1. The sweet side of catabolism: carbohydrates as cellular fuels Russian National Research Medical University Maxim A. Abakumov Moscow, 2014

2. Carbohydrates metabolism Usually comes as polysaccharides Two main polysacharides are glicogen and starch Polysacharides can not be used in native form Breakdown into monosacharides and transport from gut to blood stream and periheral tissues are needed

3. Digestion of carbohydrates Digestion – enzyme driven breakdown of large polysacharide molecules into monosacharides Usually takes plase in gastrointestinal tract Glucose polymers Starch, glycogen Disaccharides MaltoseSucroseLactose Digestion by amylase Monosaccharides 2xGlucose Glucose+Fructose Glucose+Galactose MaltaseSucraseLactase

4. Absorption of carbohydrates Process of monosacharides transport from gut to blood stream or lymph Involves special transporting proteins located on membrane of intestine cells

5. Digestion Composition of carbohydrates in your diet: ~ 70% starch (polysaccharide) ~ 20% sucrose (disaccharide) ~ 6% lactose (disaccharide) ~ 2% maltose (disaccharide) Polysacharides digestion occurs in mouth and small intestine This process is driven by salivary and pancreatic amylases

6. Digestion

7. Absorption Transmembrane transporter proteins are involved First, monosacharides are transported into cell from intestine Second, monosacharides are released into blood stream

8. Absorption

9. Glucose metabolism Glucose decomposition for energy release (ATP synthesis) called glycolysis Glucose synthesis with energy consume (ATP hydrolysis) calles gluconeogenesis Glycolysis can be diveded into: a) aerobic b) anaerobic Aerobic products are CO 2 and H 2 O Anaerobic product is lactate For both intermediate is pyruvate

10. Glucose metabolism in cell Glucose Pyruvate AcetylCoa CO 2 + H 2 O + ATP TCA Aerobic Glycolisis ETC+OP Lactate Anaerobic Glycolisis TCA

11. Glycolisis 1. Glucokinase 2. Phosphogluco isomerase 3. Phosphofructo kinase-1 4. Aldolase 5. Triosophosphate isomerase GlucoseGlucose-6-PFructose-6-P Fructose-1,6-diP ATPADP ATPADP 123 4 5 67 8 9 10 6. Glyceraldehyde phosphate isomerase 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10. Pyruvate kinase 1,3-bisphospho glycerate 3-phospho glycerate 2-phospho glycerate ATPADP H2OH2O ATP NAD + NADH Phosphoenol pyruvate Pyruvate

12. Sequence of reactions + Pyruvate Glucose Glucose metabolism in cell Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate

13. Glucose phosphorylation First step in glucose metabolism – phoshorylation of OH-group at 6th carbon atom Phosporylated glucose (glucose-6-phospate) is charged and cannot be transported out of the cell Glucose-6-P goes to metabolism Catalyzed by two types of enzyme (isozymes)

14. Glucose phosphorylation Hexokinase Glucokinase Low K m value High affinity to glucose Located in most tissue cells Three isoforms (I, II, III) High K m value Low affinity to glucose Located mostly in liver cells Actually IV isoform of hexokinase

15. Glucose-2- 18 F PET tracer Indicates glucose cosumption by cells Phosphorylates after transport in cell OH-group at 2nd carbon atom is substituted by 18 F Further metabolism is blocked Cells with more active metabolism increase glucose consumption, glucose-2- 18 F level and consequently signal on PET scanner

16. Phase I Coversion of glucose (6 carbon) to dihydroaceton phosphate and gliceraldehyde- 3-phosphate (2x3 carbon) 2 ATP are required (will be regenerated later) 1st and 3rd reaction are irreversible

17. Phase I. Preparatory phase. 1. Glucokinase 2. Phosphogluco isomerase 3. Phosphofructo kinase-1 4. Aldolase 5. Triosophosphate isomerase GlucoseGlucose-6-PFructose-6-P Fructose-1,6-diP ATPADP ATPADP 123 4 5 Dyhydroxy acetone phosphate D-glyceraldehyde -3-phosphate

18. Glucose to glucose-6-P 1st ATP is hydrolysed Total ATP count: -1 ATP Total NADH count: 0 NADH

19. Glucose-6-P to fructose-6-P Total ATP count: -1 ATP Total NADH count: 0 NADH Phosphohexose isomerase

20. Fructose-6-P to fructose-1,6-diP 2nd ATP is hydrolysed Total ATP count: -2 ATP Total NADH count: 0 NADH Phosphofructokinase-1 ATP ADP Fructose-6-P Fructose-1,6-diP

21. Fructose-2,6-diP to gliceraldehyde-3- phosphate and dihidroxyacetone-phosphate Total ATP count: -2 ATP Total NADH count: 0 NADH Aldolase Fructose-2,6-diP Dyhydroxyacetone phosphate D-glyceraldehyde- 3-phosphate

22. Phase II. Payoff phase Coversion of dihydroaceton phosphate and gliceraldehyde-3-phosphate (2x3 carbon) to pyruvate (2x3 carbon) 4 ATP are restored Last reaction is irreversible

23. Phase II. Payoff phase. 67 8 9 10 1,3-bisphospho glycerate 3-phospho glycerate 2-phospho glycerate ATPADP H2OH2O ATP NAD + NADH 6. Glyceraldehyde phosphate isomerase 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10. Pyruvate kinase Phosphoenol pyruvate Pyruvate

24. Gliceraldehyde-3-phosphate to 1,3- bisphosphoglycerate 2x Total ATP count: -2 ATP Total NADH count: 2 NADH NAD + NADH PiH+H+ Glyceraldehyde-3-phosphate dehydrogenase D-glyceraldehyde- 3-phosphate 1,3-bisphosphoglycerate

25. 1,3-bisphosphoglycerate to 3- phosphoglycerate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 2 ATP are synthesized 1.3-bisphosphoglycerate ADP ATP Phosphoglycerate kinase 3-Phosphoglycerate

26. 3-phosphoglycerate to 2- phosphoglycerate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 3-Phosphoglycerate Phosphoglycerate mutase 2-Phosphoglycerate

27. 2-phosphoglycerate to phosphoenolpyruvate 2x Total ATP count: 0 ATP Total NADH count: 2 NADH 2-Phosphoglycerate H2OH2O Phosphoenolpyruvate Enolase

28. Phosphoenolpyruvate to pyruvate 2x Total ATP count: 2 ATP Total NADH count: 2 NADH 2 ATP are synthesized Phosphoenolpyruvate ADP ATP Pyruvate kinase Pyruvate

29. Glucose→Pyruvate Total energy output 2 ATP are consumed 4 ATP are synthesized Total 2 ATP from 1 glucose 2 NADH are synthesized All ATP is synthesized without O 2 (substrate-level phosphorylation) Anaerobic glycolysis

30. Glucose→AcCoA→CO 2 + H 2 O Total energy output Total 2 ATP + 2 NADH from anaerobic glycolisis. 2 NADH from PDH 6 NADH+ 2 FADH 2 from TCA 2 GTP from TCA Total 10 NADH+4 ATP + 2FADH 2 = 32 ATP All ATP is synthesized with O 2 (oxidative phosphorylation) Aerobic glycolysis

31. Sequence of reactions + Pyruvate Glucose Glucose metabolism in cell Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate 32 ATP2 ATP

32. Glycolysis regulation Glucose Glucose-6-P Fructose-6-P Fructose-1,6-diP Phosphoenol pyruvate Pyruvate Hexokinase Glucose 6- phosphatase Fructose-1,6- bisphosphatase Phosphofructo kinase1 (PFK1) Pyruvate carboxylase Pyruvate kinase AMP ATP Citrate ATP Acetyl-CoA AMP Acetyl-CoA Inhibition Activation

33. Glycolysis regulation 3 enzymes catalyzing irreversible steps are regulated: 1) Hexokinase 2) Phosphofructokinase-1 3) Pyruvate kinase Feedback or hormonal control

34. Hexokinase regulation Feedback mechanism

35. PFK1 and PFK2. Distinguish them. Only kinase activity Phosporylates F-6-P Produces F-1,6-BP for further glycolysis Insulin activated Glucagon inhibited Both kinase and phospatase activity Regulates F-6-P and F-2,6-BP amount F-2,6-BP doesn’t go to glycolisis PFK1PFK2

36. PFK1regulation. Feedback mechanism.

37. Pyruvate kinase regulation Feedback mechanism

38. Hormonal control Insulin and glucagon are two main hormones controlling glucose methabolism Insulin – fed state hormone Insuline provides glycolysis, glicogen and fatty acid synthesis Glucagon – fasting state hormone Glucagon provides gluconeogenesis, glicogen and fatty acids decomposition

39. Hormonal control over PFK1 and pyruvate kinase PFK2 FBPase-2 PFK2 FBPase-2 ATP ADP ATPADP H2OH2O PiPi Active P H2OH2OPiPi Protein kinase-1 Protein phosphatase-1 Glucagon Insulin Activation of gluconeogenesisActivation of glycolysis Fructose-1-P Fructose-2,6-diP Fructose-1-P

40. Sequence of reactions + Pyruvate Glucose Aerobic and anaerobic glycolysis ATP production Anaerobic Glycolisis CoA 2x + CO 2 Aerobic Glycolisis TCA, ETC, OP Lactate 32 ATP2 ATP

41. In mammals Anaerobic (lactic acid fermentation Aerobic OxidationAnaerobic (alcoholic fermentation) LactatePyruvateEthanol Pyruvate fate

42. Pyruvate dehydrogenase NAD + NADH Pyruvate to AcCoA PDH

43. PDH regulation

44. Pyruvate to lactate

45. Pyruvate to oxaloacetate Pyruvate kinase reaction is irreversible In cytosol glucose and oxaloacetate can not be synthesized from pyruvate Oxaloacetate is TCA intermediate If unsufficient can be synthesized from pyruvate in mytochondria Catalyzed by pyruvatecarboxylase

46. Pyruvate carboxylase Aspartate (transamination) Citrate (TCA cycle) Phosphoenolpyruvate (gluconeogenesis)