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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. Diabetologia 36, 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 Gluconeogenesis What De novo synthesis of glucose as opposed to glycogenolysis Where 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) Glutamine CO2 Liver proximal tubule epithelial cell CO2 HCO3 When - Glucose BLOOD Glutaminase Glutamate DH GNG Glutamine Glutamate 2-Oxoglutarate Glucose H + Na+ NH3 NH3 + + Na H NH3 NH3 NH4+ NH4+ URINE After exercise, starvation, diabetes, at birth. Substrates Lactic acid (exercise / Cori cycle) 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). 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. Urea Amino acids Glutamate 2-Oxoglutarate Alanine 2-Oxo acids Alanine Pathway reverse of glycolysis except for three steps with very negative DG. Glucose-6-phosphatase instead of glucokinase (hexokinase) Fructose-1,6bisphosphatase instead of phosphofructokinase Gluconeogenesis needs NADH Gluconeogenesis needs ATP Pyruvate carboxylase plus phosphoenolpyruvate carboxykinase (PEPCK) instead of pyruvate kinase. HCO3- Uses 2 ATPs to reverse a glycolytic step that makes 1 ATP Note that pyruvate carboxylation is mitochondrial whereas PEPCK is cytosolic; hence we need oxaloacetate to cross mitochondrial inner membrane. 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). Glutamate 2-oxoglutarate Glutamate 2-oxoglutarate Cytosol Pyruvate carboxylase in mitochondria Regulation Regulation can be: Long term (e.g. starvation and diabetes) 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. # # By regulator protein. Note also that pyruvate carboxylase is regulated by allosteric effectors and substrate supply 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). Glucocorticoid response element Thyroid hormone response element cAMP response element 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 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. Stress hormone including adrenaline (a1-receptors), opiates, vasopressin and angiotensin work through activation of phospholipase c Hormone Receptor PLC activation DAG Protein kinase C PIP2 Mitochondrial metabolism IP3 Ca2+ Calmodulin-dependent protein kinases Identification of control points 1. Effects of hormones on the rates of gluconeogenesis from different substrates Glucagon Glucagon and Ca-hormones 2. Futile cycle measurements 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 Metabolite level as % control 250 L-Lactate as substrate 200 Crossover Crossover 150 100 DHA as substrate 50 0 LAC MAL PYR 3-PGA PEP G3P G6P DHA F16bisP Gluc Glucagon produces a crossover at both PEPCK / PK and PF-1-K / Fru-1,6Pase a-adrenergic agonists only produce a crossover at PEPCK / PK step 4. Flux control coefficient measurements Flux control coefficient x 100 [L-Lactate] 5mM 0.5mM 5mM 0.5mM 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 Pyruvate transport Mechanisms of short term regulation of gluconeogenesis 1. 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 2oxoglutarate, a competitive inhibitor. Glucagon and Ca-mobilising hormones decrease the concentration of 2-oxoglutarate by a Ca-mediated activation of 2-oxoglutarate dehydrogenase. 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 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). Phosphorylation by calmodulindependent protein kinase has a similar but less potent inhibitory effect and accounts for some of the effects of Camobilising hormones on gluconeogenesis. F16P2 Phosphorylation Alanine ATP For glucagons in the fed state, there is a strong correlation between phosphorylation / inhibition of PK and stimulation of gluconeogenesis. 1 [PEP] mM 2 At the levels of glucagon present in the starved state PK is already almost totally inhibited and thus does not play a role in the regulation of gluconeogenesis under these conditions. 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. 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 Fatty acid oxidation Physiological range 250 [Acetyl CoA] mM 500 Hormones Mitochondrial [Ca 2+] Matrix [PPi] Cyclic AMP PKA and CPT1 K+ entry into Matrix Ca-sensitive dehydrogenases NADH 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. [ 2-OG] NAD Matrix volume Fatty acid oxidation Sites used for inhibiting GNG Relieve inhibition of PEPCK [Glu] Activation of respiration Pyruvate carboxylase ATP ADP [Acetyl-CoA] Stimulation of gluconeogenesis 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. R COOH R CoA O ATP POCA Cl AMP + PPi COSCoA O Tetradecylglycidate CH2(CH2)4 CH3(CH2)13- Inhibitors of b-oxidation such as hypoglycin (unripe ackee fruit - Jamaican vomiting sickness) CH2 CH2 C NH2 CH2 CH-CH2-CH-COOH Hypoglycin CH2 C Transamination O CH-CH2-C-COOH Methylene-cyclopropylpropionic acid Oxidative decarboxylation CH2 CH2 C O CH-CH2-C-S-CoA Methylene-cyclopropyl-acetyl-CoA Irreversible inhibitor of butyryl-CoA dehydrogenase (Pent-4-enoate has a similar effect) B. Inhibitors of the respiratory chain The respiratory chain has a high flux control coefficient for gluconeogenesis V/J [ATP] Although [ATP] changes little the calculated ATP/ADP ratio drops a lot and calculated free [AMP] increases Rate of GNG Respiratory chain activity 0 50 100 [Respiratory chain inhibitor] Thus could mild inhibitors of the respiratory chain are potential antidiabetic 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. The diabetic drugs metformin and phenformin (biguanides) act on the respiratory chain. Phenformin Metformin CH3 CH3 NH2+ NH2+ CH2 N-C-NH2 CH3 N-C-NH2 0 [Metformin] (mM) 2.5 5 7.5 10 Incubation with 10mM metformin at 8oC Respiratory rate as % of control 100 80 Metformin 60 K0.5 14.9 ± 1.19 mM Incubation at 8oC with inhibitor for 4 hr (metformin) or 5 min (phenformin) 40 20 Phenformin K0.5 0.05 ± 0.0015 mM 0 0 0.1 0.2 0.3 [Phenformin] (mM) 0.4 Metformin inhibits immediately in sub-mitochondrial particles but requires higher concentrations 0 [Metformin] (mM) 10 20 30 40 50 CH3 100 Respiratory rate as % of control CH3 NH2+ Metformin N-C-NH2 Accumulation Dy = -180mV 80 60 K0.5 79.0 ± 3.4 mM Cf 15 mM in intact energised mitochondria 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. 40 20 K0.5 2.23 ± 0.18 mM Cf 0.05 mM 0 0 2 4 6 8 [Phenformin] (mM) 10 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. S ta te 3 resp iration ra te as % Co ntro l Prolonged exposure allows metformin to inhibit the respiratory chain at therapeutic doses 130 Glutamate / malate (4) Succinate (4) 120 24 hours 110 60 hours (3) (4) 100 (5)* Hepatoma cell incubated with metformin for the time shown and then mitochondrial respiration measured in permeabilised cells. 90 80 (4)* (4)** (5)* 70 60 50 [Me tformin] 50mM 100mM 50mM 100m M Time dependent inhibition of gluconeogenesis in rat liver cells by metformin Direct effects of metformin on GNG via changes in ATP/ADP ratio and NADH/NAD+ ratio Biguanides Inhibition of respiration and fatty acid oxidation [Triose phosphates] [Lactate] [Pyruvate] NADH NAD [Acetyl-CoA] 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. ATP ADP [2- + 3-PGA] [PEP] Pyruvate kinase Inhibition of gluconeogenesis Recent data from several labs has shown that metformin treatment activates AMP dependent protein kinase (AMPK, and that this may play a key role in its antidiabetic 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) AMPK ? LKB1 tumour supressor AMPK-P (Active) [AMP] ? Metformin Phosphorylation of target proteins Inhibition of the respiratory chain Metformin 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 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) upregulation (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 SREBP-1c (Sterol Response respiratory chain ? Element Protein)– an important insulin stimulated transcription factor [AMP] [ATP]/[ADP] implicated in the pathogenesis of insulin resistance AMPK may also phosphorylate IRS-1 leading to increased insulin sensitivity 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. Sulphonylureas stimulate insulin secretion Glucose O Inhibition of potassium efflux causes depolarisation and calcium entry + K Ca 2+ Pyruvate [ATP] mitochondrion sulphonylureas glyburide = glibenclamide Insulin 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) 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 Mechanisms of short term regulation of gluconeogenesis 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 Inhibited by ATP citrate and PEP PFK2 ADP Enzyme is 49kDa dimer with both activities on the same polypeptide Activity switches depending on its phosphorylation state Pi Inhibited by F-6-P F-2,6-bisPase Fructose-2,6-bisP (Activates PFK1 and inhibits F-1,6-bisPase) 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. 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. Glycogen storage diseases 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 Glucokinase (GK) Repressed in starvation and diabetes. Short term regulation by fructose which stimulates the conversion of glucose to glucose6-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]. GK Activity No regulatory protein With regulatory protein + 200mM F-1P Effect was lost on purification but sensitivity to inhibition by F-6P restored upon addition of an ancillary inhibitory protein (68kDa) With regulatory protein 50 [F-6P] mM In crude cytosolic extracts of liver F-1P activates GK and F-6P inhibits. 100 F-1P F-6P F-6P F-1P R’ R’ R R GK 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 Active F-6P Regulatory protein resides in the nucleus where GK is also sequestered. R GK Inactive Note that some individuals have GK deficiency and show early onset and severe Type 2 diabetes.