligated and punctured rats as compared with sham-operated rats at 48 h after the operation. Mannoheptulose injection in septic rats caused no change in blood alanine concentration at 24 h after the operation. At 48 h , however, septic rats treated with mannoheptulose had significantly decreased concentrations of blood alanine as compared to septic rats (Fig. 7.6.). Fig. 7.6. Although streptozotocin treatment tended to decrease the concentrations of blood alanine in septic rats at 48 h after the operation, this change was not statistically significant (Fig. 7.6.). Mannoheptulose treatment, therefore, caused blood concentrations of pyruvate and alanine to decrease and induced hyperglycaemia, whereas the only significant effect of streptozotocin was hyperglycaemia. The effects of mannoheptulose or streptozotocin treatment on the concentrations of blood ketone bodies during the induction of sepsis Sepsis is known to cause inhibition of the hyperketonaemia in response to starvation, as confirmed by the lower blood concentrations of ketone bodies at 24 and 48 h after the operation (Fig. 7.7.). Treatment to lower plasma insulin (mannoheptulose or streptozotocin injection) in septic rats returned the blood ketone body concentrations to the values of the sham-operated rats (Fig. 7.7.) Fig. 7.7. The effects of mannoheptulose or streptozotocin treatment on plasma insulin concentration during the induction of sepsis Sepsis caused a significant increase in plasma insulin concentration at 24 and 48 h after the operation. When mannoheptulose was administered to septic rats, at 24 h after the operation, plasma insulin concentrations were lower but not significantly different from those of caecal-ligated and punctured rats. At 48 h after the operation mannoheptulose treatment caused a significant decrease in plasma insulin concentration, as compared to that of septic rats, but these values were still significantly higher than those of sham-operated rats. Septic rats injected with streptozotocin had a significant decrease in insulin concentrations in plasma, as compared to those of untreated septic rats, at 48 h after the operation (Fig. 7.8.). Fig. 7.8. The effects of mannoheptulose or streptozotocin treatment on hepatic concentrations of lactate, pyruvate, alanine and glucose during the induction of sepsis Hepatic concentrations of lactate were significantly increased by sepsis at 24 and 48 h after the operation. The administration of mannoheptulose or streptozotocin treatment did not cause an appreciable change in the hepatic concentrations of lactate (Fig. 7.9.). Fig. 7.9. Hepatic lactate concentration ** (3) 3 µmol/g fresh wt. liver * (5) 2 1 ** (6) ** (12) * (5) (6) (9) Sham-op Septic Septic+MMannohept. Septic+Strz Streptoz. * P < 0.05 ** P < 0.001 versus Sham-op X + S.E.M. ( ) nº of observations 0 2424 48 Time after the operation (h) The hepatic concentrations of pyruvate were also increased during induction of sepsis at 24 and 48 h after the operation. Mannoheptulose injection in septic rats, however, caused a significant decrease in the concentrations of pyruvate in the liver at 48 h after the operation (Fig. 7.10.). The administration of streptozotocin caused no change on the hepatic concentrations of pyruvate in septic rats (Fig. 7.10.). Fig. 7.10. Sepsis caused an increase in hepatic concentrations of alanine at 48 h after the operation. Mannoheptulose treatment did not have a significant effect on the hepatic concentrations of alanine in septic rats at 24 or 48 h after the operation. Concentrations of alanine in livers from septic rats treated with streptozotocin were not different from those of either septic or sham-operated rats at 48 h after the operation (Fig. 7.11.). Fig. 7.11. Hepatic glucose concentration remained unchanged by sepsis at 24 and 48 h after the operation. However, septic rats treated with mannoheptulose or streptozotocin had a significant increase in glucose concentrations in the liver (Fig. 7.12.). Fig. 7.12. The effects of mannoheptulose or streptozotocin treatment on the hepatic concentration of metabolites involved in gluconeogenesis during the induction of sepsis The results of the effects of either mannoheptulose or streptozotocin administration to caecal-ligated and punctured rats on the hepatic concentration of metabolites involved in the glucose synthesis pathway, at 24 and 48 h after the operation, are presented in Table 7.1.. The effects of mannoheptulose or streptozotocin treatment on the hepatic concentration of metabolites involved in gluconeogenesis after the induction of Table 7.1. sepsis in vivo ____________________________________________________________ For full details see Chapter 2. Results are mean values ± S.E.M. with the number of observations in parentheses. Significance of difference by Student's t-test from septic rats: * P< 0.05; ** P < 0.01. The following abbreviations are used: Septic+M - Septic + Mannoheptulose; Septic+Strz - Septic + Streptozotocin; PEP Phosphoglycerate; - Phosphoenolpyruvate; 2PGA - D-2-Phosphoglycerate; 3PGA - D-3- DHAP - Dihydroxyacetone phosphate; FDP - Fructose-1,6-bisphosphate; G-6-P - Glucose-6-phosphate. ____________________________________________________________ Metabolite Concentration (µmol/g fresh wt. of liver) __________________________________________________________ 24 h _______________________ Septic Septic+M 48 h ____________________________________ Septic Septic+M Septic+ Strz _______________________________________________________________________ Alanine 1.51±0.28(5) 1.09±0.10(6) 1.24±0.16(11) 1.10±0.25(5) 1.47±0.13(6) 1.21±0.19(14) 1.46±0.34(5) 2.07±0.61(4) Pyruvate 0.093±0.014(5) 0.066±0.015(6) 0.078±0.009(9) 0.046±0.01(5)* 0.095±0.019(4) PEP 0.28±0.03(6) 1.18±0.68(3) Lactate 1.66±0.42(5) 0.32±0.02(5) 0.27±0.03(5) 2PGA 0.28±0.05(9) 0.22±0.03(4) 0.076±0.005(5) 0.065±0.004(6) 0.068±0.005(9) 0.075±0.005(5) 0.67±0.03(6) 0.73±0.06(9) 0.009±0.003(5) 0.015±0.002(6) 0.014±0.002(9) 0.014±0.004(5) 0.013±0.003(5) 0.015±0.004(6) 0.019±0.003(9) 0.018±0.003(5) 0.24±0.03(5) 0.27±0.02(6) 0.17±0.03(9) 0.068±0.007(4) 3PGA 0.73±0.05(5) 0.71±0.06(5) 0.76±0.08(4) DHAP 0.012±0.002(4) FDP 0.024±0.003(4) G-6-P 0.22±0.07(5) 0.090±0.025(4)* Glucose 5.97±0.56(6) 12.02±0.96(6) ** 6.03±0.20(8) 10.69±1.08(5)** 7.62±0.32(4)* _______________________________________________________________________ The same results are presented as percentage of hepatic metabolites in septic rats and caecal-ligated rats subjected to mannoheptulose or streptozotocin treatment, in relation to sham-operated metabolite concentrations, when the latter are set at 100% (Figs. 7.13.,7.14. & 7.15.). Sepsis caused a significant increase in the hepatic concentrations of lactate, pyruvate and glucose 6-phosphate at 24 h after the operation. When septic rats were treated with mannoheptulose the hepatic pyruvate concentration was no longer significantly increased, compared to the concentrations found in the livers of shamoperated rats. Mannoheptulose injection caused no appreciable change in the high concentrations of hepatic glucose 6-phosphate, but caused a significant increase in hepatic glucose concentration (Fig. 7.13.) Fig. 7.13. At 48 h after the operation, sepsis caused increases in the concentrations of most metabolites involved in gluconeogenesis, with the exception of D-2-phosphoglycerate and dihydroxyacetone phosphate and glucose. Septic rats, treated with mannoheptulose, showed a significant decrease in hepatic pyruvate concentration and a significant increase in hepatic glucose concentration (Fig. 7.14.). Fig. 7.14. Septic rats had increased hepatic concentrations of glucose 6-phosphate as early as 24 h after the operation, as compared to sham-operated rats (Table 3.1. - Chapter 3; Fig. 7.13.). However, when streptozotocin was administered to lower plasma insulin in septic rats, at 48 h after the operation, there was a significant decrease in the hepatic concentration of glucose-6-phosphate, as opposed to a significant elevation in hepatic glucose concentration. Glucose 6-phosphatase activity is known to be increased in livers of diabetic animals (Ashmore & Weber, 1959; Garfield & Cardell, 1979). If short-term treatment with streptozotocin causes a similar change in glucose 6-phosphatase activity, this may explain the decrease in glucose 6-phosphate leading to increased glucose concentration in the liver of septic-treated rats (Fig. 7.15.) Fig. 7.15. The pattern of metabolite changes in septic rats treated with mannoheptulose, at 24 and 48 h after the operation, suggests an increase in glucose synthesis at the expense of augmented pyruvate utilization. As the hepatic cytosolic redox state (NAD+/NADH ratio) is in equilibrium with the concentrations of lactate/pyruvate, the fall in hepatic pyruvate concentration during mannoheptulose treatment, in septic rats, may also reflect a decrease in the NAD+/NADH ratio, which in turn could lead to a stimulation of gluconeogenesis (Williamson et al , 1967). The diminished hepatic concentration of glucose-6-phosphate associated with an increase in hepatic glucose concentration, in septic rats treated with streptozotocin, suggests increased flux through the glucose 6-phosphatase step. The effects of mannoheptulose or streptozotocin treatment on the changes in hepatic concentrations of ketone bodies, acetyl-CoA and ATP during the induction of sepsis The inhibition of the hyperketonaemia in response to starvation caused by sepsis was reversed by mannoheptulose or streptoztocin (Fig. 7.7.). Ketone body concentrations in the liver were also decreased by sepsis at 48 h after the operation. Injection of mannoheptulose or streptozotocin, in septic rats, caused an elevation in the hepatic concentrations of ketone bodies to similar concentrations as those found in the livers of sham-operated rats at 48 h after the operation (Fig. 7.16.) Fig. 7.16. Sepsis or sepsis accompanied by mannoheptulose treatment did not cause appreciable change in hepatic concentration of acetyl-CoA at 24 h after the operation as compared to those of sham-operated rats. Acetyl-CoA concentrations, however, decreased significantly in the livers of septic rats as compared to those of sham-operated rats at 48 h after the operation. Treatment to lower plasma insulin (mannoheptulose or streptozotocin injection) decreased even further the hepatic concentration of acetyl-CoA to values significantly lower than those of septic animals (Fig. 7.17.). Fig. 7.17. ATP concentrations in the livers of septic rats were not significantly different from those of sham-operated animals at 24 and 48 h after the operation. Mannoheptulose treatment of septic rats did not cause appreciable change in hepatic ATP concentration at 24 or 48 h after the operation (Fig 7.18). Fig. 7.18. Administration of streptozotocin to septic rats caused a slight decrease in hepatic ATP concentrations, to values significantly lower than those of sham-operated rats (Fig.7.18.). The effects of mannoheptulose or streptozotocin treatment on the changes in rates of lipogenesis and cholesterogenesis in vivo during the induction of sepsis Sepsis stimulated the rates of fatty acid (saponifiable lipid fraction) synthesis at 24 and 48 h after the operation. Mannoheptulose treatment, given to septic rats at 24 and 48 h after the operation, caused a significant reduction in the rates of incorporation of 3H2O into fatty acids, as compared to those of caecal-ligated and punctured rats (Fig. 7.19.). The rates of tritiated water incorporation into fatty acids was lower, but did not reach statistical significance, in septic rats injected with streptozotocin at 48 h after the operation, as compared to those of caecal-ligated and punctured rats (Fig. 7.19.). Fig. 7.19. Increased rates of synthesis of total non-saponifiable lipids in vivo were found in livers of septic rats at 24 and 48 h after the operation. Administration of mannoheptulose or streptozotocin to septic rats caused a decrease in the rates of 3H2O incorporation into non-saponifiable lipids (squalene, cholesterol and other steroids) at 24 and 48 h after the induction of sepsis (Fig. 7.20.). Fig. 7.20. The non-saponifiable fraction was separated into squalene, cholesterol and other sterol fractions. The rates of 3H2O incorporation into squalene were increased, at 24 h after the operation, in livers from septic rats. Mannoheptulose treatment, given to septic rats, at 24 h after the operation tended to decrease 3H2O incorporation into squalene, as compared to that of septic rats, but this was not statistically significant (Fig. 7.21). When mannoheptulose or streptozotocin were injected in septic rats, 48 h after the operation, there was a significant decrease in the rates of 3H2O incorporated into squalene, when compared to those of septic rats (Fig. 7.21.). Fig. 7.21. The rates of cholesterogenesis in vivo in the livers of septic rats were significantly increased at 24 and 48 h after the operation. Treatment of septic rats with mannoheptulose or streptozotocin caused a significant decrease in the rates of 3H2O incorporation into hepatic cholesterol in vivo , as compared to those of septic rats, at 24 and 48 h after the operation (Fig. 7.22.). Fig. 7.22. The reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate by HMG-CoA reductase plays a key role in the regulation of the rate of cholesterol synthesis. Phosphorylation of HMG-CoA reductase leads to inactivation whereas the non-phosphorylated (expressed) form is the 'active' form of the enzyme (Gibson & Ingebritsen, 1978; Denton et al , 1981; Gibson & Parker, 1987). Sepsis did not cause appreciable change in the activity of the expressed or 'active' form of the enzyme, but increased significantly the phosphorylated form of HMG-CoA reductase, at 48 h after the operation (Fig. 7.23.). Fig. 7.23. Administration of mannoheptulose or streptozotocin to septic rats at 48 h after the operation, had no effect on this pattern of changes induced by sepsis, either in the activity of the expressed or active form or in the activity of the phosphorylated form of this enzyme, which remained higher as compared to that of sham-operated rats. There was a lack of correlation between the activity of HMG-CoA reductase (unchanged), and cholesterol synthesis (decreased) in septic rats subjected to mannoheptulose or streptozotocin treatment, as judged by the two-fold increase in the ratio of total HMGCoA reductase activity/cholesterol synthesis in septic treated rats (Septic: 10.4; Septic+Mannoheptulose: 18.8; Septic+Streptozotocin: 24.2). The effects of mannoheptulose treatment on the metabolic changes of sham-operated rats Sham-operated rats were treated with mannoheptulose in order to evaluate the effects of this compound in the control situation. Table 7.2. summarizes the changes brought about by mannoheptulose treatment on sham-operated rats, at 24 and 48 h after the operation. For purposes of comparison, the data on sham-operated rats are also included in the table. Plasma insulin concentrations in sham-operated rats, treated with mannoheptulose, were not significantly different from those of sham-operated rats (Sham-op: 9.90±1.90 (n=10) versus 13.55±2.64 (n=4) - µU/ml plasma) at 48 h after the operation. Despite plasma insulin concentrations remaining unchanged with the mannoheptulose treatment, the metabolic changes found in the treated sham-operated rats were compatible with those to be found under the influence of low insulin concentration, namely, increased blood and hepatic concentrations of glucose and ketone bodies. The sham-operated rats were hyperglycaemic in the presence of lower plasma insulin concentrations, as compared to those of septic rats (Fig. 7.8.). This suggests that both sham-operated and rats treated with mannoheptulose had a plasma insulin concentration which was inappropriate for the blood glucose. The increase in hepatic ketone bodies was accompanied by either unchanged (24 h) or increased hepatic acetylCoA concentration. Table 7.2. The effects of mannoheptulose treatment on the metabolic changes of sham-operated rats in vivo ____________________________________________________________ For full details see Chapter 2. Results are mean values ± S.E.M. with the number of observations in parentheses. Significance of difference by Student's t-test from septic rats: * P< 0.05; ** P < 0.01; *** P < 0.001. The following abbreviations are used: Sham-op - Sham-operated; Sham-op+M - Sham-operated + Mannoheptulose; PEP - Phosphoenolpyruvate; 2PGA - D-2-Phosphoglycerate; 3PGA - D-3- Phosphoglycerate; DHAP - Dihydroxyacetone phosphate; FDP - Fructose-1,6-bisphosphate; G-6-P - Glucose-6-phosphate. ____________________________________________________________ Metabolite Concentration (µmol/g fresh wt. of liver) _____________________________________________________ 24 h 48 h ____________________________ Sham-op _____________________________ Sham-op+M Sham-op Sham-op+M _______________________________________________________________________ Blood Lactate 1.56±0.19(9) 1.20±0.13(4) 1.14±0.14(9) 1.41±0.089(4) Pyruvate 0.058±0.009(3) 0.040±0.007(3) 0.057±0.010(7) 0.044±0.012(4) Alanine 0.24±0.015(9) 0.16±0.016(4)** 0.14±0.020(6) 0.16±0.014(4) Glucose 4.27±0.32(9) 9.51±0.29(4)*** 4.10±0.26(8) 8.97±0.95(4)*** Ketone bodies 0.79±0.13(9) 1.46±0.17(4)* 1.08±0.14(11) 2.02±0.33(4)** Hepatic Lactate 0.63±0.095(6) 0.28±0.080(4) * 0.36±0.064(11) 0.56±0.068(4) Pyruvate 0.036±0.004(6) 0.029±0.008(4) 0.038±0.007(8) 0.043±0.002(3) Alanine 0.94±0.19(6) 0.25±0.012(4)* 0.39±0.074(10) 0.043±0.062(4)* PEP 0.26±0.052(6) 0.15±0.093(4) 0.14±0.032(8) 0.077±0.002(4) 2PGA 0.059±0.007(6) 0.045±0.050(4) 0.050±0.007(8) 0.022±0.011(4) 3PGA 0.67±0.14(6) 0.38±0.069(4) 0.43±0.075(8) 0.011±0.002(6) 0.010±0.006(4) DHAP 0.010±0.003(8) 0.15±0.010(4)* 0.004±0.002(3) FDP 0.012±0.003(6) 0.016±0.007(4) 0.007±0.001(8) 0.009±0.002(3) G6P 0.093±0.037(6) 0.16±0.025(4) 0.075±0.017(8) 0.074±0.015(4) 6.19±0.55(6) 12.24±0.81(4)** Glucose 5.66±0.32(7) 13.39±1.50(4)*** Ketone bodies 0.73±0.089(6) 1.68±0.14(4)*** 1.65±0.14(12) 1.99±0.35(4)* Acetyl-CoA 0.088±0.015(6) 0.053±0.016(4) 0.100±0.008(4) 0.189±0.023 (4)* 2.35±0.29(6) 1.66±0.15(4) 2.11±0.074(8) 2.88±0.51(4) ATP _______________________________________________________________________ In sham-operated rats hepatic lipogenesis was very low, and unaffected by the mannoheptulose treatment (Fig. 7.24.) Fig. 7.24. Cholesterogenesis, on the other hand, was significantly decreased by mannoheptulose treatment at 24 and 48 h after sham- operation (Fig.7.25.). Fig. 7.25. The effects of insulin replacement given to septic rats treated with mannoheptulose on the metabolic changes induced by sepsis To investigate whether the metabolite changes observed in septic rats treated with mannoheptulose, were likely to be due to insulin deficiency, insulin was administered to septic rats (48 h) treated with mannoheptulose. However, it must be emphasised, that a pharmacological dose of insulin was used, and therefore, the resulting hyperinsulinaemia was greater than that found in septic rats before the treatment with mannoheptulose. A summary of the results of insulin replacement in septic rats treated with mannoheptulose is shown on Table 7.3.. For comparison, results obtained from septic rats and septic rats treated with mannoheptulose are also included in the table. Insulin replacement to septic rats previously treated with mannoheptulose caused profound hypoglycaemia associated with a decrease in hepatic glucose concentration. Ketone body concentration in blood and liver also decreased. The replacement with pharmacological doses of insulin was, however, unable to increase the rates of either lipogenesis or cholesterogenesis to levels comparable to those in septic rats. Table 7.3. The effects of insulin administration on the metabolic changes of septic rats treated with mannoheptulose in vivo at 48 h after the operation ____________________________________________________________ For full details see Material and Methods (Chapter 2). Results are mean values ± S.E.M. with the number of observations in parentheses. Significance of difference by Student's t-test from septic rats : ** P < 0.01 . * P< 0.05; From septic rats treated with mannoheptulose: ∆ P < 0.05; ∆∆ P < 0.01. The following abbreviation is used: Septic+M - Septic + Mannoheptulose. ____________________________________________________________ Metabolite Concentration (µmol/g fresh wt. of liver) _________________________________________________ Septic Septic+M Septic+M+Insulin _______________________________________________________________________ Plasma Insulin 35.83±3.88(10) 24.71±3.42(9)* 409.9±117.2(4)**∆∆ Blood Lactate 2.37±0.22(12) 2.19±0.41(5) 0.120±0.019(10) 0.53±0.0.19(5)* 0.34±0.076(7) 0.19±0.017(5)* 3.90±0.11(9) 7.34±0.81(5)* 1.29±0.55(4)* Pyruvate 0.076±0.027(4) Alanine 0.17±0.049(4) Glucose 0.54±0.061(4)**∆∆ Ketone bodies 0.59±0.055(14) 1.60±0.086(5)** 1.21±0.19(14) 1.46±0.34(5) 0.078±0.009(9) 0.046±0.010(4)* 0.37±0.027(4) ∆∆ Hepatic Lactate 0.78±0.20(4) Pyruvate 0.099±0.013 (4) ∆ Alanine 1.24±0.17(11) 1.10±0.25(5) 6.03±0.20(8) 10.69±1.08(5)* 0.55±0.25(4) Glucose 1.54±0.14(4)**∆∆ Ketone bodies 0.78±0.052(14) 1.72±0.19(5)** 0.53±0.069(4) *∆∆ Hepatic Lipogenesis 3.04±0.41(18) 1.61±0.15(14)** 1.48±0.13(4)* Cholesterogenesis 1.73±0.22(18) 0.94±0.15(14)* 0.87±0.18(4) * _______________________________________________________________________ Discussion The aim of these experiments was to see whether lowering the raised plasma insulin in septic rats, by treatment with mannoheptulose or streptozotocin, would return the metabolic profile to that seen in the sham- operated animals. The treatment did lower plasma insulin (Fig. 7.8.), particularly at 48 h after the operation. It must, however, be emphasized that the plasma insulin was not returned to sham-operated values. A summary of the significant changes brought about by mannoheptulose or streptozotocin treatment of septic rats (48 h after the operation) are listed in Fig. 7.26. Fig. 7.26. The decrease in plasma insulin, after administration of mannoheptulose or streptozotocin to septic rats, resulted in the expected metabolic changes (hyperglycaemia, hyperketonaemia and decreased lipogenesis). This finding supports the view that it is the raised plasma insulin which is, in part, responsible for the anomalous response of septic rats to starvation. The fact that the hepatic glucose concentration was increased, by giving septic rats mannoheptulose treatment, may reflect increased hepatic glucose synthesis. The development of hyperglycaemia may be the result of either increased hepatic synthesis or decreased glucose utilization by peripheral tissues. However, insulin stimulated glucose uptake in vitro to the same extent in muscles from rats subjected to caecal ligation and double puncture (needle gauge-18) or sham-operated rats, at 16 and 24 h after the operation (Wichterman et al , 1979). The high lactate concentrations in the blood and in the liver of septic rats remained unchanged after mannoheptulose or streptozotocin treatment, supporting the view of continued lactate availability from glycolysis in the periphery. The lower concentrations of pyruvate in the blood and liver of septic rats, subjected to mannoheptulose injection, may be the result of increased hepatic pyruvate utilization or a change in the cytosolic NADH/NAD ratio (Williamson et al , 1967). Increased utilization of pyruvate can occur to meet the demand for the stimulated hepatic glucose production, as indicated by the elevated hepatic glucose concentrations. The pattern of changes in the concentrations of metabolites involved in gluconeogenesis suggests that one effect of mannoheptulose is to increase glucose synthesis, with the consequent decrease in hepatic pyruvate concentration, and an increase in glucose concentration in the liver. Streptozotocin treatment of caecal-ligated and punctured rats, on the other hand, caused a decrease in the hepatic concentration of glucose-6-phosphate together with an elevation in the concentration of glucose in the liver, suggesting increased glucose synthesis from this immediate precursor. It must, however, be emphasised that as the concentrations of plasma glucose and hepatic glucose rapidly equilibrate, any increase in the latter may be due to a decrease in peripheral utilization. Streptozotocin treatment, therefore, may have overcome the lower activity of glucose 6-phosphatase found during sepsis (Canonico et al ; 1977). This may have been achieved if the effect of short-term streptozotocin treatment increases the activity of glucose 6-phosphatase, as it does in streptozotocin induced diabetic rats (Garfield & Cardell, 1979). Therefore, in septic rats treated to lower plasma insulin the glucose 6-phosphate/glucose cycle appears to operate preferentially towards glucose formation (Fig. 7.27.). In contrast, Wannemacher et al (1980) measuring 14C-alanine incorporation into glucose in vivo , in rats pre-treated with mannoheptulose and injected with Streptoccocus pneumoniae (104) showed no increase in 14C incorporation into glucose, as compared to controls. In addition, rats receiving 107 Streptoccocus pneumoniae had a significantly lower incorporation of 14C into glucose, when compared to heat-treated controls. Therefore, the milder bacterial treatment caused no appreciable change in gluconeogenic rates in vivo with mannoheptulose treatment, whereas with the more severe bacterial injection gluconeogenesis in vivo was decreased in mannoheptulose treated rats. Insulin deficiency, with mannoheptulose treatment, was therefore not able to help to overcome the decreased gluconeogenic rates found in very severe sepsis. These results illustrate the importance of the severity of infection in the modulaton of the metabolic response to sepsis. Fig. 7.27. The injection of mannoheptulose or streptozotocin increased the concentration of blood and hepatic ketone bodies during sepsis. Treated septic rats (mannoheptulose or streptozotocin), had their ketone bodies increased to concentrations similar to those found in sham-operated rats. This suggests the impairment in the utilization of long-chain fatty acids, in the livers of septic rats (see Chapter 6), was overcome by lowering plasma insulin concentration. Mannoheptulose or streptozotocin treatment had also a significant effect in decreasing the rates of lipogenesis, and presumably lowering the concentration of malonyl-CoA. The lower concentrations of malonyl-CoA would, in turn, activate carnitine acyl-transferase I resulting in increased flux of long-acyl CoA into the mitochondria for ß-oxidation leading to formation of acetyl-CoA, the immediate precursor for ketogenesis. Treatment to lower plasma insulin in septic rats, caused a further decrease in the hepatic concentration of acetyl-CoA. As there appears to be increased flux of acylCoA into the mitochondria for ß-oxidation leading to increased formation of acetyl-CoA, the lower hepatic concentrations of acetyl-CoA could be the result of either increased total oxidation in the Krebs cycle, partial oxidation to yield ketone bodies, or a combination of both. However during long-term insulin deprivation (diabetes) or in starvation, when ketogenesis is increased, hepatic concentrations of acetyl-CoA are increased (Fig. 3.8. - Chapter 3). Lowering plasma insulin during a shorter period of time (3h), in the sham-operated rats treated with mannoheptulose, caused an increase in hepatic acetyl-CoA at 48 h after the operation (Table 7.2.). Sepsis, therefore, appears to have an independent effect on decreasing the hepatic acetyl-CoA concentration, as opposed to the effect of starvation (increase) or trauma and starvation accompanied by mannoheptulose treatment (increase), the latter represented by the increase in acetylCoA in sham-operated rats treated with mannoheptulose (Table 7.2.). The reason for this difference is not clear. The ATP concentration in the livers of septic or sham-operated rats was not significantly affected by the administration of mannoheptulose, whereas there was a slight decrease in ATP concentration in septic rats treated with streptozotocin, as compared to that found in the livers of sham-operated rats at 48 h after the operation. The rates of incorporation of 3H2O into fatty acids and non-saponifiable lipids were significantly increased by sepsis. Treatment to lower plasma insulin in septic rats, decreased these rates, and livers from septic rats injected with mannoheptulose or streptozotocin, produced fatty acids, and non-saponifiable lipids at significantly lower rates when compared to those of septic rats; some of these rates were very similar to those of sham-operated rats. Sepsis caused a significant increase in the rates of cholesterogenesis (12, 24 and 48 h after the operation) and squalene formation (24 h after the operation). Mannoheptulose or streptozotocin treatment also caused a decrease in the rates of cholesterol and squalene synthesis in the liver, measured by the decreased incorporation of 3H2O into these sterols. To evaluate the regulation of cholesterol metabolism under these conditions, the total (phosphorylated) and the expressed or 'active' form of HMG-CoA reductase were measured in septic rats subjected to treatment to lower plasma insulin. There was a lack of correlation between enzyme activity, which remained high, at rates comparable to those of septic rats, and the decreased rate of cholesterol synthesis. The higher activity of HMG-CoA reductase in septic rats at 48 h after the operation suggests higher plasma insulin concentration may have had its longer-term action by increasing enzyme concentration in the liver. During the short-term mannoheptulose or streptozotocin treatment, insulin appeared not to exert its action by promoting covalent phosphorylation -dephosphorylation leading to changes in the activation state of the enzyme, because the activity of the expressed form of the enzyme remained unchanged by sepsis associated with either mannoheptulose or streptozotocin injection. Easom & Zammit (1984) developed a technique for rapid sampling of rat liver, by injecting ice-cooled sucrose buffer into the portal system to try to clear the liver from red cells, and to provide immediate ice-cooling of the organ. By using this technique they claim the phosphorylation state of HMG-CoA reductase in vivo is better maintained in the microsomal preparation for determination of enzyme activity. We have not used this technique and, therefore, a portion of the expressed form may have been lost, but it is likely that this loss was similar in sham-operated and septic rats. In addition, the changes induced by sepsis were mainly in the total form of the enzyme (Fig. 7.25.). The treatment to lower plasma insulin, in septic rats, therefore, was able to promote the reversal of the changes in ketone body concentration in blood and liver, in pyruvate concentration in liver and blood, in hepatic glucose-6-phosphate concentration , and to induce an increase in hepatic and blood glucose. Reversal of the rates of cholesterogenesis, squalene synthesis and lipogenesis were also obtained. The administration of insulin, in pharmacological dose, to septic rats treated with mannoheptulose (Table 7.3.), caused a partial return of the metabolic profile of septic mannoheptulose-treated rats to that found in septic rats. The present results, therefore, support the view that the higher insulin concentration is involved in the development of the changes which occur during the induction of sepsis. Consequently, in many respects, septic rats subjected to mannoheptulose or streptozotocin treatment, behaved more like sham-operated rats than septic rats. CHAPTER 8 General Discussion The metabolic response to infection is part of a concerted physiological and immunological response, which is dependent on a number of factors, including the nutritional status, the hormonal milieu, and the stage and degree or severity of sepsis. The interrelation between these factors is reflected in the large spectrum of the metabolic changes observed in septic patients (Cerra, 1982; Wilmore, 1983; Forse & Kinney, 1985). The severity of infection plays an important part in the septic process and can modulate the metabolic changes induced by sepsis, as illustrated in Fig. 8.1. The experimental model used in the work presented in this Thesis induced a moderate form of sepsis, as compared with that of others (Chaudry et al , 1979; Wichterman et al , 1980). The septic rats did not become hypothermic nor shocked. The blood lactate in the septic group was increased as early as 12 h after the operation but never reached values defined as those for lactic acidosis (5 mmol/l). They did not go through the late hypodynamic late septic state (Chaudry et al , 1979), demonstrated by the fact that they did not develop hypoglycaemia or hypoinsulinaemia at any time point studied. Fig. 8.1. The regulation of the metabolic response to sepsis The exact cascade of events which leads to the establishment and evolution of the septic response is starting to become clearer. Immunological, inflammatory and endocrine mediators appear to act in concert to mount the metabolic and physiological response, which appears to be fairly uniform and partially independent of the infective agent (Wiles et al , 1980; Frayn, 1986). In Chapter 1, various "mediators" of the response to sepsis were discussed, including interleukin-1 or its sub-products (Dinarello & Wolff, 1982; Takemura & Werb, 1984), and cachectin (Tracey et al , 1986; Beutler & Cerami, 1987). These "mediators" may represent the link between the immunological and the metabolic response. In support of this view is the action of interleukin-1 (leucocytic endogenous mediator; proteolysis inducing factor) in promoting the muscle catabolism and stimulating the ß-cells to secrete insulin during sepsis (George et al , 1977; Clowes et al , 1983 and 1985). The elevation in plasma insulin appears to be a key factor in the hepatic metabolic changes induced by caecal-ligation and puncture in the rat. This led to the proposal of a unified hypothesis for the regulation of the changes in hepatic metabolism by the action of the raised plasma insulin (Chapter 7). Furthermore, treatment to lower plasma insulin reversed many of the metabolic changes induced by sepsis, giving support to the important modulatory role of insulin (Chapter 7). Whether the leucocyte-derived "mediators" also play a direct role in the regulation of the metabolic response to sepsis, in the liver, requires further investigation. Recent evidence suggests that interleukin-1 injected into the rat in vivo , has a stimulatory effect on hepatic metabolism in isolated hepatocytes (Roh et al , 1986). The metabolic changes induced by interleukin-1 adminstration had a time-dependent pattern and included an increase in glucose synthesis from alanine, stimulation of protein synthesis and an increase in oxygen consumption. Interleukin-1 appears to exert its effect on the liver indirectly, for no action was observed when interleukin-1 was added to the hepatocytes' incubation medium. The infusion of cachectin into rats caused some of the metabolic changes found during sepsis, such as hyperglycaemia and hyperlactataemia and led to the development of changes in target organs similar to those found in the multiple organ failure syndrome (Beutler & Cerami, 1987). Nevertheless, the regulatory role of cachectin upon the metabolic changes in the liver still needs clarification. Further research involving in vivo turnover studies might help to elucidate the action of these leucocytederived 'mediators'. The administration for example of monoclonal antibodies, anticachectin and/or anti-interleukin-1, to caecal-ligated and punctured rats may clarify the specific action of both 'hormones' upon hepatic metabolism in vivo , and their role in the modulation of the metabolic response. Is the metabolic response to sepsis adaptive ? The magnitude of the stress imposed by sepsis is a function of the size of the infectious process, the number of invading organisms and their virulence. The severity of infection modulates the metabolic response, for example increasing the resting energy expenditure and the extent of the negative nitrogen balance (Elwyn, 1980). The effort to contain and eliminate invading microorganisms is directly related to their number and virulence, so that whereas a few thousand microorganisms will be contained by phagocytosis, many million require mobilisation of both cellular and humoral immune responses. Wiles et al (1980) hypothesized that the systemic response to sepsis is largely independent of the type of invading organism. In favour of this view is the similarity in many of the metabolic and physiological changes in gram-positive, gram-negative, anaerobic, fungal and even viral sepsis (Deutschman et al , 1987), suggesting that the series of responses to infection are primarily a function of the septic animal. Therefore, if the metabolic response to sepsis is considered to be uniform, despite the nature of the infective agent, it would appear to be adaptive and protective. It is probable that an injured or infected animal in the wild, under conditions where food is not immediately available, could starve. It seems to be adaptive, that in order to maintain energy production and cell function throughout the body, protein, especially from skeletal muscle, is catabolised. However, prolonged proteolysis can lead to an impaired immunological capacity and to perpetuation of the infectious process (Chapter 1; Kettlewell et al , 1979). Ketone bodies are able to decrease skeletal muscle amino acid release, therefore decreasing the availability of precursors for gluconeogenesis (Williamson et al , 1978). The fact that ketogenesis is depressed by sepsis during the starved state (Chapter 5) might also be regarded as detrimental. The administration of exogenous ketone bodies was, however, unable to reduce proteolysis and gluconeogenesis in fasted-infected sheep (Radcliffe et al , 1981 and 1983). The progression of the septic process to the development of multiple organ failure (Duff, 1985) and death, might be considered "maladaptive" (Wilmore et al , 1983), but more likely represents the effects of overwhelming the adaptive responses to sepsis. Manipulation of the metabolic response to sepsis The attenuation of many of the classical postoperative metabolic responses, such as increased cortisol release and negative nitrogen balance, does not seem to cause harmful effects following elective operative procedures (Kehlet et al , 1980). With modern intensive therapy, supported by high-technology medicine, the possibility of manipulating the metabolic response to sepsis may become part of the therapeutic strategy. The exaggerated and possibly harmful metabolic response could be attenuated if the patient's temperature and substrate flow were adjusted by provision of exogenous heat and substrates, the latter in the form of adequate nutritional support (Wilmore et al , 1983). The development of non-invasive techniques, such as bed-sided 'metabolic carts', make it possible to monitor energy substrates' kinetics (Elwyn et al , 1981; Stein, 1986). Some of the possible sites for modifying the metabolic response to sepsis are illustrated in Fig. 8.2. The use of a number of biological response modifiers has been discussed in the introductory Chapter, and considerable research has been devoted to assess their effect in the metabolic response to injury/sepsis (Nohr & Meakins, 1985). The administration of large doses of steroids in sepsis is common practice, especially in septic shock (Forse & Kinney, 1985). Recent evidence supports, at least in theory, the use of steroids because of their actions, such as the ability to induce key gluconeogenic enzymes (Shackelford et al , 1986), to inhibit oxygen-free radical generation (Karakusis, 1986), to block prostaglandin and leukotriene synthesis (HallaAngeras et al , 1986; Samuelsson, 1983), Fig. 8.2. and to block cachectin formation by macrophages (Cerami et al , 1986; Beutler & Cerami, 1987). In order to be able to block cachectin synthesis in vitro , however, macrophages have to be exposed to the effects of steroids before their activation by the bacterial product (LPS) (Fig. 8.1.). Support for the use of steroids at an early stage of infection, is the finding that an early intravenous bolus of methylprednisolone administered at the time of the operation, significantly improved the survival of caecal-ligated and punctured rats, compared to infusing the drug continuously over the 7 day study period (Hollenbach et al , 1986). In contrast to the experimental situation, however, patients almost always present themselves with well established infection when many machrophages have already been activated. Cachectin has been proposed as an important mediator of the changes brought about by infection (Beutler et al , 1986; Beutler & Cerami, 1987). It would seem therefore conceivable that neutralizing monoclonal antibodies against human cachetin might prove useful in the treatment of sepsis in its early stages (Fig. 8.2.). In the present study many of the metabolic changes in response to sepsis were reversed by the treatment to lower the plasma insulin (Chapter 7), either by mannoheptulose or streptozotocin (Fig. 8.2.). Streptozotocin leads to permanent damage of pancreatic ß-cells (Weiss, 1986), and its clinical use is, therefore, impossible. On the other hand, mannoheptulose has been studied in man (Viktora et al , 1969; Johnson et al , 1969; Johnson & Wolff, 1970; Lev-Ran et al , 1970). Whether its use in septic patients will cause reversal of some of the metabolic changes in the liver is not known. Mannoheptulose causes hyperglycaemia, which could prohibit its use in septic patients with established glucose intolerance. One advantage in the possibility of manipulating the plasma insulin concentrations during sepsis, is the fact that, as opposed to the activation of macrophages and the consequent release of interleukin-1 or cachectin, which are early events in the establishment of the septic state, the increased plasma insulin concentration, induced by sepsis, is present in the hyperdynamic septic state, falling only in the hypometabolic late sepsis (Chaudry et al , 1979). Thus, insulin modulation could theoretically be used as a way to modify the metabolic response in moderately and severely septic patients (Fig. 8.1.). Nevertheless, any attempt to modify the metabolic response to sepsis must consider the stage and severity of infection, the nutritional and hormonal status of the patient, and which of the unwanted metabolic changes have been induced. CHAPTER 8 General Discussion The septic rat model and its metabolic implications The experimental model used in the work presented in this Thesis induced a moderate form of sepsis, as compared with that of others (Chaudry et al , 1979; Wichterman et al , 1980). The septic rats did not become hypothermic nor shocked. The blood lactate in the septic group was increased as early as 12 h after the operation but never reached values defined as those for lactic acidosis (5 mmol/l). They did not go through the late hypodynamic septic state (Chaudry et al , 1979), demonstrated by the fact that they did not develop hypoglycaemia or hypoinsulinaemia at any time point studied. The results demonstrate that gluconeogenesis and ketogenesis in rats made septic by caecal ligation and puncture are impaired in vitro , whereas, there are increased rates of lipogenesis and cholesterogenesis in vivo in the septic rats. Therefore, although deprived of food, the septic rats, behaved more like fed than fasted rats in terms of hepatic metabolism. Increased concentrations of blood and liver metabolites involved in gluconeogenesis - the new steady state The catabolism induced by sepsis increased the availability of substrates, such as pyruvate, lactate and alanine to the liver. The concentration of most of the metabolites involved in the gluconeogenic pathway rose within the liver, but no change was observed in either blood or hepatic glucose concentrations. The pattern of accumulation of glucose precursors up to the level of glucose-6phosphate, without changes in hepatic glucose concentration, suggests a new steady state has been achieved with higher concentrations of precursors. Hepatocyte behavior during sepsis The results clearly demonstrated that there is impairment in the hepatic capacity for gluconeogenesis and ketogenesis in vitro in septic rats. In the case of gluconeogenesis, the inhibition in vitro was progressive with time, and affected all substrates tested (Chapter 4). There is evidence that pneumoccocal infection in the rat decreases the hepatic activity of glucose 6phosphatase by 50% (Canonico et al , 1977), and that endotoxin, injected in mice, decreases the hepatic activity of the gluconeogenic enzymes, namely, glucose 6phosphatase, fuctose 1,6-bisphosphatase and phosphenolpyruvate carboxykinase (Shackleford et al , 1986). The hepatic concentration of acetyl-CoA, an important activator of pyruvate carboxylase, is decreased at 48 h after the induction of sepsis (Chapter 3). These alterations in key gluconeogenic enzymes may, therefore, explain the decreased glucose synthesis in vitro . The absence of hypoglycaemia in the septic rats suggests that this impaired gluconeogenesis may be overcome in vivo , at least during moderate sepsis, by increased concentrations of precursors, especially lactate and alanine (Chapter 3), which are supplied in increased amounts from muscle (Beisel & Wannemacher, 1980). The studies incubating isolated hepatocytes with lactate, at concentrations similar to those found in the blood of the septic and sham-operated rats in vivo , also supported the view that the depression in glucose synthesis can be overcome in vivo (Chapter 4). Whether this effect of sepsis reducing the hepatocyte ability to promote glucose synthesis may play a homeostatic role, leading to the new steady state described (Chapter 3) and avoiding uncontrolled hyperglycaemia, is still not clear. However, several turnover studies in vivo have shown increased gluconeogenic rates in sepsis, which conflicts with the findings in this study (Wannemacher et al , 1980; Kelleher et al , 1982; Lang et al , 1984; Shaw et al , 1985). The increase in glucose synthesis may be due to inappropriately low insulin levels for the metabolic circunstances prevailing, for example the presence of hyperglycaemia, in these turnover studies. Furthermore, some of these studies report either mild or initial forms of sepsis, almost always associated with injury. Therefore, as there is a lag period before the gluconeogenic enzymes become inhibited (Canonico et al , 1977) leading to the depression in gluconeogenesis (Chapter 4); substrate drive, at a time when gluconeogenic precursors are available from peripheral catabolism, may become the predominant effect, as in pure injury. During moderate forms of sepsis, as described in this study, gluconeogenesis is impaired, but this can be overcome, so that glucose synthesis can be maintained (Chapter 4). In severe forms of sepsis, the capacity to overcome the gluconeogenic inhibition no longer exists; the compensatory mechanisms, for example increased precursor availability, are overwhelmed leading to profound hypoglycaemia and death. Therefore the severity of sepsis, and the degree of stress induced by injury to which sepsis is often associated, appear to be of fundamental importance in the modulation of the metabolic response to sepsis. This view is supported by studies measuring rates of gluconeogenesis in vivo from 14C-alanine (Wannemacher et al , 1980). In these studies gluconeogenesis was increased in fasted rats which received a smaller dose of Streptoccocus pneumonie , whereas glucose synthesis was decreased in vivo when the dose of this microorganism was increased. The importance of the severiy of infection is also illustrated by Wilmore's study (1976), in which nonshocked patients with burns and gram-negative sepsis exhibited decreased gluconeogenesis in vivo leading to hypoglycaemia. Sepsis also depressed ketogenesis in vitro (Chapter 5). Decreased ketone body formation from long-chain fatty acids (oleate) in hepatocytes from septic rats was observed 12 h after the operation, and the inhibition was not enhanced at the later time points (Chapter 5). As this decrease in ketogenesis was not observed with the short-chain fatty acid, butyrate, it is likely that entry of long-chain acylCoA into the mitochondria for oxidation is diminished during sepsis. Whether this is induced by modulation of carnitine acyltransferase I activity, via increased malonyl-CoA (McGarry & Foster, 1980; Vary et al , 1986) is still an open question. Changes in hepatic lipogenesis and cholesterogenesis in vivo Sepsis stimulated the rates of hepatic lipogenesis and cholesterogenesis in vivo . Increased hepatic lipogenesis in vitro has been reported in pneumoccocal infection in the rat (Canonico et al , 1977). Sepsis has also been claimed to increase the activities of acetyl-CoA carboxylase and lipid synthetase (Pace et al , 1981 ), the former enzyme is subject to short-term activation by raised insulin concentrations (Brownsley & Denton, 1987). In the present study, sepsis increased the rate of synthesis of the non-saponifiable as well of the saponifiable lipids (Chapter 6). The increased hepatic cholesterogenesis in vivo may reflect the increased demand for cholesterol in the catabolic state of sepsis, for membrane synthesis and the proliferation of cells in the reticuloendothelial system. Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterogenesis, and its activity is increased by sepsis (Chapter 6; Canonico et al , 1977). This increased HMG-CoA reductase activity appeared to be related to total enzyme concentration, rather than changes in the enzyme activation state (Chapter 6). A unified hypothesis for the regulation of the changes in hepatic metabolism in response to sepsis - the action of insulin. The key factor which may prevent the septic rats responding in the same way as the sham-operated rats to starvation is the raised plasma insulin. This increase in plasma insulin is likely to be due to stimulation of pancreatic secretion by bacterial products, such as LPS (Yelich & Filkins, 1980) and leucocyte endogenous mediator, which contains interleukin 1 (George et al , 1977). Insulin is known to stimulate lipogenesis and cholesterogenesis and decrease gluconeogenesis and ketogenesis by its short-term actions on the activation state of key enzymes, such as acetyl-CoA carboxylase, and by longerterm regulation of enzyme concentration. Although plasma glucagon is also increased by sepsis (Beisel & Wannemacher, 1980), its stimulatory effects on ketogenesis and gluconeogenesis and its inhibitory effects on lipogenesis may be antagonised by the raised plasma insulin. The results from septic rats treated with either mannoheptulose or streptozotocin (Chapter 7), with the aim of decreasing the plasma insulin, support the view that hyperinsulinaemia is a key factor in the metabolic changes in the liver induced by sepsis. Treatment to lower plasma insulin produced a decrease in hepatic pyruvate concentration, and changes in the new steady state of gluconeogenic intemediates in sepsis by activating the glucose 6-phosphate/glucose cycle, leading to a decrease in glucose 6-phosphate concentration and the consequent hyperglycaemia (Chapter 7) suggesting increased glucose synthesis. On the other hand, Wannemacher et al (1980) measuring 14C-alanine incorporation into glucose, in vivo , in rats pre-treated with mannoheptulose and injected with Streptoccocus pneumoniae (104), showed no increase in 14C incorporation into glucose, as compared to controls. However, rats receiving 107 Streptoccocus pneumoniae had a significantly lower incorporation of 14C into glucose and when compared to heat-treated controls. Therefore mannoheptulose treatment caused no appreciable change in gluconeogenic rates in vivo with the milder bacterial treatment, whereas with the more severe bacterial injection gluconeogenic rates were decreased in mannoheptulose treated rats in vivo . Mannoheptulose was therefore not able to help to overcome the decreased gluconeogenic rates found in very severe sepsis. Septic rats, deprived of food, and treated to lower plasma insulin levels had blood and hepatic ketone body concentrations similar to those of shamoperated rats (Chapter 7). As the hepatic lipogenic rates were decreased by the same treatment (Chapter 7), possibly decreasing malonyl-CoA concentration, it is possible that ketogenesis was reactivated in the septic treated rat, perhaps as a result of more long-chain acyl-CoA entering the mitochondria for oxidation. Lowering plasma insulin concentration also induced a reduction in the rates of cholesterogenesis in vivo in the liver of septic rats (Chapter 7). Is the metabolic response to sepsis adaptive ? The magnitude of the stress imposed by sepsis is a function of the size of the infectious process, the number of invading organisms and their virulence. The severity of infection modulates the metabolic response, for example increasing the resting energy expenditure and the extent of the negative nitrogen balance (Elwyn, 1980). The effort to contain and eliminate invading microorganisms is directly related to their number and virulence, so that whereas a few thousand microorganisms will be contained by phagocytosis, many million require mobilisation of both cellular and humoral immune responses. Wiles et al (1980) hypothesized that the systemic response to sepsis is largely independent of the type of invading organism. In favour of this view is the similarity in many of the metabolic and physiological changes in gram-positive, gram-negative, anaerobic, fungal and even viral sepsis (Deutschman et al , 1987), suggesting the series of responses after the septic insult are primarily a function of the septic animal. Therefore, if the metabolic response to sepsis is considered to be uniform, despite of the nature of the infective agent, it would appear to be adaptive and protective. It is probable that an injured or infected animal in the wild, under conditions where food is not immediately available, could starve. It seems to be adaptive, that in order to maintain energy production and cell function throughout the body, protein, especially from skeletal muscle, is catabolised. However, prolonged proteolysis can lead to an impaired immunological capacity and to perpetuation of the infectious process (Chapter 1; Kettlewell et al , 1979). As sepsis sometimes may be associated with renal failure, the protein catabolism induced by sepsis under these circunstances might be considered as maladaptive (Wilmore et al , 1983), or more properly, that the adaptive mechanisms were overwhelmed. Ketone bodies are able to decrease skeletal muscle amino acid release, therefore decreasing the availability of precursors for gluconeogenesis (Williamson et al , 1977). The fact that ketogenesis is depressed by sepsis during the starved state (Chapter 5) might also be regarded as detrimental. The administration of exogenous ketone bodies was, however, unable to depress proteolysis and gluconeogenesis in fasted-infected sheep (Radcliffe et al , 1981 and 1983). The progression of the septic process to the development of multiple organ failure (Duff, 1985) and death, might be considered a mal adaptation, but more likely represents the effects of overwhelming the adaptive responses to sepsis. Manipulation of the metabolic response to sepsis The attenuation of many of the classical postoperative metabolic responses, such as increased cortisol release and negative nitrogen balance, does not seem to cause harmful effects following elective operative procedures (Kehlet et al, 1980). With modern intensive therapy, supported by high-technology medicine, the possibility of manipulating the metabolic response to sepsis may become part of the therapeutic strategy. The exaggerated and possibly harmful metabolic response could be attenuated if the patient's temperature and substrate flow were adjusted by provision of exogenous heat and substrates, the latter in the form of adequate nutritional support (Wilmore et al , 1983). The development of non-invasive techniques, such as bed-sided 'metabolic carts', make possible to monitor energy substrates kinetics (Elwyn et al , 1981; Stein, 1986). Some of the possible sites for modifying the metabolic response to sepsis are illustrated in Fig. 8.1. Fig. 8.1. The use of a number of biological response modifiers has been discussed in the introductory Chapter, and intense research has been devoted to assess their effect in the metabolic response to injury/sepsis (Nohr & Meakins, 1985). The administration of large doses of steroids in sepsis is common practice, especially in septic shock (Forse & Kinney, 1985). Recent evidence supports, at least in theory, the use of steroids because of their actions, such as the ability to induce key gluconeogenic enzymes (Shackelford, 1986), to inhibit oxygen-free radical generation (Karakusis, 1986), to block prostaglandin and leukotriene synthesis (Halla-Angeras et al , 1986; Samuelsson, 1983), and to block cachectin formation by macrophages (Cerami et al , 1986). In order to be able to block cachectin synthesis in vitro , however, macrophages have to be exposed to the effects of steroids before their activation by the bacterial product (LPS) (Fig. 8.1.). Support for the use of steroids at an early stage of infection, is the finding that an early intravenous bolus of methylprednisolone administered at the time of the operation, significantly improved the survival of caecal-ligated and punctured rats, compared to infusing the drug continuously over the 7 day study period (Hollenbach et al , 1986). In contrast to the experimental situation, however, patients almost always present themselves with well established infection when many machrophages have already been activated. Cachectin has been proposed as an important mediator of the changes brought about by infection (Cerami et al , 1986; Beutler & Cerami, 1987). It would seem therefore conceivable that neutralizing monoclonal antibodies against human cachetin might prove useful in the treatment of sepsis in its early stages (Fig. 8.1.). In the present study many of the metabolic changes in response to sepsis were reversed by the treatment to lower the plasma insulin (Chapter 7), either by mannoheptulose or streptozotocin (Fig. 8.1.). Streptozotocin leads to permanent damage of pancreatic ß-cells (Weiss, 1986), and its clinical use is, therefore, impossible. On the other hand, mannoheptulose has been studied in man (Viktora et al , 1969; Johnson et al , 1969; Jonhson & Wolff, 1970; Lev-Ran et al , 1970). Whether its use in septic patients will cause reversal of some of the metabolic changes in the liver is not known. Mannoheptulose causes hyperglycaemia, which could prohibit its use in septic patients with established glucose intolerance. Diazoxide did not have any appreciable effect on the metabolic changes in the septic rats (Chapter 7), perhaps because of its indirect hyperglycaemic effect from stimulation of catecholamines, which in turn, inhibit insulin secretion (Henquin et al , 1982). One advantage in the possibility of manipulating with the plasma insulin concentrations during sepsis, is the fact that, as opposed to the activation of macrophages and the consequent release of interleukin-1 or cachectin, which are early events in the establishment of the septic state, the increased plasma insulin concentration, induced by sepsis, is present along with the hyperdynamic septic state, falling only in the hypometabolic late sepsis (Chaudry et al , 1979). Thus, insulin modulation could theoretically be used as a way to modify the metabolic response in moderately and severely septic patients (Fig. 8.1.). Nevertheless, any attempt to modify the metabolic response to sepsis must consider the stage and severity of infection, the nutritional and hormonal status of the patient, and which of the unwanted metabolic changes have been induced. CHAPTER 9 Common features of the changes in hepatic metabolism induced by sepsis and by total parenteral nutrition in the human Introduction Total Parenteral Nutrition (TPN) is an accepted treatment for patients with malnutrition and intestinal failure (for review see: Grant & Todd, 1982; Silk, 1983; Phillips & Odgers, 1986). Controversy surrounds the choice of nutrients and the rates of administration (Elwyn, 1980; Wolfe et al , 1980; Kettlewell, 1982). Recent evidence, from experimental work (including the results already presented in this Thesis) and clinical studies, suggest some similarity in the effects of sepsis on hepatic metabolism compared to those of the fed state promoted by intravenous feeding (Fig. 9.1.). Fig. 9.1. Hepatic dysfunction, including increases in serum levels of liver enzymes, has been consistently reported in experimental animals (Meurling & Roos, 1981; Boelhouwer et al, 1983) and patients receiving Total Parenteral Nutrition (Grant et al, 1977; Sitges-Serra et al , 1983; Robertson et al , 1986). Hepatic dysfunction has also been reported during sepsis (Silen & Skillman, 1976; Royle & Kettlewell, 1980; Forse & Kinney, 1985). In addition, parenterally fed patients with major sepsis were found to have almost double the incidence of abnormal liver function tests compared with patients with no evidence of sepsis, but also receiving intravenous feeding (Robertson et al , 1986). Sepsis increases substrate availability and elevates plasma insulin (Chapter 3) concentrations which, in turn, stimulate hepatic lipogenesis in vivo (Chapter 6). Esterification of the newly formed non-esterified fatty acids is also increased by sepsis (Chapter 5), and perhaps, the accumulation of lipids found during sepsis (Guckian et al , 1973; Campion , 1976) may well reflect an imbalance in the liver between production and the capacity to export triacylglycerol. Total Parenteral Nutrition also increases hepatic lipogenesis and fat accumulation in the liver particularly when excess carbohydrate intake is transformed into fat (Elwyn et al , 1981; Stein & Mullen, 1985). This becomes the steatosis seen in liver biopsies (Benotti et al , 1976; Maini et al , 1976; Grant et al , 1977). Hall et al (1984) have shown that the liver steatosis in rats, induced by TPN, was due to enhanced hepatic synthesis of fatty acid and reduced triacylglycerol secretion. Hepatic acetyl-CoA carboxylase activity increases in parenterally nourished rats, and is positively correlated with hepatic lipid content (Hall et al, 1984). It is also interesting that acetyl-CoA carboxylase and fatty acid synthetase have increased activity in the fasted-infected rat (Pace et al , 1981). It is important to emphasize that acetyl-CoA carboxylase is subject to short-term regulation by insulin, the latter increasing its activity in the liver both in vivo and in vitro (Brownsey & Denton, 1987) Sepsis impairs ketogenesis in vitro (Chapter 4) and causes a decrease in blood ketone bodies despite starvation (Chapter 3). Blood ketone bodies are also low in patients fed parenterally, probably due to inhibition of lypolysis by elevated plasma insulin concentrations (Maini et al , 1976). Therefore, the factor bringing about these shared changes (Fig. 9.1.) is probably the increased plasma insulin concentrations present during sepsis (Beisel & Wannemacher, 1980; Neufeld et al , 1982; Shaw et al , 1985; Long et al , 1985; Frayn et al , 1986) and in parenterally fed patients (Maini et al , 1976; Page & Clibon, 1980). The hyperinsulinaemia of sepsis may be induced by stimulation of the ß-cell by bacterial products (endotoxin, leucocytic endogenous mediator) causing increased insulin secretion (George et al , 1977; Yelich & Filkins, 1980), whereas, patients receiving intravenous feeding appear to increase their plasma insulin concentrations in response to the considerable amount of glucose infused continuously to meet the caloric needs (Sanderson & Deitel, 1974; Maini et al , 1976). It may be beneficial, therefore, to lower the elevated concentrations of plasma insulin, by diminishing the exogenous-glucose stimulatory effect on ßcells, in patients fed parenterally. This was attempted in a clinical study either by reducing glucose intake, by offering lipid as part of the caloric source, or by promoting a period free of glucose infusion during intermittent or cyclical TPN. Abnormalities of liver funtion developing in parenterally fed patients during sepsis were also studied. CYCLICAL PARENTERAL NUTRITION "When we are forced to do something artificial we must aim at remainning as close as possible to the natural situation." Professor Sir Hans Krebs [1900-1981] Continuous infusion of nutrients throughout 24 h imposes important changes in the hormonal milieu comparable with a "steady fed state" (Page & Clibon, 1980). Man is a meal eater and physiologically alternates periods of feeding with post-absorptive periods. Classical continous TPN may reasonably be criticised on the grounds it does not allow for any post-absorptive period, and therefore maintains patients in a store mode, inhibiting the state where nutrients are redistributed. This practice evolved following the classical studies of Dudrick et al (1969), who demonstrated that growth and nitrogen balance could be achieved with the continuous infusion of glucose and amino acids. Recently, it is becoming common practice to provide intravenous nutrition intermittently (Cyclical Parenteral Nutrition), to selected groups of hospital patients, usually over 12-14 h (Maini et al , 1976; Page & Clibon, 1980; Matuchanski et al , 1981 and 1985). In addition to allowing a period of freedom from intravenous appendages, enhancing patients' mobility and morale, Cyclical TPN also provides a 'post-absorptive' period which is theoretically closer to the physiological meal pattern. The criteria for selection of hospital patients to receive Cyclical Parenteral Nutrition (adapted from Page & Clibon, 1980) are illustrated in Fig. 9.2. Fig. 9.2. This form of TPN administration was originally developed for home patients requiring intravenous feeding. In 1973 Jeejeebhoy et al reported a successful case of home parenteral nutrition, when cyclical intravenous feeding was the method of administration. The introduction of a Dacron-cuffed, silicone ruber catheter (Broviac, 1973) to deliver TPN solutions into a large vein for long periods of time was an important step for home parenteral feeding. Today, selected patients with intestinal failure can be successfully maintained in good health and full activity (For review on Home Parenteral Nutrition see: Lees et al , 1981; Johnston, 1982; Silk, 1983; Phillips & Odgers, 1986). To date some 200 patients have been fed parenterally at home in the United Kingdon and Ireland, between 1977 and 1986 (Mughal & Irving, I987), with the main indications illustrated in Fig. 9.3. Despite being fairly widely used, many of the metabolic effects of intermittent intravenous feeding, as well as, some of the basic information, such as the optimal essential trace element provision to home TPN patients (Shenkin et al, 1986), are not yet clear. Messing et al (1983) have shown that in mildly catabolic patients, cyclical TPN promoted a gain in visceral and lean body mass, similar to that obtained by continuous TPN. Matuchansky et al (1981) also reported that nocturnal cyclical TPN was safe, efficient and psychologically well tolerated in malnourished patients with severe gastrointestinal disorders. Cyclical TPN has also been used successfully in children undergoing bone marrow transplantation, when an infusion-free period is required for the adminstration of drugs and blood transfusions without interfering with the nutritional support (Reed et al , 1983). Fig. 9.3. Some of the possible metabolic differences between Cyclical Parenteral Nutrition and Continuous TPN are illustrated in Fig. 9.4., as described by Page and Clibon (1980). Fig. 9.4. Recour et al (1980) reported changes in blood and plasma metabolites in two children on nocturnal cyclical TPN. They showed increases in insulin after the start of the feeding period. During infusion, in contrast to fasting, there was an increase in pyruvate and lactate, but decreases in free fatty acids and ketone bodies. Benotti et al (1977) have shown improvement in hepatic function test values during intermittent intravenous feeding, in patients fed initially continuously followed by an identical period on cyclical TPN. In other studies, glucose and hypertonic amino acid infusion was stopped for 8-10 hours during the night, and blood and plasma metabolites were measured 3-4 hours after the suspension of the infusion. There was a decrease in glucose and an increase in insulin, but no change in fatty acids, alanine, lactate or ß-hydroxybutyrate levels (Maini et al , 1976). However, during the glucose-free period, patients received either 3% amino acids or oral protein (40-90 g / day). These investigators suggested that during the 'post-absorptive', or infusion-free period, stored energy such as fat and glycogen can meet the energy requirements and the infused amino acids can be incorporated and preferentially used for visceral protein synthesis, which suggests that Cyclical TPN is best for preserving visceral protein. The mobilization of nutrients during the post-absorptive period of intermittent intravenous feeding has been studied in children, using indirect calorimetry. Glycogen was used and lipid stores mobilised during the post absorptive period (Putet et al , 1984). It is interesting that, during cyclical TPN, patients may show marked losses of calcium in the urine and develop negative calcium balance as compared to patients on continous TPN. What implications this might have on the metabolic bone disease described on long-term home TPN patients requires further investigation (Wood et al , 1985). Essential fatty acid defficiency is a well known complication in patients receiving lipid-free TPN regimens (Wene et al , 1975; Riella et al , 1975; Gooodgame et al , 1978), but it may be corrected by either the inclusion of lipid in the TPN regimen (Elwyn, 1980) or, alternatively, by the cutaneous application of sunflower seed oil (Press et al , 1974). Mascioli et al (1979) have shown that cyclical TPN, possibly due to the periodical reduction of insulin levels stimulating fat mobilization, may be effective in preventing and treating fatty acid defficiency. Lanza-Jacoby et al (1985) demonstrated changes in the circadian rhythmicity of liver weight, and protein and glycogen content in rats fed with cyclical TPN. These rhythm patterns were similar in rats receiving cyclical TPN as compared to those of rats fed with intermittent enteral feeding. Matuchansky et al (1985) studied the effects of cyclical nocturnal TPN on circadian rhythms of blood lipids, lipoproteins and alipoproteins in six patients who were receiving cyclical intravenous feeding for at least a month. Although these rhythms may represent endogenous bioperiodic phenomena, nocturnal cyclical TPN was associated with the persistence of the circadian rhythms detected in control hospital patients fed 3 meals a day. Despite the increased interest in Cyclical TPN, it does not appear to be clear whether the changes in blood metabolites and plasma insulin, during the 12 hour infusion-free period, can enhance the development of a 'post-absorptive' period sufficient to produce the expected metabolic changes (Fig. 9.3.), to confirm Cyclical TPN as the more physiological form of intravenous feeding administration. Experimental Design To assess the contribution of sepsis to liver dysfunction during Total Parenteral Nutrition, a large number of parenterally fed patients with sepsis was studied. In addition, in an attempt to lower plasma insulin, and perhaps avoid some of the unwanted effects of Total Parenteral Nutrition on hepatic metabolism, two studies were designed. Each study was approved by the Ethics Committee of the John Radcliffe Hospital, Oxford, and an informed consent was obtained from each patient. STUDY ON LIVER DYSFUNCTION DEVELOPING IN PARENTERALLY FED PATIENTS DURING SEPSIS Although abnormalities of liver function developing in patients on TPN are common (Roberson et al , 1986), the time-course of the effect of sepsis on liver function tests in TPN patients requires clarification. Therefore, in order to evaluate how common altered liver function test values are, and how they progress during continuous Total Parenteral Nutrition and during sepsis, a survey was conducted of liver function tests in parenterally fed patients who were or were not septic during their TPN course. We have studied the serum albumin, bilirubin, aspartate transaminase (AST), and alkaline phosphatase concentrations in adults receiving TPN. The data were collected prospectively and the results analysed in two groups: nonseptic versus septic patients. Patients 285 patients referred to the Oxford TPN Team in the period between 1978 and 1986 were studied. Septic patients Included in the septic group were 126 patients with clinical evidence of sepsis, such as the discharge of pus from wounds or drainage tubes, or a pyrexia greater than 37.5°C with positive bacterial cultures from pus and blood. Many of the patients developed sepsis as a post-operative complication. Non-septic patients 159 patients without any clinical evidence of sepsis were compared to the septic group. Parenteral Nutrition Regimen Approximately 70% of the patients received the standard Oxford fat-based TPN regimen (Harper et al , 1983). The other patients either received glucose as their sole calorie source, or special regimens to meet their clinical situation, for example patients in renal failure (Grant & Todd, 1982). Measurements of liver function tests (LFTs) Liver function tests were measured three times a week during the first week on TPN, and twice a week thereafter. The mean of the results of LFTs measured during weeks 1,2,3&4 of intravenous feeding were analysed in the study. RANDOMIZED CONTROLLED STUDY ON STANDARD VERSUS TAILORED TOTAL PARENTERAL NUTRITION In this study the non-protein calorie intake was given either entirely as glucose, or glucose and fat. An "individualized" or a "standard" regimen was provided to those who had glucose as their sole calorie source. The results obtained from these patients were compared to those obtained from patients who received an equicaloric "standard" lipid TPN regimen containing fat, as half of the non-protein calorie source. Criteria for selection of patients and informed consent Patients refered to the Oxford TPN team, requiring intravenous feeding were randomly allocated to receive a standard glucose-based regimen, or a tailored glucose-based regimen, or the standard Oxford lipid-based TPN regimen (Harper et al , 1983). Patients with renal or liver impairment, patients with diabetes mellitus, or critically ill patients were excluded from the trial. All patients agreed to take part in the study after being informed on all the procedures involved, as well as, receiving a detailed explanatory note. Patients Fifty patients were studied. Table 9.1. & 9.2. give the details about their age, sex and TPN regimen received, and the main indications for TPN. Table 9.1. Patients fed on tailored or standard TPN regimens ____________________________________________________________ Nº of Patients Age Sex (M/F) TPN Regimen _____________________________________________________________ 8 11 59.0±5.1 46.9±7.3 4:4 7:4 31 51.3±6.4 12:19 Tailored Glucose Regimen Standard Glucose Regimen Standard Lipid Regimen Total = 50 _____________________________________________________________ Values for age expressed as Mean±S.E.M. _____________________________________________________________ Main indications for TPN _____________________________________________________________ Table 9.2. Nº of Patients ___________________________________________ Standard Glucose Tailored Glucose Standard Fat _____________________________________________________________ Inflammatory bowel disease 4 4 9 Post-operative complications 2 1 6 Injury 1 0 5 2 6 Other conditions 10 _____________________________________________________________________ __ Total Parenteral Nutrition Regimens 1. The Tailored Glucose Regimen was defined based on the estimated resting energy expenditure, calculated using the Harris-Benedict (1919) equation which takes into account the following variables: age, height, weight and sex (Table 9.3.). Table 9.3. The Harris-Benedict Equation __________________________________________________________ Daily REE for males = 66.4230 +013.7516 W + 5.0033 H - 6.7750 A Daily REE for females = 655.0955 + 9.5634 W + 1.8496 H - 4.6756 A __________________________________________________________ Where W = weight in Kg, H = height in cm, and A = age in years Although differences are found between the the "calculated" values and those obtained with indirect calorimetry using a "metabolic cart", the differences are not large enough to invalidate the equation, especially when the estimated values are corrected acording to the patients' clinical conditions (Elwyn, 1980; Stein, 1985). Therefore, patients received 1.10 (post-operative patients) or 1.25 (depleted patients) of their calculated Resting Energy Expenditure. The protein intake also was adjusted to either 160 or 250 mg/kg body wt./day. 2. The Standard Glucose Regimen consisted of: Nitrogen 14 g Glucose 500 g Sodium 100 mmol Potassium Chloride 100 mmol 124 mmol Calcium 14 mmol Phosphate 30 mmol Zinc Magnesium 0.2 mmol 19 mmol Folic acid 15 mg Fat emulsion 20% 100 g Vitamin and mineral supplements were given as described by Harper et al (1983). This standard glucose-based regimen supplied 4.2 MJ / 24 h (2000kcal / 24 h) of glucose (500 g). The glucose-amino acid solution was made up in sterile conditions under a laminar flow hood to volumes from 1.5 to 3 litres in a three litre bag. In order to avoid the development of essential fatty acid deficiency both standard and tailored glucose-based regimens included the infusion of one bottle of a 10% lipid emulsion, once a week, starting at the end of the first week of feeding. 3. The Standard Lipid Regimen (Oxford Regimen) differed from the Standard Glucose Regimen only in the non-nitrogen calorie source. Half the calories were provided as a 20% lipid emulsion (Fat - 100 g). This lipid-based regimen has been proved to be efficient in approximately 70% of patients who received TPN in Oxford over a three year period (Harper et al , 1983). Blood sampling and measurements Blood samples were taken at about 10 a.m. three times during the first week of feeding (Days 1,3,6), and twice a week thereafter (Days 10,14,18,etc.). Betwen 1 and 2 ml of blood were immediately deproteinized at the bedside in a previously weighed tube containing 5 ml of ice-cold perchloric acid (10% weight/volume), and used for metabolite determination. Pyruvate, acetoacetate, ßhydroxybutyrate, lactate, alanine and glucose were assayed using standard enzymatic methods in patients receiving both glucose-based regimens (For details see Chapter 2). Ketone bodies were measured in patients receiving the lipidbased regimen. Other measurements were those required for normal clinical management of TPN patients (Harper et al , 1983). Daily 24 h urine collection was performed to measure urinary urea, and total urinary nitrogen loss estimated (Lee & Hartley, 1975) using the formula: ___________________________________________________________ Urine urea (mmol/l) X 24 h urine volume (litres) X 0.028 X 6/5 = nitrogen (grams/24h) ___________________________________________________________ Blood samples were also analysed to determine liver function tests, including albumin, bilirubin, aspartate transaminase and alkaline phosphatase. CONTROLLED STUDY ON CONTINUOUS VERSUS CYCLICAL TPN In this study TPN was given initially continuously, followed by a period in which the nutritents were infused during the day only, and finally TPN was again offered continuously (Fig. 9.5). Changes in blood metabolites and plasma insulin were measured 3 hourly over 24 h. The aim of this study was therefore to assess how physiological Cyclical TPN is, in providing a 'postabsorptive' period and lowering plasma insulin during the infusion-free period, to study whether this might resolve some of the unwanted metabolic changes induced by TPN. Fig. 9.5. Criteria for selection of patients and informed consent: Patients who had received continuous TPN for not less than two weeks and required at least a further 7 days more TPN, who were clinically and metabolically stable, were included in the study. All patients agreed to take part in the study after being informed on all the procedures involved, as well as, receiving a detailed explanatory note. Patients Six patients were studied. Table 9.4. shows details about their age, sex and underlying condition. Table 9.4. Patients who received cyclical parenteral nutrition _____________________________________________________________ Patient Age Sex Diagnosis _____________________________________________________________ J.A. 64 F Ca of pancreas - Whipple operation G.S. 45 F Ca of oesophagus - oesophagogastrectomy B.W. 21 M R.T.A. - Multiple fractures G.S. 39 M Superior mesenteric artery thrombosis - Short gut V.G. 75 F Duodenal perforation + intestinal obstruction P.W. 44 W Crohn's Mean±S.E.M. 48±7.7 M/F=1 _____________________________________________________________ The study period was 7 days. On day one, patients remained on continuous TPN. On days 2-6 TPN was given during the day only (9A.M. to 9 P.M.), and on day 7 patients were fed again continuously. Blood sampling Blood was sampled 3-hourly on days 1,2,6,7 (total of 32 ml a day) and twice-a-day on days 3,4,5 (total of 8 ml a day) (Fig. 9.4.). The sequential blood samples every 3 hours over 48 h were taken via a cannula (Venflon G 18 - Viggo) inserted into a forearm vein under local anaesthesia, using a two syringe method. The cannula was securely taped and attached via an extension tubing to a 10 ml syringe with 5 ml of 0.9% sterile sodium chloride containing heparin (Hepsal 10units/ml). The dead space of the plastic cannula and the extension tubing was 1.2ml. Two ml of the dilute blood were therefore withdrawn and discarded before each blood sample was taken with a new syringe. The cannula system was flushed with Hepsal following each sampling to prevent blood clotting in the cannula or tubing, and an end-tap fitted to the extension tubing, which was therefore locked, bent into a loop, and taped to the forearm. Between 1 and 2 ml of blood were immediately deproteinized at the bedside in a previously weighed tube containing 5 ml of ice-cold perchloric acid (10% weight / volume), and used for metabolite determination. The remaining blood, from the 4 ml withdrawn in each sample, was immediately centrifuged at 5°C at 3000 rpm for 10 min to separate the plasma, which was then transferred to a new tube and stored at -20°C for the insulin determination. TPN Regimen All patients received the same sandard TPN Oxford regimen (Harper et al , 1983) already described. Measurements Measurements were those required for normal clinical management of TPN patients (Harper et al , 1983). Daily 24 h urine collection was performed to measure urinary urea, and total urinary nitrogen loss estimated using the Lee & Hartley (1975) equation, already described. Twice weekly blood samples were analysed to determine liver function tests, including albumin, bilirubin, AST and alkaline phosphatase. The deproteinized blood samples were used to determine the concentrations of pyruvate, acetoacetate, ß-hydroxybutyrate, lactate, glutamate, alanine and glucose using standard enzymatic methods (For details see Chapter 2). Plasma immunoreactive insulin was determined by a single anti-body technique using charcoal binding (Albano et al , 1972 - For details see Chapter 2). STATISTICAL ANALYSIS Results were expressed as the mean ± the standard error of the mean (S.E.M.) accompanied by the number of observations in parenthesis. The representation used for mean values, and the mathematical definition of S.E.M. are shown below: Results were analysed using the Mann-Whitney U Test. Results The effect of sepsis on liver function tests in patients receiving total parenteral nutrition A survey was undertanken of ill patients receiving TPN in order to assess the incidence of liver dysfunction induced not only by TPN but also the contribution from sepsis. The percentage of parenterally fed patients, who were septic or not, with altered serum albumin concentration is shown in Figs. 9.6. Fig. 9.6. Patients with abnormal albumin during sepsis and TPN 80 * Abnormal albumin (%) 60 ² ² Non-Septic Septic 40 * P < 0.01 versus non-septic 20 ² P < 0.01 versus week 1 0 1 2 3 4 Time of intravenous feeding (week) The administration of Total Parenteral Nutrition had a beneficial effect in nonseptic patients by decreasing, with time, the percentage of patients with abnormal (low) serum albumin concentration. More septic patients, however, remained with abnormal albumin levels, perhaps, reflecting a higher degree of malnutrition in these patients (Young & Hill, 1978), or the fact that severe malnutrition can be perpertuated by sepsis (see Chapter 1), despite nutritional support. The percentage of patients with abnormal serum bilirubin in shown in Figs. 9.7. Fig. 9.7. Patients with abnormal bilirubin during sepsis and TPN 50 Abnormal bilirubin (%) 40 * 30 Non-septic Septic 20 P < 0.01 versus non-septic 10 0 1 1 Week 2 2 Week 3 3 Week 4 4 Week Time of intravenous feeding (week) The percentage of non-septic patients with abnormal serum bilirubin increased with time. More septic patients than non-septic patients presented abnormal bilirubin during the first week of TPN. The percentage of septic patients on parenteral nutrition who presented with abnormal serum alkaline concentrations is illustrated on Fig. 9.8. Fig. 9.8. Patients with abnormal alkaline phosphatase during sepsis and TPN 100 ² Abnormal alk. phosph. (%) 80 ² * Non-septic Septic 60 * 40 * P < 0.01 versus non-septic ² P < 0.01 versus week 1 20 0 1 1 Week 2 2 Week 3 3 Week Time of intravenous feeding (week) 4 4 Week The proportion of both non-septic and septic parenterally fed patients with abnormal alkaline phosphatase (elevated) levels, increased with time during the first two weeks of feeding. More septic patients had raised alkaline phosphatase than non-septic patients. However, as more non-septic patients developed abnormal alkaline phosphatase levels with time, the percentage of patients with this abnormality converged at the fourth and fifth week. This suggests sepsis appears to promote the changes in alkaline phosphate at an earlier stage than TPN alone. The changes in the proportion of parenterally fed patients with serum aspartate transaminase alterations, in the presence or absence of sepsis, is shown in Fig. 9.9. Fig. 9.9. The proportion of septic patients receiving TPN with altered serum asparate transaminase was higher than that of non-septic patients only in the second week of intravenous feeding. Tailored Glucose versus Standard Glucose TPN Regimen Patients who received the tailored glucose regimen required slightly longer period of feeding (21.5±6.21 days) than those who either received the standard glucose TPN (17±2.42 days) or standard fat TPN (16.4±4.1 days Mean±S.E.M.). The nitrogen and caloric intake are presented in Table 9.5 . Nitrogen and energy intake ___________________________________________________________ Table 9.5. ___ Nitrogen(g / d) Glucose(g /d) Kcal (d) Kcal / N ___________________________________________________________ ___ Glucose Tailored TPN * 12.28±1.10 375.2±26.21 1506.4±64.7 126.3±11.1 Glucose Standard TPN 14 500 2000 Lipid Standard TPN ** 14 250 2000 142 142 ______________________________________________________________________ ___ * Results expressed as Mean value±S.E.M. ; ** In order to provide 2000 kcal/day , the other half the non-nitrogen calories (1000 kcal/day) were offered as fat. ______________________________________________________________________ _ Table 9.6. gives the same data according to the body weight at the start of the TPN course. Nitrogen and energy intake according to the body weight ______________________________________________________________ Table 9.6. Nitrogen (g/kg/d) Glucose(g/kg/d) Energy (kcal/kg/d) ______________________________________________________________ Glucose Tailored TPN 0.232±0.017 6.84±0.411 27.3±1.65 Glucose Standard TPN 0.424±0.165 8.93±0.568 * 37.7±2.27 * ______________________________________________________________________ ___ * P < 0.05 ; compared to glucose tailored TPN Patients who received the individualized glucose TPN regimen therefore received significantly less glucose and calories than those who were on the standard glucose regimen (Table 9.6). Some of the patients receiving glucose as their sole non-nitrogen caloric source required exogenous insulin at some stage of their feeding (Tailored Glucose TPN - 3 / 8 (37.5%); Standard Glucose TPN - 3 / 11 (27.2%). This compares unfavourably with only 9% in patients fed a standard lipid regimen requiring insulin supplementation (Harper et al , 1983). Providing tailored TPN did not cause appreciable change in the concentration of blood glucose, lactate, pyruvate, alanine and ketone bodies (Figs 9.10., 9.11. and 9.12.). There was no appreciable day to day difference in the results for each day. Therefore, data are presented as the mean for each day studied (Day 1+3+6+10+14). Results from patients receiving exogenous insulin were excluded. Fig. 9.10. Standard versus Tailored TPN- Blood Metabolites 6 µmol/ml whole blood 5 (24) (16) 4 Standard TPN Glucose Tailored TPN Glucose 3 2 (16) (23) 1 0 Glucose Lactate X + S.E.M. ( ) Nº of observations Blood alanine concentrations in patients receiving an individualized regimen were slightly higher than those of patients receiving the standard glucose regimen, perhaps reflecting the lower energy / nitrogen ratio in tailored glucose patients (Table 9.5.). Fig. 9.11. Standard versus Tailored TPN - Blood Metabolites 0.5 (16) µmol/ml whole blood 0.4 (24) 0.3 Standard TPN Glucose Tailored TPN Glucose 0.2 (23) (16) 0.1 X + S.E.M. ( ) Nº of observations 0.0 Alanine Pyruvate Ketone body concentrations were also compared to those in patients receiving the standard lipid regimen (Fig.9.12.) Fig. 9. 12. Standard versus Tailored TPN- Blood ketone bodies (98) µmol/ml whole blood 0.10 0.08 0.06 (16) Standard GGlucose Tailored GGlucose Standard Fat (23) 0.04 X + S.E.M. ( ) Nº of observations 0.02 0.00 Ketone bodies Providing half the non-nitrogen calories as fat increased ketone body concentration, however, the difference did not reach statistical significance. The fact that the ketone body levels in the lipid fed patients remained low was probably due to the high carbohydrate load increasing plasma insulin, which in turn, inhibited lipolysis and reduced the availability of fatty acids , the ketone body precursors for the liver. Standard versus Tailored TPN - Effect on Nitrogen Balance Varying the non-protein caloric intake or individualizing the TPN regimen did not produce any significant change in nitrogen balance, which was positive overall (Fig. 9.13.). Patients receiving the higher carbohydrate intake had slightly higher nitrogen retention. Fig. 9.13. Tailored versus Standard TPN - Effect on Nitrogen Balance 6 Nitrogen retained (g/day) 5 (34) Standard Glucose G Taylored Tailored glucose Standard Standard Lipid Fat (45) 4 (91) 3 X + S.E.M. ( ) Nº of observations 2 1 0 Nitrogen Balanc Continuous versus Cyclical Parenteral Nutrition - Changes in blood metabolites and plasma insulin over 24 h Delivering TPN over 12 h during the day only (Cyclical TPN) produced small changes in blood glucose concentration (Fig. 9.14.) which were not statistically significant. Fig. 9.14. None of the patients had glycosuria and, therefore, did not require exogenous insulin. Blood glucose concentrations were variable during the day and reached a steady plateau during the night on continuous feeding. Blood glucose concentrations fell during the infusion-free period of Cyclical TPN, but this fall was not statistically significant. Similar results have been reported by Byrne et al (1981). During Cyclical Parenteral Nutrition, blood concentrations of pyruvate were similar to those during Continuous TPN. The provision of a 'post-absorptive period' had no detectable effect on venous pyruvate concentration (Fig. 9.15.). Fig. 9.15. Increases in blood pyruvate concentration during the infusion period of two children receiving Cyclical TPN have been reported by Recour et al (1980). Blood glutamate was measured but no change in concentration was observed during Cyclical TPN as compared to Continuous TPN (Fig. 9.16.) Fig. 9.16. Blood lactate concentration changes over 24 h of Cyclical versus Continuous TPN are illustrated in Fig. 9.17. Lactate concentrations were slightly higher during Continuous TPN compared to those during the infusion-free period (Cyclical TPN OFF) but again the differences were not statisticaly significantly significant. Fig. 9.17. Maini et al (1976) found no change in blood lactate during a 8-10 hour glucosefree period, whereas Recour et al (1980) reported increased blood lactate concentration during the the infusion period in two children receiving Cyclical TPN. Changes in alanine concentration during 24 h are shown in Fig. 9.18. Fig. 9.18. Blood alanine levels were generally slightly higher during the infusion period and fell during the post-absorptive period of Cyclical TPN, but this fall was not statistically significant. Alanine concentration remained stable during Continuous TPN. The changes in plasma insulin concentration, over 24 h, induced by offering TPN only during the day are shown on Fig. 9.19. Fig. 9.19. Plasma insulin concentration was fairly uniform during Continuous TPN. During the feeding period of Cyclical TPN, when the infusion of nutrients doubles, insulin concentration increased. During the 'post-absorptive' period insulin concentrations decreased to concentrations as low as those found after an overnight fast (5 mU/l plasma - Record et al , 1973). Ketone bodies remained very low at both Continuous and Cyclical TPN (Fig. 9.20.). The decrease in insulin levels during the infusion-free period of Cyclical TPN had no effect on blood ketone body concentration. However, during the feeding period of Cyclical TPN, when the the rate of infusion is doubled as compared to that of Continuous TPN, ketone body concentrations were slightly lower than those found during Continuous TPN, although the differences were not statistically significant. Fig. 9.20. Small numbers and marked individual variability in the metabolic response tends to mask the overall effects of cyclical TPN. Therefore, all the data from the "infusion" or "infusion free" period were aggregated and compared with the aggregated data for continuous TPN. Pyruvate and glutamate concentrations did not change (Figs. 9.21. & 9.22). Fig. 9.21. Fig. 9.22. Lactate concentrations, however, were significantly decreased by promoting a 'post-absorptive' period during Cyclical TPN (Fig. 9.23.). Fig.9.23. Blood alanine concentration increased when the rate of infusion was doubled, during the feeding period of Cyclical TPN. The 'post-absorptive' period decreased alanine concentrations (Fig. 9.24.). Similar increases in amino acid concentration, by a factor of 1.5 with refeeding, have been reported (Newshome & Leech, 1983). Fig. 9.24. Blood glucose concentrations remained unchanged during the feeding period of Cyclical TPN, however, they decreased significantly during the 'postabsorptive' period, but not sufficiently to induce hypoglycaemia (Fig. 9.25.). Fig. 9.25. Plasma insulin concentrations increased significantly during the 'feeding period' of Cyclical TPN, compared to those during Continuous TPN. On the other hand they decreased dramatically in the infusion-free period (Fig. 9.26.). Fig. 9.26. Similar responses of insulin concentration to continuous TPN infusion (Dudrick et al , 1970; Sanderson & Dietel, 1974; Byrne et al , 1981) and to intermittent intravenous feeding (Maini et al , 1976; Byrne et al , 1981) have been reported. The decrease in plasma insulin during the 'post-absorptive' period of Cyclical TPN did not produce any appreciable change in the very low concentrations of blood ketone bodies (Fig. 9.27.) Fig. 9.27. Urea excretion and nitrogen balance remained unchanged by Cyclical TPN compared to Continuous TPN (Fig. 9.28.). Fig. 9.28. Continuous versus Cyclical TPN - Effect on Nitrogen Balance 2.4 (16) Nitrogen retained (g/day) 2.0 (13) Continuous TPN Cyclical TPN 1.6 1.2 0.8 X + S.E.M. ( ) Nº of observation 0.4 0.0 Nitrogen Changes in liver function during standard versus tailored Total Parenteral Nutrition Decreasing the carbohydrate intake by tailoring TPN did not produce an appreciable effect on the concentrations of albumin or bilirubin (Fig. 9.29. & 9.30.). Fig.9.29. Fig. 9.30. The bilirubin concentrations increased with time, and, in patients receiving the standard lipid regimen were always slightly higher than those of patients on the glucose-based regimens, but these differences never reached statistical significance. Tailoring TPN also did not alter the concentrations of alkaline phosphatase and aspartate transaminase (Figs. 9.31. & 9.32.). Fig. 9.31. Fig. 9.32. The same data are presented in a composite form in Table 9.7. Table 9.7. Tailored versus Standard TPN - Effects on liver function tests ___________________________________________________________ Results expressed as Mean±S.E.M. with number of observations in parenthesis. ___________________________________________________________ Glucose Tailored Glucose Standard Lipid Standard ______________________________________________________________________ Albumin (g / l) 31.1±0.52 (70) 31.0±1.0 (49) 29.5±0.61 (92) Bilirubin (µmol / ml) 11.8±1.1 (64) 8.8±0.70 (49) 19.7±3.0 (90) Alkaline phosphatase (IU / l) 300±25.5 (63) 377±37.3 (50) 228±11.1 (87) Aspartate transaminase (IU / l) 27.9±2.1 (58) 49.9±8.8 (50) 46.3±8.5 (87) ______________________________________________________________________ Changes in liver function during continuous versus cyclical Total Parenteral Nutrition Liver function test values obtained from patients whilst on Continuous TPN were compared to those when they were on Cyclical TPN. No statistically significant changes were observed (Table 9.8.). Table 9.8. Continuous versus Cyclical TPN - Effects on liver function ___________________________________________________________ Results expressed as Mean±S.E.M. with number of observations in parenthesis. ___________________________________________________________ Continuous TPN Cyclical TPN ___________________________________________________________ Albumin (g / l ) 32.6±1.9 (15) 36.2±1.3 (8) Bilirubin (µmol / ml) 17.2±2.4 (21) 9.6±1.3 (10) Alkaline phosphatase (IU /l) 411±38.6 (23) 316±51.7 (9) Aspartate transaminase (IU / l) 28.9±3.9 (20) 40.0±10.1 (9) ___________________________________________________________ _ Discussion Most patients can handle fairly large quantities of glucose, fat and amino acids infused intravenously, and retain nitrogen, maintain or gain weight and avoid glycosuria (Harper et al , 1983). In the present study, patients receiving glucose or glucose and fat as their source of non-nitrogen calories, also retained nitrogen and demonstrated a positive nitrogen balance (Fig. 9.13.). Nevertheless, parenteral nutrition is invasive and unphysiological and not surprisingly, therefore, the literature abounds with the problems caused by this form of nutrition (Grant & Todd, 1982; Silk, 1983; Philips & Odgers, 1986). Perhaps hepatic dysfunction with altered biochemical liver function tests (LFTs) is the commonest recorded metabolic complication of intravenous nutrition (Robertson et al ,1986). There is also a common association between sepsis and abnormal liver function tests (Royle & Kettlewell, 1980). In the present survey, the proportion of septic patients with low albumin concentrations at the start of their parenteral nutrition course was similar to that of non-septic patients, but significantly more septic patients had abnormal bilirubin concentrations over the first week of intravenous feeding (Figs. 9.6.& 9.7). TPN caused a progressive increase, with time, in the proportion of patients with alkaline phosphatase abnormalities in both septic and non-septic patients. However a significantly higher proportion of septic patients had raised alkaline phosphatase levels in the early stages of TPN, when compared to the non-septic patients (Fig. 9.9.). Modulating the carbohydrate intake of parenterally fed patients did not prevent the alterations in hepatic liver function tests (Table 9.6.), such as progressive rises in liver enzymes (Harper et al , 1983; Robertson et al , 1986), indicating that there was no advantage in decreasing the glucose load (approximately 25% in patients receiving individualized glucose prescriptions Tables 9.5. & 9.6.; and 50% in patients receiving half their non-protein calories as fat) in this respect. Offering a 'post-absorptive' period during Cyclical TPN also did not alter liver function tests (Table 9.8.), compared to those measured during Continuous TPN. Possibly the period of Cyclical TPN was too short (5 days) to promote such changes. Benotti et al (1976) claim to have improved liver function tests of patients who were receiving continuous TPN for more than 3 weeks, and were converted to cyclical TPN for a further 3 weeks. Against this view is the fact that alterations in liver function tests are also reported in home TPN patients (Müller et al , 1982). Furthermore, other factors have been implicated in these hepatic alterations, including nitrogen sources, the lack of any oral intake, intestinal overgrowth by bacteria, sepsis, and additives used in the preparation of some intravenous regimens (Robertson et al , 1986). Decreasing the carbohydrate load, by tailoring the TPN regimen, caused no changes in blood metabolites, particularly glucose, lactate, pyruvate, alanine, and ketone bodies. This absence of a rise in ketone body concentrations may be due to inhibition of lypolysis by the hyperinsulinaemia induced by the carbohydrate load given to tailored TPN patients. Urea excretion and nitrogen balance were not significantly different with any of the forms of TPN studied (Fig. 9.13. & 9.28.). No tapering off period was used when stopping the infusion in Cyclical TPN patients for this study. One episode of rebound hypoglycaemia was observed, which has been described by others (Dudrick et al , 1970; Sanderson & Dietel, 1974). However, in a recent study involving 48 patients, Wagman et al (1986) reached the conclusion that acute discontinuation of TPN infusion was a fairly safe procedure. The faster infusion of nutrients during cyclical TPN (12 hours - feeding period) did not induce hyperglycaemia because plasma insulin levels rose "pari passu", which in turn, illustrates a normal insulin secretory response to the glucose load in these patients. This elevation in plasma insulin during the 12hinfusion period of Cyclical TPN was accompanied by an increase in blood alanine concentration (Fig. 9.24.) to values comparable with those measured in the fed state (400 µM) (Renold et al , 1978). In addition substrate cycles, such as the glucose/glucose 6-phosphate and glycogen/glucose 1-phosphate cycle in the liver, the protein/amino acid cycle in muscle, and the triacylglycerol/fatty acid cycle in adipose tissue, may have a role during the absorptive period in amplifying the sensitivity to small changes in concentrations of fuels and hormones (Newsholme & Leech, 1983). The promotion of a 'post-absorptive' period during the 12 h infusion-free Cyclical TPN is supported by the decreases in plasma insulin and in the blood concentrations of lactate, alanine and glucose, to levels similar to those found after an overnight fast, such as the values measured in 21 normal volunteers fasted overnight, used as controls in a metabolic study on glucose tolerance by Record et al (1973). A comparison between the metabolic data published in the latter study and those measured in this present work is shown in Table 9.9. Cyclical Total Parenteral Nutrition, therefore, promotes a 'post-absorptive' period, and therefore appears more physiological than Continuous Parenteral Nutrition in terms of substrate metabolism. In addition it allows more mobility for patients without, apparently, sacrificing efficacy. Although Cyclical TPN patients presented a normal insulin secretory response to the intermittent infusion of nutrients, about 30% of our patients receiving TPN glucose-based regimens required exogenous insulin. demonstrates the glucose intolerance and the This resistance to the action of insulin which can occur in parenterally fed patients (Fig. 9.14.; Fig. .9.21. - Dudrick et al , 1970; Sanderson et al , 1974). Table 9.9. A comparison between blood metabolite values obtained during the 'post- absorptive' Cyclical TPN ("OFF" or infusion free period) and those found in normal controls after an overnight fast. _____________________________________________________________ Results expressed as Mean±S.E.M. . * Data extracted from Record et al , 1973. __________________________________________________________ ___ Cyclical TPN "OFF" Overnight fast * __________________________________________________________ ___ Insulin (µUI / ml ) 10.7±0.91 (50) 6.4±0.5 (21) Lactate (µmol / ml ) 0.82±0.03 (54) 0.72±0.04 (21) Pyruvate (µmol / ml ) 0.091±0.003 (52) 0.072±0.005 (21) Ketone bodies (µmol / ml ) 0.053±0.006 (56) 0.057±0.004 (21) __________________________________________________________ ____ Despite some controversy, hyperinsulinaemia has been implicated in the development of insulin resistance in man, probably causing a defect at a postbinding site (Rizza et al , 1985). This view has been supported in a recent study (Marangou et al , 1986) involving normal volunteers subjected to a 20 h insulin infusion in order to promote a moderate sustained hyperinsulinaemia (30mUI / l). This level of hyperinsulinaemia appears to be within the range of that observed in insulin-resistant states, such as in sepsis (Fig. 3.5. - Chapter 3; Clemens et al , 1982), injury (Black et al , 1982), chronic renal failure (DeFronzo et al , 1981), obesity (Kolterman et al , 1980), type II diabetes (DeFronzo et al , 1982), cirrhosis (Proietto et al , 1980), in patients with insulinoma (Bar et al , 1977), and patients receiving Continuous TPN (Fig. 9.26. - Maini et al , 1976; Wagman et al , 1986). This moderate hyperinsulinaemic state led to a decrease in insulin action, despite the presence of normal monocyte-insulin receptor binding, and normal insulin secretion Therefore, if the binding of insulin to monocytes reflects insulin binding in key insulin-sensitive tissues, this impairment in insulin action probably involves a post-receptor mechanism (Crettaz & Jeanrenaud, 1980). A defect at the post-receptor level has also been suggested as the cause of insulin resistance in injured patients (Black et al , 1982). By contrast, Byrne et al (1981) studying parenterally fed patients with gastrointestinal disorders, also showed no alteration in insulin binding to circulating monocytes in patients receiving either 17 hours/day or continuous TPN infusions, but, in a group of patients receiving Cyclical TPN (over 12 hours), total insulin-monocyte binding was significantly diminished. This was also accompanied by a decrease in high-affinity receptors, possibly "down-regulating" the response to hyperinsulinaemia with each 12 h-TPN infusion. These changes in insulin receptors (Kahn, 1980) could, therefore, help to explain why some patients develop carbohydrate intolerance. They might also produce a protective effect from post-infusion hypoglycaemia. The importance of the role of insulin in this adaptation to the infusion, and cessation of nutrients is further supported by the fact that there were no apparent changes in counter regulatory hormones in these studies (Byrne et al , 1981). The resistance to insulin action may also be regarded as yet another common metabolic feature between sepsis and total parenteral nutrition. Further investigation on the role of hyperinsulinaemia in promoting metabolic changes during Total Parenteral Nutrition, especially in the ill septic patient, is therefore required. 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