1. Glycolysis and Gluconeogenesis Alice Skoumalová

3. 1. Glycolysis

4. Glucose: the universal fuel for human cells Sources:  diet (the major sugar in our diet)  internal glycogen stores  blood (glucose homeostasis) Glucose oxidation:  after a meal: almost all tissues  during fasting: brain, erythrocytes

5. Glycolysis:  oxidation and cleavage of glucose  ATP generation (with and without oxygen)  all cells  in the cytosol (the reducing equivalents are transferred to the electron-transport chain by the shuttle) ATP is generated: 1. via substrate-level phosphorylation 2. from NADH 3. from oxidation of pyruvate Regulation of glycolysis: 1. Hexokinase 2. Phosphofructokinase 3. Pyruvate Kinase Generation of precursors for biosynthesis:  fatty acids  amino acids  ribosis-5-P

6. Anaerobic glycolysis  a limited supply of O 2  no mitochondria  increased demands for ATP Lactic acidemia  in hypoxia

7. Phosphorylation of glucose:  irreversible Glucose 6-P:  cannot be transported back across the plasma membrane  a precursor for many pathways that uses glucose Hexokinases Glucokinase (liver, β-cell of the pancreas)  high K m

8. Michaelis-Menten kinetics

9. 1. Conversion of glucose 6-P to the triose phosphates 2. Oxidation and substrate-level phosphorylation

10. 1. Conversion of glucose 6-P to the triose phosphates irreversible regulation essential for the subsequent cleavage

11. Substrate-level phophorylation 2. Oxidation and substrate-level phosphorylation

12. Summary of the glycolytic pathway: Glucosis + 2 NAD + + 2 P i + 2 ADP 2 pyruvate + 2 NADH + 4 H + + 2 ATP + 2 H 2 O ∆G 0´ = - 22 kcal (it cannot be reversed without the expenditure of energy!)

13. Clinical correlations: Hypoxemia (lack of oxygen in tissues)  Acute hemorrhage (hypotension, lost of erythrocytes) - anaerobic glycolysis - lactate formation, metabolic acidosis  Chronic obstructive pulmonary disease (an insuficient ventilation) - anaerobic glycolysis, lactate formation, metabolic acidosis - accumulation of CO 2, respiratory acidosis  Myocardial infarction (lack of oxygen in myocardium) - anaerobic glycolysis, lactate formation - lack of ATP

14. Aerobic glycolysis:  involving shuttles that transfer reducing equivalents across the mitochondrial membrane

15. Glycerol 3-phosphate shuttle:

16. Malate-aspartate shuttle:

17. Anaerobic glycolysis: Energy yield 2 mol of ATP dissociation and formation of H +

18. Daily lactate production115 (g/d) Erythrocytes29 Skin20 Brain17 Sceletal muscle16 Renal medulla15 Intestinal mucosa8 Other tissues10 Major tissues of lactate production: (in a resting state)

19. Cori cycle: Lactate can be further metabolized by:  heart, sceletal muscle Lactate dehydrogenase: a tetramer (subunits M and H)

20. Lactate dehydrogenase Pyruvate + NADH + H + lactate + NAD + LD 5 isoenzymes: Heart (lactate) Muscle (pyruvate)

21. Biosynthetic functions of glycolysis:

22. Clinical correlations: Long-intensity exercise (for example a sprint) - the need for ATP exceeds the capacity of the mitochondria for oxidative phosphorylation, anaerobic glycolysis → lactate formation, muscle fatigue and pain - a training → the amounts of mitochondria and myoglobin increase

23. Regulation

24. Fructose 2,6-bis-phosphate:  is not an intermediate of glycolysis!  Phosphofructokinase-2:inhibited through phosphorylation - cAMP-dependent protein kinase (inhibition of glycolysis during fasting-glucagon) tissue-specific isoenzymes (low K m, a high afinity) glucokinase (high K m ) the rate-limiting, allosteric enzyme tissue-specific isoenzymes

25. the liver isoenzyme - inhibition by cAMP-dependent protein kinase (inhibition of glycolysis during fasting) Lactic acidemia: increased NADH/NAD + ratioinhibition of pyruvate dehydrogenase

26. 2. Gluconeogenesis

27. Gluconeogenesis:  synthesis of glucose from noncarbohydrate precursors → to maintain blood glucose levels during fasting  liver, kidney  fasting, prolonged exercise, a high- protein diet, stress Specific pathways: 1.Pyruvate → Phosphoenolpyruvate 2.Fructose-1,6-P → Fructose-6-P 3.Glucose-6-P → Glucose

28. Precursors for gluconeogenesis 1.lactate (anaerobic glycolysis) 2.amino acids (muscle proteins) 3.glycerol (adipose tissue)

29. Conversion of pyruvate to phosphoenolpyruvate 1. Pyruvate → Oxaloacetate  Pyruvate carboxylase 2. Oxaloacetate → PEP  Phosphoenolpyruvate- carboxykinase

30. Conversion of phosphoenolpyruvate to glucose 3. Fructose-1,6-P → Fructose-6-P  Fructose 1,6-bisphosphatase (cytosol) 4. Glucose-6-P → Glucose  Glucose 6-phosphatase (ER)

31. Clinical correlations: Alcoholism - excessive ethanol consumption → increase NADH/NAD + ratio that drive the lactate dehydrogenase reaction toward lactate - lack of precursors for gluconeogenesis → its inhibition - insuficient diet - reduced glucose in the blood, consumption of glycogen in the liver → hypoglycemia

32. Regulation of gluconeogenesis:  concomitant inactivation of the glycolytic enzymes and activation of the enzymes of gluconeogenesis 1. Pyruvate → PEP Phosphoenolpyruvate carboxykinase - induced by glucagon, epinephrine, and cortisol 2. Fructose 1,6-P → Fructose 6-P Fructose 1,6-bisphosphatase - inhibited by fructose 2,6-P 3. Glucose 6-P → Glucose Glucose 6-phosphatase - induced during fasting

34. Summary Glycolysis Generation of ATP (with or without oxygen) The role of glycolysis in different tissues Lactate production Regulation Gluconeogenesis Activation during fasting, prolonged exercise, after a high- protein diet Precursors: lactate, glycerol, amino acids 3 key reactions:Pyruvate → PEP Fructose-1,6-P→ Fructose-6-P Glucose-6-P → Glucose Regulation

35. Pictures used in the presentation: Marks´ Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M. Lieberman, A.D. Marks)