1 Carbohydrate metabolism Intermediary Metabolism Elizabeth F. Neufeld Suggested reference: Champe, Harvey and Ferrier, Lippincott’s Illustrated Reviews – Biochemistry, 3 rd Edition

2 Kinetic properties of glucose transporters Uptake in liver and pancreas  -cells is proportional to plasma concentration GLUT-2 GLUT-3 GLUT-1 Uptake in brain is independent of plasma concentration over physiological range Km = concentration at which half maximum rate of transport occurs (1/2 Vmax)

3 Intracellular pool of GLUT4 in membranous vesicles translocate to the cell membrane when insulin binds to its receptor. The presence of more receptors increases the V max for glucose uptake (does not affect Km). When insulin signal is withdrawn, GLUT4 proteins return to their intracellular pool. GLUT4 is present in muscle and adipose tissue. GLUT4 activity is regulated by insulin- dependent translocation

4 Fate of glucose in the liver GLUT2 Glucose Glucose-6-P Glucokinase Glycogen synthesis Pentose phosphate Glycolysis

5 Glucokinase vs. Hexokinase Glucokinase: Km = 10 mM, not inhibited by glucose 6-phosphate. Present in liver and in pancreas  cells. Hexokinase: Km= 0.2 mM, inhibited by glucose 6- phosphate. Present in most cells.

6 Glucokinase vs. Hexokinase Glucokinase is found in liver and  -cells of pancreas Glucokinase allows liver to respond to blood glucose levels At low glucose levels, very little taken up by liver, so is spared for other tissues. Not inhibited by glucose 6-phosphate, allowing accumulation in liver for storage as glycogen It has a high Km, so it does not become saturated till very high levels of glucose are reached Hexokinase has low Km and therefore can efficiently use low levels of glucose. But is quickly saturated.

7 Glucose action in the  -cell Glucose enters the  -cell as blood glucose concentration rises. Glycolysis to generate ATP closes K+ channels in the cell membrane, stopping outward transport, and opening Ca+ channels. Inward flux of Ca+ causes exocytosis of insulin- containing secretory vesicles. Glucose also stimulates synthesis of new insulin.

8 Fate of glucose in muscle GLUT4 Glucose Glucose-6-P Hexokinase Glycogen synthesis Glycolysis Insulin +

9 Glycogen accumulation in muscle

10 Fate of glucose in adipocytes GLUT4 Glucose Insulin + Glucose-6-P Hexokinase LPL Insulin + Glycerol-3-P Triglycerides Fatty acids Insulin - Lipoproteins

11 How is metabolism regulated? Two broad classes of pathways Catabolic – break down molecules to generate energy Anabolic - require energy for synthesis of molecules The two pathways are kept distinct by regulatory mechanisms and/or sequestration in different cell compartments. Pathways contain recurring enzymatic mechanisms Oxidation-reduction reactions Isomerization reactions Group transfer reactions Hydrolytic reactions Addition or removal of functional groups

12 Movement Active transport Signal amplification Biosynthesis Oxidation of fuel molecules High ATP concentrations inhibit catabolic pathways and stimulate anabolic pathways How is metabolism regulated?

13 How is metabolism regulated? Fast mechanisms, for immediate changes Substrate concentration Allosteric regulation (feedback, feed forward) Phosphorylation-dephosphorylation Signals emanating from hormone action Slow mechanisms, for long-term changes Genetic regulation Response to diet and other environmental variables

14 long term effects How is metabolism regulated? Rapid effect Rapid effects

15 Overview of glucose metabolic pathways Glycolysis: from G6P to pyruvate Gluconeogenesis: from oxaloacetate to G6P Glycogen synthesis: from G6P to glycogen Glycogenolysis: from glycogen to G6P TCA cycle The pathways must be carefully regulated to keep pathways going in opposite directions from proceeding simultaneously.

16 Regulation of glycolysis Glycolytic flux is controlled by need for ATP and/or for intermediates formed by the pathway (e.g., for fatty acid synthesis). Control occurs at sites of irreversible reactions Phosphofructokinase- major control point; first enzyme “unique” to glycolysis Hexokinase or glucokinase Pyruvate kinase Phosphofructokinase responds to changes in: Energy state of the cell (high ATP levels inhibit) H+ concentration (high lactate levels inhibit) Availability of alternate fuels such as fatty acids, ketone bodies (high citrate levels inhibit) Insulin/glucagon ratio in blood (high fructose 2,6- bisphosphate levels activate)

17 Control points in glycolysis hexokinase Glucose-6-P - *

18 Why is phosphofructokinase, rather than hexokinase, the key control point of glycolysis? Because glucose-6-phosphate is not only an intermediate in glycolysis. It is also involved in glycogen synthesis and the pentose phosphate pathway. PFK catalyzes the first unique and irreversible reaction in glycolysis.

19 Phosphofructokinase (PFK-1) as a regulator of glycolysis fructose-6-phosphate fructose-1,6-bisphosphate PFK-1 PFK allosterically inhibited by: High ATP lower affinity for fructose-6-phosphate by binding to a regulatory site distinct from catalytic site. High H+ reduced activity to prevent excessive lactic acid formation and drop in blood pH (acidosis). Citrate prevents glycolysis by accumulation of this citric acid cycle intermediate to signal ample biosynthetic precursors and availability of fatty acids or ketone bodies for oxidation.

20 Phosphofructokinase (PFK-1) as a regulator of glycolysis PFK-1 activated by: Fructose-2,6-bisphosphate (F-2,6-P 2 ) F-6-P F-1,6-P 2 F-2,6-P 2 glycolysis + PFK-2 PFK-1 Activates PFK-1 by increasing its affinity for fructose-6-phosphate and diminishing the inhibitory effect of ATP. F-2,6-P 2

21 Phosphofructokinase-2 (PFK-2) is also a phosphatase (bifunctional enzyme) Bifunctional enzyme has two activities: 6-phosphofructo-2-kinase activity, decreased by phosphorylation Fructose-2,6-bisphosphatase activity, increased by phosphorylation fructose-6-phosphate fructose-2,6-bisphosphate phosphatase kinase ATP ADP PiPi

22 Hormonal control of F-2,6-P 2 levels and glycolysis Hormonal regulation of bifunctional enzyme Glucagon (liver) or epinephrine (muscle) increase cAMP levels, activate cAMP- dependent protein kinase. In liver, this leads to decreased F-2,6-P and inhibits glycolysis. The effect is opposite in muscle; epinephrine stimulates glycolysis. Insulin decreases cAMP, increases F-2,6-P  stimulates glycolysis. Phosphorylation of PFK2 by protein kinase activates its phosphatase activity on F2,6P in liver.

23 GLUCOSE G-6-Pase GK G-6-P F-6-P P-ENOLPYRUVATE PEPCK PK PYRUVATE OXALOACETATE FBPase 1 PFK 1 F-1,6-P 2 GlycolysisGluconeogenesis

24 GLUCOSE G-6-Pase GK G-6-P F-6-P P-ENOLPYRUVATE PEPCK PK PYRUVATE OXALOACETATE FBPase 1 PFK 1 F-1,6-P 2 Glycolysis Gluconeogenesis Increase Hepatic Glucose Utilization Decrease Hepatic Glucose Output

25 GLUCOSE G-6-Pase GK G-6-P F-6-P P-ENOLPYRUVATE PEPCK PK PYRUVATE OXALOACETATE FBPase 1 PFK 1 F-1,6-P 2 GlycolysisGluconeogenesis Decrease Hepatic Glucose Utilization Increase Hepatic Glucose Output

26 + F-6-P / F-1,6-P 2 SUBCYCLE FBP ase 1 PFK 1 F -1,6-P 2 FBPase 2PFK 2 PK - + F-6-P G-6-P F-2,6-P 2

27 The bifunctional enzyme FBPase 2PFK 2 Fructose-6-P Fructose-2,6-bis-P Fructose-6-P P

28 The bifunctional enzyme FBPase 2PFK 2 Fructose-6-P Fructose-2,6-bis-P Fructose-6-P P Phosphorylation of PFK2 by PKA promotes gluconeogenesis

29 The bifunctional enzyme FBPase 2PFK 2 Fructose-6-P Fructose-2,6-bis-P Fructose-6-P Double mutant, blocks phosphorylation of PFK2 and phosphatase activity of FBPase2

30 The bifunctional enzyme FBPase 2PFK 2 Fructose-6-P Fructose -2,6-bis-P Fructose-6-P Increased PFK1, Increased glycolysis, Fed State Hepatic overexpression of the double mutant results in a gene expression profile consistent with the fed state, and protection from Type I and II diabetes

31 Gluconeogenesis Mechanism to maintain adequate glucose levels in tissues, especially in brain (brain uses 120 g of the 160g of glucose needed daily). Erythrocytes also require glucose. Occurs exclusively in liver (90%) and kidney (10%) Glucose is synthesized from non-carbohydrate precursors derived from muscle, adipose tissue: pyruvate and lactate (60%), amino acids (20%), glycerol (20%)

32 Gluconeogenesis takes energy and is regulated Converts pyruvate to glucose Gluconeogenesis is not simply the reverse of glycolysis; it utilizes unique enzymes (pyruvate carboxylase, PEPCK, fructose-1,6- bisphosphatase, and glucose-6-phosphatase) for irreversible reactions. 6 ATP equivalents are consumed in synthesizing 1 glucose from pyruvate in this pathway hexokinase Glucose-6-P - Glucose 6-phosphatase

33 Irreversible steps in gluconeogenesis First step by a gluconeogenic-specific enzyme occurs in the mitochondria pyruvate oxaloacetate Pyruvate carboxylase Once oxaloacetate is produced, it is reduced to malate so that it can be transported to the cytosol. In the cytosol, oxaloacetate is subsequently dexcarboxylated/phosphorylated by PEPCK (phosphoenolpyruvate carboxykinase), a second enzyme unique to gluconeogenesis. The resulting phosphoenol pyruvate is metabolized by glycolysis enzymes in reverse, until the next irreversible step

34 Irreversible steps in gluconeogenesis (continued) Fructose 1,6-bisphosphate + H 2 O fructose-6-phosphate + P i Fructose 1,6- Bisphosphatase (FBPase ) In liver, glucose-6-phosphate can be dephosphorylated to glucose, which is released and transported to other tissues. This reaction occurs in the lumen of the endoplasmic reticulum. Requires 5 proteins! 2) Ca-binding stabilizing protein (SP) 1) G-6-P transporter 3) G-6-Pase 4) Glucose transporter 5) P i transporter

35 Gluconeogenesis and Glycolysis are reciprocally regulated Fructose 1,6-bisphosphatase is main regulatory step in gluconeogenesis. Corresponding step in glycolysis is 6-phosphofructo-1-kinase (PFK-1). These two enzymes are regulated in a reciprocal manner by several metabolites. Fructose-6-phosphate Fructose 1,6-bisphosphate 6-phosphofructo -1-kinase Fructose 1,6-bisphosphatase + Citrate - AMP - F 2,6-BP Citrate - AMP + F 2,6-BP + Reciprocal control—prevents simultaneous reactions in same cell.