GLUCONEOGENESIS

1. GLUCONEOGENESIS ASSOC. PROF. DR.CEMİLE KOCA YBU & ANKARA ATATÜRK TRAINING AND RESEARCH HOSPITAL

2. GLUCONEOGENESIS • The process of synthesizing glucose from noncarbohydrate precursors • Takes place in cytosol, requires both cytosolic and mitochondrial enzymes • Major substrates: pyruvate, glucogenic amino acids (alanin), lactate, glycerol, and propionate • Source: Liver and kidney cortex (+ small intestine in fasting)

3. Gluconeogenesis Glycolysis GLUCONEOGENESIS FROM TRIGLYCERIDES Glycerol, generated by hydrolysis of triacylglycerols(fat) to yield free FAs + glycerol can also provide a raw material for gluconeogenesis Acetyl CoA, the main breakdown product of fatty acids, cannot be used to feed gluconeogenesis

4. GLUCONEOGENESIS FROM PROTEINS When glycogen reserves are low, (muscle) protein breakdown to amino acids provides raw material for glucose synthesis by gluconeogenesis. Alanine aminotransferasetransfers amino groups from amino acids to pyruvate to form alanine. Alanine is transferred to the liver where the reverse reaction feeds gluconeogenesis. Other amino acids are converted to intermediates of the citric acid cycle

5. Oxaloacetate is the starting material in TCA for gluconeogenesis

6. PHYSIOLOGICAL SIGNIFICANCE OF GLUCONEOGENESIS • Glucose is a major fuel source for many animal tissues: • Embryo • Nervous system • Kidney medulla • Brain (120 g/day! 75% of all) • RBC • lens, cornea of the eye • testes • exercising muscle • Gluconeogenesis allows synthesis of glucose for times when liver glycogen reserves are substantially depleted; during fasting and during starvation • Humans consume 160 g gluc/day • 75%of that is in the brain • Glycogen stores yield 180-200 gof glucose • So the body must be able to make its own glucose

7. Three steps of glycolysis are accompanied by large changes in free energy and so are effectively irreversible in vivo To reverse glycolysis, alternative enzymes are required to catalyse reactions that by-pass these steps

8. GLUCONEOGENESIS GLYCOLYSIS glucose hexokinase glucose 6-phosphatase glucose 6-phosphate fructose 6-phosphate Phosphofructokinase-1 fructose bisphosphatase fructose 1,6-bisphosphate phosphoenolpyruvate carboxykinase phosphoenolpyruvate oxaloacetate pyruvate kinase pyruvate carboxylase pyruvate

14. GLUCONEOGENESIS: ‘By-Pass’ Reactions (1) Pyruvate to phosphoenolpyruvate Pyruvate is first converted to oxaloacetate by the enzyme pyruvatecarboxylase Oxaloacetate is then converted to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase Pyruvate Kinase reversed by Pyruvate Carboxylase and PEPCK

15. Bypass 1 for pyruvate kinase • PEP can be synthesized either from pyruvate or lactate that are considered the glucogenic precursor a) Pyruvate pathway 1. Transport to Mitochondria as: a) pyruvate b) alanine (followed by transdeamination to pyruvate)

16. Bypass 1 for pyruvate kinase-a 2. Pyruvate Carboxylase: Pyruvate + HCO3 + ATP  oxaloacetate + ADP + Pi Localized in mitochondria usesbiotin as prosthetic group PC stimulated by acetyl CoA, inhibited by ADP

17. Biotin is a coenzyme

18. Bypass 1 for pyruvate kinase-a 2. Pyruvate Carboxylase: Pyruvate + HCO3 + ATP  oxaloacetate + ADP + Pi 3. Mitochondrial Malate DH: Oxaloacetate + NADH + H+L-malate + NAD+ 4. Malate--ketoglutarate transporter (IMM): transport of malate to the cytosol 5. Cytosolic Malate dehydrogenase: L-malate + NAD+ Oxaloacetate + NADH + H+

19. Bypass 1 for pyruvate kinase-a 6. Phosphoenolpyruvatecarboxykinase (Mg2+ and GTP dependent): Both in cytosolandmitochondria Oxaloacetate + GTP  phosphoenolpyruvate + CO2+ GDP OAA The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism PEP

20. Bypass 1 for pyruvate kinase-a What is the metabolic logic for this pathway through mitochon. ? • [NADH]/[NAD+] ratio is ~ 105 x lower in the cytosolas compared to the mitochondria • Gluconeogenesis requires NADH & can not proceed without it • Transport of malate to the cytosol moves reducing equivalents that are normally scarce in the cytosol • This pathway provides balance for NADH produced and consumed inthe cytosol during glucose synthesis

21. Bypass 1 for pyruvate kinase-b • b) via Lactate • Important in vertebrates after exercise or erythrocytes. Lactate is produced after vigorous exercise since oxygen can not be carried fast enough to tissues to regenerate NAD+ A build-up of lactate causes acidosis and muscle soreness Clearing the lactate will eliminate this problem.

22. Bypass 1 for pyruvate kinase-b • Pathway • 1. Lactate dehydrogenase: lactate  pyruvate • 2. Pyruvate transport into the mitochondria • 3. Pyruvate carboxylase: pyruvate  OA • 4. Mitochondrial PEP carboxykinase: OA  PEP • 5. PEP transport to the cytosol • Overall: Lactate + NAD+ PEP + NADH + H+

23. The Cori Cycle

25. GLUCONEOGENESIS: ‘By-Pass’ Reactions (2) Fructose 1,6-bisphosphate to Fructose 6-phosphate Catalysed by fructose bisphosphatase. PFK is bypassed by Fructose-1,6-bisphosphatase Allosteric regulation: inhibited by Fru-2,6-P2

26. Bypass 2 of phosphofructokinase-1 (*kinase adds a phosphoryl group, a phosphatase takes it off) Fructose-1,6-phosphate  fructose-6-phosphate Mg2+-dependent fructose-1,6-bisphosphatase (FBPase-1): catalyzes an irreversible hydrolysis of the C1 phosphate Fructose-1,6-bisphosphate + H2O  fructose-6-phosphate + Pi G' = 16.3 kJ/mol

28. GLUCONEOGENESIS: ‘By-Pass’ Reactions (3) Glucose 6-phosphate to glucose Catalysed by glucose 6-phosphatase. Glucose 6-phosphatase is chiefly found in liver cells where it is important for producing glucose to ‘top-up’ blood glucose levels It is absent in muscle cells Hexokinase is bypassed by Glucose-6-phosphatase G6Pase localized to the ER G6Pase is a complex: transporters plus enzyme

29. Bypass 3 of hexokinase • Glucose-6-phosphate  Glucose • Mg2+ activated Glucose-6-phosphatase: • catalyzes an irreversible hydrolysis • Is found in the liver and kidney (lumenal side of ER) • Glucose-6-phosphate + H2O  glucose + Pi • G' = 13.8 kJ/mol • Glucose is transported to the brain and muscle that are • unable to make glucose for lack of this enzyme

31. The overall equation for gluconeogenesis is: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 4 H2Oglucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+ G' = 16 kJ/mol (irreversible) For each molecule of glucose produced, 6 high energy phosphate groups are required as are 2 molecules of NADH Thus “Gluconeogenesis Costs” Glycolysis Glucose + 2 ADP + 2 Pi + 2 NAD+ 2 pyruvate + 2 ATP + 2NADH + 2H+ + 2 H2O G' = 63 kJ/mol

32. Regulation of carbonhydrate metabolism: • Three mechanisms: regulating the activity of enzymes in carbohydrate metabolism • 1. changes in the rate of enzyme synthesis • 2. covalent modification by reversible phosphorylation • 3. allosteric effects

33. Regulation of Gluconeogenesis Glycolysis & Gluconeogenesisareboth spontaneous If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy Three strategies to prevent futile cycling: • High energy intermediate • Compartmentation • Allosteric regulation To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated

34. Regulation of Gluconeogenesis Reciprocal control with glycolysis • When glycolysis is turned on, gluconeogenesis should be turned off • When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose • When energy status is low, glucose should be rapidly degraded to provide energy • The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

35. Regulation of Gluconeogenesis Reciprocal Regulation of Glucose Metabolism • What happens when the cells energetic needs are met? • Oxidative phosphorylation slows and NADH accumulates • (recall excess ATP will inhibit oxidative phosphorylation) • If there is a high concentration of acetyl-CoA, citrate or ATP, glucose synthesis will be favored • Inhibition of the TCA causes accumulation of acetyl-CoA • Net result: excess pyruvate is converted to glucose

37. BYPASS- 1

38. Reciprocal Regulation of Glucose Metabolism – Bypass 1 The fate of pyruvate depends on acetyl-CoA • Acetyl-CoA stimulates pyruvate carboxylase and inhibits the pyruvate dehydrogenase complex • Inhibition of the TCA and stimulation of gluconeogenesis

39. BYPASS- 2

40. Reciprocal Regulation of Glucose Metabolism – Bypass 2 Fructose-1,6-bisphosphatase and PFK-1 are coordinately controlled and reciprocally regulated F-1,6-bisPase is inhibited by AMP, activated by citrate - the reverse of glycolysis Fructose 2,6-bisphosphate is the most important regulator of glycolysis and gluconeogenesis

41. Fructose-2,6-bisphosphate (F26BP) is a potent allosteric regulator of PFK-1 and FBPase-1 Fructose 2,6-bisP is not an intermediate of either pathway is synthesised from F 6-P by a dual function enzyme known as phosphofructokinase-2/fructose 2,6-bisphosphatase Fructose-2,6-bisP is an allosteric inhibitor of F-1,6-bisPase

42. F2,6BP is a mediator of hormonal regulation of glycolysis and gluconeogenesis Bifunctional protein • Cellular [F2,6BP] is regulated by glucagon and insulin • Glucagon raises blood glucose, insulin lowers blood glucose • F2,6BP is produced under normal glucose levels, but its formation by PFK-2 is inhibited by glucagon

43. Hormonal Regulation of Glycolysis / Gluconeogenesis • The pancreatic hormone glucagon stimulates gluconeogenesis and inhibits glycolysis by 3 mechanism: • Changes in allosteric effectors: level ofF2,6 BP (activ. of F1,6-BPase and inh. PFK, thus favoring gluconeogenesis over glycolysis • Covalent modification of enzyme activity: via in cAMP level and cAMP-dependent protein kinase activity, stim. the conversion of PK to its inactive (phosphorylated) form This decreases the conversion of PEP to pyruvate • Induction of enzyme synthesis: transcription of the PEP-carboxykinase gene, thereby increasing the availability of this enzyme's activity as levels of its substrate rise during fasting. [Note: Insulin causes decreased transcription of the mRNA for this enzyme]

44. Hormonal Regulation of Glycolysis / Gluconeogenesis • The pancreatic hormone glucagon stimulates gluconeogenesis and inhibits glycolysis glucagon G-protein / cAMP signalling cascade protein kinase A PFK-2/fructose 2,6-bisphosphatase – OH PFK-2/fructose 2,6-bisphosphatase – P fructose 6-bisphosphate fructose 2,6-bisphosphate Removal of fructose 2,6-bisphosphate stimulates gluconeogenesis and inhibits glycolysis

45. Hormonal Regulation of Glycolysis / Gluconeogenesis • The pancreatic hormone insulin inhibits gluconeogenesis and stimulates glycolysis insulin protein kinase A PFK-2/fructose 2,6-bisphosphatase – OH PFK-2/fructose 2,6-bisphosphatase – P fructose 2,6-bisphosphate fructose 6-bisphosphate Presence of fructose 2,6-bisphosphate inhibits gluconeogenesis and stimulates glycolysis

46. Glucagon and insulin mediate long-term effects by inducing andrepressing the synthesis of key enzymes Glucagon induces the synthesis of: PEP-carboxykinase Gluconeogenic fructose 1,6-bisphosphatase enzymes glucose 6-phosphatase certain aminotransferases Glucagon represses the synthesis of: glucokinase Glycolytic PFK1 enzymes pyruvate kinase Insulin generally opposes these actions

47. BOTTOM LINE: WHEN BLOOD GLUCOSE IS LOW: GLYCOLYSIS AND GLUCONEOGENESIS SO THAT YOU MAKE MORE GLUCOSE IN THE LIVER!!!!

49. Regulation of Glycolysis and Gluconeogenesis • High glucose levels and insulin promote glycolysis • Low glucose levels and glucagon promote gluconeogenesis