Regulation of glycolysis and gluconeogenesis, and the mechanism of anti-diabetic drugs

1. Regulation of glycolysis and gluconeogenesis, and the mechanism of anti-diabetic drugs Prof. A. P. Halestrap References Pilkis, S. J. & Granner, D. K. (1992). Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54 885-909. Van Schaftingen, E. (1993). Glycolysis Revisited. Diabetologia36, 581-588. Moller, D. E. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821-827 Rutter, G. A.; Xavier, G. D., and Leclerc, I. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochemical Journal. 2003; 3751-16

2. Gluconeogenesis CO2 CO2 HCO3- Na+ H+ Liver and proximal convoluted tubules of the kidney (late in starvation - pH regulation in acidosis involves conversion of glutamine to ammonia (excreted) and 2-oxoglutarate which forms glucose by gluconeogenesis) Where Glucose Glutamine BLOOD Na+ H+ Liver proximal tubule epithelial cell NH4+ NH4+ Glutaminase Glutamate DH GNG Glutamine Glutamate 2-Oxoglutarate Glucose NH3 NH3 When After exercise, starvation, diabetes, at birth. NH3 NH3 URINE What De novo synthesis of glucose as opposed to glycogenolysis

4. Substrates Some amino acids and especially alanine and glutamine (alanine cycle and glutamine cycle used to transfer amino groups from muscle to liver for urea synthesis). Urea Amino acids Glutamate 2-Oxo acids Alanine Alanine 2-Oxoglutarate Lactic acid (exercise / Cori cycle) Fructose (from sucrose) Glycerol and propionate (from odd chain fatty acid b-oxidation) are the only components of triglycerides that can be used for glucose production.

5. Pathway reverse of glycolysis except for three steps with very negative DG. Glucose-6-phosphatase instead of glucokinase (hexokinase) Fructose-1,6-bisphosphatase instead of phosphofructokinase

6. Gluconeogenesis needs NADH Gluconeogenesis needs ATP

7. Uses 2 ATPs to reverse a glycolytic step that makes 1 ATP Pyruvate carboxylase plus phosphoenolpyruvate carboxykinase (PEPCK) instead of pyruvate kinase. HCO3-

8. Note that pyruvate carboxylation is mitochondrial whereas PEPCK is cytosolic; hence we need oxaloacetate to cross mitochondrial inner membrane. Glutamate Glutamate 2-oxoglutarate 2-oxoglutarate For most substrates oxaloacetate crosses as malate and effectively transfers NADH from the mitochondria (where it is abundant from fatty acid oxidation and citric acid cycle activity) to the cytosol (Route 2) Where L-lactate is the substrate this occurs as aspartate since lactate conversion to pyruvate produces NADH to drive glycolysis backwards (Route 1 in diagram).

9. Cytosol Pyruvate carboxylase in mitochondria

10. Regulation can be:Long term (e.g. starvation and diabetes) Regulation Medium term (birth and acidosis) Short term (e.g. during and after exercise and other stresses - Cori cycle). Long and medium term regulation involve changes in gene expression whilst short term regulation involves a change in enzyme activity or substrate supply. Note that both long and short term regulation involves the those enzymes that can participate in futile cycles.

11. # # By regulator protein. Note also that pyruvate carboxylase is regulated by allosteric effectors and substrate supply

12. Long and medium term regulation Primarily mediated through an increased glucagon/insulin ratio causing induction of gluconeogenic enzymes (especially PEPCK, but also other key GNG enzymes in Table 1) with permissive effect of glucocorticoids such as cortisol. Glycolytic enzymes such as GK and PK are repressed. Starvation and Diabetes both induce a large decrease in glucagon / insulin ratio and cause a 5-10 fold increase in PEPCK in liver and 2-3 fold increase in kidney. In kidney PEPCK induction also occurs in response to acidosis. In the liver it can be shown that PEPCK protein synthesis induced by glucagon follows a rise in cyclic AMP and mRNAPEPCK synthesis. After 20 min mRNA increased 5-fold: After 90 min 9-fold) mRNA degradation is not affected (addition of a-amanitin to block RNA synthesis promotes the same rate of PEPCK degradation in controls and glucagon- treated livers). The mechanism involves a range of regulatory elements in the PEPCK promoter including cAMP, gluocorticoid and thyroid hormone response elements. (Other promoters have similar regulatory elements).

13. Glucocorticoid response element cAMP response element Thyroid hormone response element

14. Note the immense increase in PEPCK activity seen at birth are also brought about by large changes in glucagons/insulin ratios. Transgenic mice in which the PEPCK promoter is linked to the growth hormone gene greatly enhances the production of growth hormone at birth, leading to very large mice that grow at twice normal rate! GH PEPCK

15. Stress hormone including adrenaline (a1-receptors), opiates, vasopressin and angiotensin work through activation of phospholipase c PLC activation Hormone Receptor PIP2 Mitochondrial metabolism DAG IP3 Ca2+ Protein kinase C Calmodulin-dependent protein kinases Short term regulation This involves both substrate supply and hormones. Note that alcohol reduces gluconeogenesis by increasing NADH/NAD+ and hence decreasing [oxaloacetate]. Stimulation by glucagon and other hormones that increase cyclic AMP (adrenaline via b -receptors in some species) regulate enzyme activity through the activation of protein kinase A. These effects are antagonised by insulin which lowers cyclic AMP.

16. Glucagon Glucagon and Ca-hormones Identification of control points 1. Effects of hormones on the rates of gluconeogenesis from different substrates

17. Futile cycling only occurs to a significant extent in the fed state and is insignificant in the starved state. Glucagon inhibits futile-cycling at both PEPCK / PK and PF-1-K / Fru-1,6-Pase whilst Ca-mobilising hormones (e.g.vasopression and a-adrenergic agonists) only inhibit futile-cycling at PEPCK / PK and to a lesser extent than glucagon. 3. Crossover plots Glucagon induced changes in metabolite concentration 250 Glucagon produces a crossover atboth PEPCK / PK and PF-1-K / Fru-1,6-Pase L-Lactate as substrate Crossover 200 Crossover 150 Metabolite level as % control 100 100 DHA as substrate 50 0 LAC MAL 3-PGA G3P G6P PYR PEP DHA F16bisP Gluc 2. Futile cycle measurements a-adrenergic agonists only produce a crossover at PEPCK / PKstep

18. 4. Flux control coefficient measurements Most rate determining These data show that pyruvate carboxylase is the most rate limiting process And that regulation by glucagon at both PEPCK / PK and PF-1-K / Fru-1,6-P2ase Flux control coefficient x 100 [L-Lactate] 5mM 0.5mM 5mM 0.5mM

19. Pyruvate transport

20. Pathologically, the enzyme is inhibited if tryptophan levels are high. Tryptophan is broken down to quinolinate which chelates Fe2+, an essential cofactor. COO Fe2+ COO • Mechanisms of short term regulation of gluconeogenesis • Pyruvate to phosphoenolpyruvate step • a) PEPCK Short term regulation is primarily through the supply of oxaloacetate whose cytosolic concentrations are less than the enzymes Km (about 9 mM). There may also be regulation through changes in the concentration of 2-oxoglutarate, a competitive inhibitor. Glucagon and Ca-mobilising hormones decrease the concentration of 2-oxoglutarate by a Ca-mediated activation of 2-oxoglutarate dehydrogenase.

21. b)    Pyruvate kinase The liver isoform of PK is a key regulator of gluconeogenesis in the FED state. It is inhibited by protein kinase A mediated phosphorylation , which decreases the substrate affinity of the enzyme. (The kidney M2 isoform can also be regulated in this way). F16P2 Phosphorylation Alanine Activity ATP 2 1 [PEP] mM Phosphorylation by calmodulin-dependent protein kinase has a similar but less potent inhibitory effect and accounts for some of the effects of Ca-mobilising hormones on gluconeogenesis. For glucagons in the fed state, there is a strong correlation between phosphorylation / inhibition of PK and stimulation of gluconeogenesis. At the levels of glucagon presentin the starved state PK is already almost totally inhibited and thus does not play a role in the regulation of gluconeogenesis under these conditions.

22. PC is critically dependent on acetyl-CoA which acts as an allosteric activator over the physiological range of concentrations, and this provides a regulatory link pyruvate carboxylation to fatty acid oxidation. Enzyme in mitochondria Activity Fatty acid oxidation Physiological range 500 250 [Acetyl CoA] mM d) Pyruvate carboxylase Exclusively mitochondrial enzyme with Km for pyruvate of about 200mM. This is in the physiological range and regulation through substrate supply is important.

23. Hormones 2+ Mitochondrial [Ca ] Matrix Ca-sensitive [PPi] dehydrogenases Cyclic AMP PKA and CPT1 + K entry into [2-OG] NADH Matrix NAD Relieve inhibition of PEPCK [Glu] Matrix volume Activation of respiration Pyruvate Fatty acid carboxylase oxidation ATP ADP Stimulation of Sites used for inhibiting GNG gluconeogenesis [Acetyl-CoA] PC is inhibited by glutamate and by increases in the ADP/ATP ratio. These provide a mechanism by which glucagon and Ca-mobilising hormones can stimulate pyruvate carboxylase.

24. Hypoglycaemic agents and antidiabetic drugs A. Inhibitors of fatty acid oxidation Inhibitors of carnitine palmitoyl transferase 1, especially cyclo-oxirane derivatives which are activated by fatty-acyl CoA synthetase to their CoA derivative which inhibits CPT1 with Ki values of less than 1mM. CoA ATP AMP + PPi POCA Tetradecylglycidate

25. Oxidative decarboxylation CH2 O CH2 C CH-CH2-C-S-CoA Methylene-cyclopropyl-acetyl-CoA Irreversible inhibitor of butyryl-CoA dehydrogenase (Pent-4-enoate has a similar effect) Inhibitors of b-oxidation such as hypoglycin (unripe ackee fruit - Jamaican vomiting sickness) CH2 NH2 CH2O CH2 C CH-CH2-C-COOH CH2 C CH-CH2-CH-COOH Methylene-cyclopropyl-propionic acid HypoglycinTransamination

26. V / J [ATP] Rate of GNG Respiratory chain activity 0 100 50 [Respiratory chain inhibitor] B. Inhibitors of the respiratory chain The respiratory chain has a high flux control coefficient for gluconeogenesis Although [ATP] changes little the calculated ATP/ADP ratio drops a lot and calculated free [AMP] increases Thus could mild inhibitors of the respiratory chain are potential anti-diabetic agents? The surprising answer is yes and the most commonly prescribed antidiabetic drug, metformin, probably works this way. Owen, M. R.; Doran, E., and Halestrap, A. P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochemical Journal. 2000; 348607-614.

27. The diabetic drugs metformin and phenformin (biguanides) act on the respiratory chain. Phenformin + + 2 2 Metformin Incubation at 8oC with inhibitor for 4 hr (metformin) or 5 min(phenformin) Phenformin Metformin Incubation with 10mM metformin at 8oC

28. Metformin inhibits immediately in sub-mitochondrial particles but requires higher concentrations + 2 Metformin Accumulation • Positive charge allows slow accumulation in mitochondria where they act as weak inhibitors of complex 1. • Uptake is self-limiting: if excessive inhibition occurs Dy drops preventing further accumulation. • Phenformin is much more potent than Metformin because it is more hydrophobic and enter the mitochondria more rapidly. It has a much higher risk of causing the rare side-effect of severe lactic acidosis. Dy = -180mV Cf 15 mM in intact energised mitochondria Cf 0.05 mM

29. Prolonged exposure allows metformin to inhibit the respiratory chain at therapeutic doses Hepatoma cell incubated with metformin for the time shown and then mitochondrial respiration measured in permeabilised cells.

30. Time dependent inhibition of gluconeogenesis in rat liver cells by metformin

31. and fatty acid oxidation [Triose phosphates] [2- + 3-PGA] [Acetyl-CoA] [PEP] Pyruvate kinase Direct effects of metformin on GNG via changes in ATP/ADP ratio and NADH/NAD+ ratio Biguanides Inhibition of respiration [Lactate] ATP NADH ADP NAD [Pyruvate] Pyruvate carboxylase The evidence for the proposed mechanism of action comes from measurements of metabolite levels in hepatocytes and whole animals treated with metformin, and from studies on isolated mitochondria. Inhibition of gluconeogenesis

32. Recent data from several labs has shown that metformin treatment activates AMPdependent protein kinase (AMPK, and that this may play a key role in its anti-diabetic effects. (AMPK inhibitor blocks effects but not very specific). Activation of AMPK is through an indirect mechanism - (no effect on isolated AMPK). Metformin increases the calculated free [AMP] which could account for this but no increase in total [AMP] can be measured. Either total [AMP] measurements mask changes in free [AMP] (quite likely) or metformin acts via some unidentified mechanism. Metformin fails to activate AMPK in cells from an LKB1 knockout mouse AMPKK (AMPK Kinase) ? ? LKB1 tumour supressor Metformin Phosphorylation of target proteins AMPK-P (Active) AMPK Inhibition of the respiratory chain Metformin [AMP] Zhou, G et al. (2001) Role of AMP-activated protein kinase in mechanism of metformin action J Clin. Invest. 108: 1167-1174. Also papers from Grahame Hardie’s group

33. AMPK activation can account for effects on metformin on gene transcription (down regulation of fatty acid oxidation and gluconeogenesis genes) and glucose transporter (GLUT-4) up-regulation (expression and translocation) in muscle. Inhibition of acetyl-CoA carboxylase in liver also occurs by this mechanism and may help explain the decrease in plasma free fatty acids and triglycerides. Inhibition of the respiratory chain [AMP] [ATP]/[ADP] SREBP-1c (Sterol Response Element Protein)– an important insulin stimulated transcription factor implicated in the pathogenesis of insulin resistance ? AMPK may also phosphorylate IRS-1 leading to increased insulin sensitivity

34. Problems with the AMPK activation theory Some of the enzyme activities modulated through changed gene expression (e.g. fatty acid synthetase and liver pyruvate kinase) or direct phosphorylation (acetyl CoA carboxylase) are in the opposite direction to insulin. Many experiments have been performed at concentration of metformin and phenformin far in excess of those used to treat Diabetes Note that the liver is exposed to much higher [Metformin] than other tissues (except the gut) since it receives the drug from the gut via the portal blood supply. This may be why ingestion of metformin is without major side-effects on tissues such as the heart and brain that are highly dependent on an active respiratory chain.

35. sulphonylureas glyburide = glibenclamide Sulphonylureas stimulate insulin secretion Inhibition of potassium efflux causes depolarisation and calcium entry + Glucose Insulin K O 2+ Ca Pyruvate [ATP] mitochondrion

36. D. Insulin Sensitizers Thiazolidinediones such as ciglitazone act as insulin sensitizers, reducing the peripheral insulin resistance that occurs in type 2 diabetes. They are agonists of the peroxisome proliferatory-activated receptor g (PPARg), an orphan member of the nuclear hormone receptor superfamily that is expressed at high levels in adipocytes. PPARg is a central regulator of adipocyte gene expression and differentiation one of whose effects is to decrease Resistin secretion. Resistin works in opposition to leptin and increases insulin resistance (Nature 2001 Jan 18;409(6818):307-12)

37. Moller, D. E. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821-827 Acrp30 is adiponectin PDK4 is PDH kinase 4

38. Mechanisms of short term regulation of gluconeogenesis Pi P ATP ADP PKA Glucagon cAMP Glucagon [F-2,6-bisP] hence stimulating F-1,6-bisPase and inhibiting PFK1. Calmodulin-dependent protein kinase does not phosphorylate the enzyme, accounting for the lack of effect of Ca-mobilising hormones on this step. 2. Phosphofructokinase / Fructose-1,6-bisphosphatase step Key regulation is by fructose 2,6-bisphosphate (F-2,6-bisPase). Activates , phosphofructokinase 1 (PFK1) and inhibits fructose-1,6-bisphosphatase F-1,6-bisPase. Fructose-6-P ATP Inhibited by Inhibited by Pi Pi Enzyme is 49kDa dimer with both activities on the same polypeptide F-6-P citrate and PEP F-2,6-bisPase PFK2 Activity switches depending on its phosphorylation state ADP Fructose-2,6-bisP (Activates PFK1 and inhibits F-1,6-bisPase)

39. 3.     Glucose-6-phosphatase / glucokinase Glucose-6-phosphatase (G-6-Pase) is a microsomal enzyme that is induced in starvation and diabetes but for which there is no good evidence for short-term regulation.

40. Deficiency of G-6-Pase causes glycogen storage disease (Von Gierke’s Disease) since the elevation of G-6-P in the liver inhibits glycogen phosphorylase leading to massive glycogen accumulation in the liver (which is enlarged).Mutations in any of the G-6-Pase constituent proteins have been shown to produce the disease.Patients also show severe hypoglycaemia after a short fast because they cannot mobilize their liver glycogen which represents the first source of blood glucose on starvation Glycogen storage diseases

41. In crude cytosolic extracts of liver F-1P activates GK and F-6P inhibits. Effect was lost on purification but sensitivity to inhibition by F-6P restored upon addition of an ancillary inhibitory protein (68kDa) Glucokinase (GK) Repressed in starvation and diabetes. Short term regulation by fructose which stimulates the conversion of glucose to glucose-6-P in isolated hepatocytes by about 2-4 fold in a reversible fashion. Van Schaftingen - the effect correlated with an increase in tissue [Fructose-1-P] and a decrease in [Fructose-6-P].

42. F-1P F-6P F-1P R F-6P F-6P R’ R’ R R Active GK is released from the regulatory protein in response to F-1P or glucose (by some ill-defined mechanism,) and translocated to the cytosol GK GK Active Regulatory protein resides in the nucleus where GK is also sequestered. Inactive Note that some individuals have GK deficiency and show early onset and severe Type 2 diabetes.