1. Metabolism

2. Overview of metabolism most vertebrates eat periodically during absorption monosaccharides, amino acids, lipoproteins are present in the blood in high concentration the problem is storage: e.g. glucose appears in the urine above a blood sugar concentration of 200 mg% (11 mmol/l) importance of the hepatic portal circulation between meals the problem is mobilization some cells can store nutritients others rely on the blood supply (e.g. neurons, blood cells) liver (glycogen) and adipose tissue (triglycerides) store nutritients for the whole body muscle cells store for themselves (glycogen) these tissues have decisive role in regulation transport nutritients: glucose, free fatty acids (FFA), ketone bodies, amino acids – these substances are decisive in regulation 2/34

3. Regulation of metabolism regulation targets key enzymes selecting between alternate routes enzymes are regulated partly by metabolites. partly by hormones in mobilization period appropriate level of glucose is very important as neurons can only use this nutrients (after a longer fasting, ketone bodies as well) therefore, concentration should be kept between narrow limits: minimum 4,5 – 5 mmol/l, maximum 9-10 mmol/l in this regulation, hormones of the pancreatic islets of Langerhans, insulin and glucagon, are the most important glucose can enter several different metabolic pathways 3/34

4. Membrane transport of glucose glucose enters enterocytes and renal epithelial cells through indirect active transport (Na + co-transporter) through the basolateral membrane and into other cells it is transported by facilitated diffusion GLUT family 12TM transporter proteins: –GLUT 1 – blood-brain-barrier endothelium, red blood cells – high affinity, independent from insulin –GLUT 2 – basolateral membrane of enterocytes and renal epithelial cells, hepatic cells, B-cells in pancreas – low affinity, independent from insulin –GLUT 3 – neurons, liver cells – independent from insulin –GLUT 4 – muscles and adipose tissue – dependent on insulin –GLUT 5 – fructose transporter –GLUT 6 – ??? 4/34

5. Glucose metabolism I. transported glucose is transformed into glucose- 6-phosphate (using ATP) inside the cell – cannot diffuse - strong concentration gradient different enzyme in the other direction (glucose- 6-phosphatase) yielding glucose and P – no such enzyme in the muscle – no glucose release glucose-6-phosphate can be reversibly transformed to glucose-1-phosphate – with UTP forms UDP-glucose – glycogen synthesis different enzyme in the other direction using inorganic P and yielding glucose-1-phosphate glucose-6-phosphate can be reversibly transformed also to fructose-6-phosphate, both can enter pentose phosphate cycle yielding NADPH, or in the glycolysis 5/34

6. Glucose metabolism II. fructose-6-phosphate is transformed using ATP to fructose-1,6-diphosphate (phosphofructo- kinase) – enters into glycolysis – reaction is facilitated by ADP, AMP, P, inhibited by ATP, citrate, fatty acids different enzyme in the other direction (fructose-1,6-diphosphatase), yielding inorganic P – last but one step of gluconeogenesis – reaction is facilitated by ATP, citrate, fatty acids, inhibited by ADP, AMP, P glycolysis runs in the cytoplasm down to pyruvate pyruvate enters mitochondrion if O 2 is available – citrate cycle (in matrix), terminal oxidation (inner membrane) – 38 ATP/glucose if no O 2 is available, pyruvate is transformed to lactic acid using up NADH – 2 ATP/glucose after intense physical exercise, lactic acid is transported to the liver and synthesized to glucose (Cori-cycle) – energy consuming process – oxygen debt 6/34

7. Gluconeogenesis gluconeogenesis means synthesis of glucose during fasting nervous system needs glucose - it is produced by gluconeogenesis from amino acids gluconeogenesis is also important in turning accumulated lactic acid into glucose there are 3 irreversible steps in glycolysis: formation of glucose-6-phosphate, fructose- 1,6-diphosphate and pyruvate   first two are reversed by dephosphorylation – see above phosphoenolpyruvate synthesis from pyruvate through oxaloacetate gluconeogenesis cannot use acetyl-CoA, thus fatty acids as two CO 2 are released in the citrate cycle before it runs to oxaloacetate glucogenic and ketogenic amino acids 7/34 Alberts et al.: Molecular biology of the cell, Garland Inc., N.Y., London 1989, Fig. 2-38.

8. Transformations of glucose glu glu-6-P ATPP GLUT transporter glu-1-PUDP-glu UTP P glycogenfru-6-Pfru-1,6-P ATP pentose-P cycle glycolysis 8/34

9. Lipid metabolism absorbed lipids are transported as lipoproteins in the circulation lipoproteins are also synthesized by the liver and the enterocytes between absorptive phases using building blocks in the blood in the endothelium of capillaries lipoprotein lipase enzyme is located cutting off free fatty acids from triglycerides – easily enter the cells in the mitochondria β-oxidation – NADH, acetyl-CoA are formed synthesis in the ER; acetyl-CoA exits the mitochondria as citrate and forms acetyl-CoA again acetyl-CoA enters the cyclic synthesis as malonyl-CoA - NADPH is also necessary fatty acids form triglycerides with glycerol-1- phosphate coming from the glycolysis acetyl-CoA can be used to form ketone bodies 9/34

10. Islets of Langerhans pancreas is 70-80 g, 1-2% of the gland gives the 1-2 million islands   50-300 cells/island A, B, D, F cells B-cells forming groups surrounded by A-, and D-cells interaction through paracrine means and through the local circulation A-cells: 20-25%, producing glucagon B-cells: 60-75%, producing insulin D-cells: 10%, producing somatostatin F-cells: ?, producing pancreatic peptide (?) 10/34 Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 11-2. B cell D cell A cell

11. Regulation of insulin production insulin is synthesized as preproinsulin (signal + proinsulin) on the rough ER signal is cleaved off, proinsulin is packaged into vesicles in Golgi – C-peptide is removed, A and B chains remain connected by 2 disulfide bridges   stored in the vesicles, it is released by exocytosis (Ca ++ ) when needed facilitatory effects: –increase of blood glucose level – transported in by GLUT-2 – producing ATP in glycolysis – ATP closes K + channel – depolarization – Ca ++ enters the cell –amino acids (arginine, leucine, lysine) –vagal effect – sweet taste in the mouth –gut hormones (incretins: GIP, CCK) inhibitory effects: –somatostatin –sympathetic effect through α 2 -receptors – hyperglycemia in stress is not diminished by insulin 11/34 Berne and Levy, Mosby Year Book Inc, 1993, Fig. 46-3

12. Insulin secretion B-cell incretins CCK, GIP hyper- glycemia amino acids (arg, leu, lys) vagal stimuli α 2 -receptor somato- statin NA Adr proinsulin gene mRNA proinsulin insulin 12/34

13. Details of glucose effect B-cell glucose GLUT-2 ATP K + -channel Ca + + -channel Ca ++ pyruvate glucose-6-P proinsulin gene mRNA proinsulin insulin vesicles 13/34

14. Insulin effects I. binds to tyrosine-kinase receptors   autophosphorylation, then phosphorylation of other proteins – termination through internalization response types (depending on the given cell): –GLUT-4 is added to the membrane from storage vesicles (adipose and muscle cells) – intake of glucose increases several fold –phosphorylation and dephosphorylation of enzymes – e.g. activation of phosphodiesterase, thus blockade of the effect of various hormones acting through cAMP: glucagon, catecholamines, etc. –modulation of gene expression, e.g. inhibition of proglucagon transcription in A-cells insulin facilitates synthetic processes, decreases the level of transport nutritients (glucose, FFA, ketone bodies, amino acids) inhibits the effect of hormones promoting catabolism 14/34

15. Insulin effects II. effects on liver cells –glycogen synthesis increases –glycogenolysis decreases –gluconeogenesis decreases –synthesis of fatty acids increases – triglycerides are transported in the circulatory system bound to lipoproteins –production of ketone bodies decreases effects on muscle cells –glucose uptake increases –glycogen synthesis increases –glycogenolysis decreases –amino acid uptake and protein synthesis increases –K+ uptake increases – cause is unknown effects on adipose cells –glucose uptake increases – glycerol is available for triglyceride synthesis –amount of lipoprotein lipase increases – FFA uptake – triglyceride synthesis increases –lipolysis (facilitated by cAMP) is inhibited 15/34

16. Insulin effect in adipose cells capillary adipose cell lipoprotein lipase FFA LDL GLUT-4 glucose FFA glycerol-1-P trigliceride β-receptor lipase cAMP AMP phospho- diesterase FFA + glycerol 16/34

17. Regulation of glucagon production proglucagon is a member of the secretin family produced by A-cells in the pancreas and in the alimentary canal it is not known whether the latter has glucagon effect in humans, but in dogs it does (see classic experiment of Best and Banting) inhibitory effects: –high glucose level –insulin by inhibiting the transcription of the proglucagon gene –somatostatin facilitatory effects: –arginine, and to some extent other amino acids as well – after a protein-rich meal hypoglycemia might develop as insulin secretion is increased – sweetness after a large meal –stress reaction – catecholamines, growth hormone, glucocorticoids – the role of the latter is permissive, enabling proglucagon transcription 17/34

18. Glucagon production A-cell proglucagon gene mRNA proglucagon glucagon catecholamines hGH glucocorticoids (permissive) insulin receptor insulin GLUT-4 amino acids (arg) glucose somato- statin 18/34

19. Glucagon effects all important effects of glucagon influence liver cells through cAMP and PKA –glycogenolysis increases –gluconeogenesis increases –glucose release increases –synthesis of ketone bodies increases insulin antagonizes all effects (enhanced degradation of cAMP) outcome depends on the ratio of the two hormones gluconeogenesis and ketogenesis require substrates (amino acids and fatty acids) – these are provided from the muscles and adipose tissue by the low insulin level 19/34

20. Hormonal background of fasting following the absorptive phase tissues and organs have to rely on stored energy not all cells and tissues have their own stores brain is unique as it can only use glucose until the level of ketone bodies is not very high brain uses 6 g glucose/hour – glucose stores of liver would not last for long - gluconeogenesis maximal tolerable length of fasting depends on how long gluconeogenesis can continue and how long triglyceride stores can provide energy for circulation, respiration, and renal functions adaptation requires: –decrease of insulin/glucagon ratio –presence of growth hormone (STH/GH) – reason? –presence of glucocorticoids (cortisol) – synthesis of enzymes for gluconeogenesis, lipolysis, secretion of glucagon – permissive role 20/34

21. Phases of fasting I. most of the recent data concerning metabolic changes during fasting have been obtained in patients undergoing drastic diet protocols (null- calorie) in the 60’s-70’s – following unexplainable fatal cases this method was discontinued post-absorptive state – max. 24 hours, occurs every day –insulin level decreases, glucagon slightly increases –blood sugar level is maintained by glycogenolysis in the liver (75%), and by gluconeogenesis (25%) using lactic acid, glycerol and some amino acids –glucose consumption decreases in tissues capable to use other nutritients, FFA and glycerol release from the adipose tissue increases – muscle cells use that 21/34

22. Phases of fasting II. short-term fasting – 24-72 hours –insulin level decreases even further, glucagon and GH concentration increases because of the low blood sugar level caused by the depletion of glycogen stores in the liver –gluconeogenesis increases using amino acids mostly from the muscles – N-excretion is rising –lipolysis increases (low insulin level, GH), most cells (but not nerve and blood cells) are using fatty acids, ketogenesis increases in the liver, muscles are burning ketone bodies chronic fasting – after 72 hours –insulin/glucagon ratio decreases further, GH increases, lipolysis, ketogenesis is enhanced –total energy consumption decreases (inactivity, decrease of thyroid activity), brain can use ketone bodies now, glucose demand decreases, proteolysis decreases – life can go on for weeks 22/34

23. Stress state stress state means the collection of the reactions of the body to various challenges catabolic state similarly to fasting, but blood sugar level is high: glycogenolysis, lipolysis gluconeogenesis a further difference is the high sympathetic activation, the increased catecholamine synthesis and glucocorticoid secretion in the adrenal gland (cortex and medulla, respectively) catecholamines inhibit insulin and enhance glucagon secretion; increase glycogenolysis, gluconeogenesis and ketogenesis in the liver, as well as lipolysis in the adipose tissue glycogenolysis in the muscles might increase lactic acid release, facilitating gluconeogenesis 23/34

24. Diabetes mellitus diabetes mellitus (mellitus=sweet as honey) – the court physician of Charles I. tasted the urine of a patient and found it sweet – this method was used for a long time in patients comatose for unknown reasons 1920 – Banting and Best induced diabetes in dogs by removing the pancreas, then alleviated the symptoms with pancreas extraction 1922 – successful trial in a diabetic child 1923 – Nobel-prize for Banting and McLoed… classical method in physiology: lesion + replacement this was the first identified hormone and hormone effect type I diabetes (juvenile) – lack of insulin type II diabetes – heterogeneous, unknown mechanism, insulin is present 24/34

25. Type I diabetes mellitus B-cells are destroyed by autoimmune reaction first antibodies only, no symptoms, later decreased glucose tolerance, then endogenous hyperglycemia in insulin sensitive tissues (muscles, adipose tissue) no glucose uptake, overproduction of glucagon glycogenolysis, gluconeogenesis, lipolysis, ketogenesis, lipemia (liver is synthesizing lipoproteins, but lipoprotein-lipase level is low glycosuria, osmotic diuresis, NaCl and water excretion, polyuria, polydipsia, dehydration, hematocrit increases, circulation deteriorates, hypoxia ketoacidosis – hyperventilation, loss of water, diabetic coma 25/34

26. Symptoms of diabetes direct effect Lack of insulin gluco- neogenesis hyperglycemia glucosuria osmotic diuresis dehydration circulatory insufficiency brain hypoxia coma glucose use hyperosmolarity overproduction of glucagon lipolízis ketogenesis ketoacidosis hyperventilation vomiting ketonuria 26/34

27. Type II diabetes mellitus insulin is usually present heterogeneous causes, not well-known exogenous and endogenous hyperglycemia is characteristic in some patients lack of insulin receptor or resistance against insulin insulin secretion sometimes can be induced with arginine, but not with glucose – lack of GLUT 2 transporter no glucagon inhibition in most cases – symptoms are strengthened by hyperglucagonemia some patients are obese, others not relatively benign, but might cause complications: atherosclerosis, myocardial infraction, blindness, renal insufficiency in the USA-ban 3-5% of whites are diabetic, in 80% type II 27/34

28. Energy turnover I. there are some related terms that should not be confused turnover of materials indicates chemical transformations and reactions only; these changes are accompanied by changes in energy – turnover of energy turnover of materials and energy together is called metabolism anabolism is when synthesis, i.e. building up of materials is the principal process difficult to measure, but it is characterized by positive nitrogen balance – less N is excreted than taken up catabolism is when degradation is the principal process – complex molecules are broken up to smaller ones 28/34

29. Energy pathways chemical energy of food absorbed chemical energy useable energy basal metabolism HEAT growth and storage external work excreted with feces urine, skin, hair, secretion 29/34

30. Energy turnover II. chemical transformations have a certain efficiency – part of the energy is lost in the form of heat serving also for the maintenance of body temperature if there is no external work, digestion or absorption, growth or storage, and the organism is in thermal balance with the surroundings, then basal metabolic rate can be determined by measuring heat production chemical energy mobilized from the stores is completely transformed into heat, and its amount do not depend on the route – Hess’ law the rate of enzymatic reactions change with temperature low temperature – low metabolic activity, decreased heat production – freezing to death high temperature – high metabolic rate, increased heat production – death by overheating 30/34

31. Basal metabolic rate I. direct calorimetry: measurement of dissipated heat – complicated, perspiration should be also measured   not reliable method if metabolic rate is low, appropriate for small birds and mammals indirect calorimetry: decrease of stores is determined by measuring O 2 consumption composition of combusted materials can be determined from the respiratory quotient (RQ=CO 2 /O 2 ) and the N-excretion sugar: RQ=1, fat: RQ=0.7, proteins: RQ=0.8 as O 2 energy-equivalence is almost independent from the combusted materials, thus determination of RQ is not absolutely necessary basal metabolic rate increases with body mass, but not linearly – MR=a*M b b=0.75 for vertebrates, invertebrates and unicellular organisms   mass-specific metabolic rate – MR/M=a*M (b-1)   31/34 Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 12-2. thermometer insulation water H 2 O absorbent CO 2 absorbent H 2 O absorbent oxygen Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 16-5b,c. Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 16-5a.

32. Basal metabolic rate II. the explanation for the precise relationship between body mass and metabolic rate is not known Rubner (1883) – surface hypothesis heat produced during metabolism is dissipated through the body surface – surface increases as the 2/3 power of the mass popular hypothesis, but the exponent is 0.75 not 0.67 equation also applies to animals with variable body temperature - that is not expected from the hypothesis – no explanation yet metabolic rate depends on the temperature: Q 10 value = 2-3 32/34

33. Basal metabolic rate III. basic metabolic rate is lower in women, and decreases with age – elderly people are more sensitive to cold malfunctioning of the thyroid gland can shift basic metabolic rate by -40 and +80%, respectively specific dynamic action: 25-30% increase in basal metabolic rate following consumption of proteins metabolic rate depends mostly on the activity of skeletal muscles mental activity acts also through the skeletal muscles – energy requirement of 1 hour intensive mental activity can be met by the consumption of a half salted peanut bean 33/34

34. Regulation of food intake food intake is motivated behavior – depends on the interplay of complex processes in addition to the need for nutritients, many other regulating factors: circadian rhythm, light-dark periods, in humans psychosocial interactions as well “centers” in hypothalamus: –ventromedial nucleus – satiation –lateral hypothalamus – hunger however, lesions of these “centers” have temporary effects only the limbic system and various brainstem nuclei also participate in the regulation of food intake facilitation: NA, GABA, NPY other peptides inhibition: 5-HT, DA, leptin stimuli: –glucose-sensitive neurons, hunger contractions –gastric distension, CCK 34/34

35. End of text

36. Glycolysis Alberts et al.: Molecular biology of the cell, Garland Inc., N.Y., London 1989, Fig. 2-38.

37. Islet of Langerhans Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 11-2. B cell D cell A cell

38. Structure of the insulin Berne and Levy, Mosby Year Book Inc, 1993, Fig. 46-3

39. Insulin receptor Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 11-6. ins ”empty” insulin receptor activated, phosphorylated insulin receptor IRS - insulin receptor substrate protein phosphorylated IRS protein

40. Direct calorimetry Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 12-2. thermometer insulation water H 2 O absorbent CO 2 absorbent H 2 O absorbent oxygen

41. Mass-specific metabolic rate I. Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 16-5b,c.

42. Mass-specific metabolic rate II. Fonyó: Orvosi Élettan, Medicina, Budapest, 1997, Fig. 16-5a.