Regulation of glycolysis and g luconeogenesis

1. Regulation of glycolysis and gluconeogenesis Dr. Vér Ágota

2. Gluconeogenesis(Synthesis of glucose) from pyruvate utilizes many of the same enzymes as Glycolysis. Three Glycolysis reactions have such a large negative DG that they are essentially irreversible. These steps must be bypassed in Gluconeogenesis. Glycolysis occurs in all tissues Gluconeogenesis occurs mainly in liver (to a more limited extent in kidney & small intestine under some conditions). 

3. Irreversible glycolytic stepsbypassed glycolysis gluconeogenesis by Glucose-6-phosphatase by Fructose 1,6-bisphosphatase (FBP-1) by Pyruvate Carboxylase (PC) & Phosphoenolpyruvate carboxykinase (PEPCK) • Glucokinase (GK) • Phosphofructokinase-1 (PFK-1) • Pyruvate kinase (PK)

4. Glucose production vs utilisation • Production: gluconeogenesis (6 high energy bound is required) • Utilization: glycolysis ( net output 2 ATP) • Futile cycling very wasteful • Direction and magnitude of substrate movements controlled

5. Control of glycolysis and gluconeogenesis glucose Regulated steps of glycolysis (glucose transport and the irreversible reactions) GLUT glucose GK G6P G6Pase F6P PFK1 F1,6BP FBPase plasma membrane Regulated steps In gluconeogenesis PEPCK PEP PK EC space oxalacetate pyruvate cytosol PC

6. Reaction of glucokinase and glucose 6-P phosphatase glucose GK G-6-Pase Glucose-6-P

7. Properties of Glucokinase and Hexokinase Substrate specificity Glucose Hexose

8. GK regulation • Protein-protein interaction (F-6-P, F-1-P, glucose) • Gene transcription: insulin, feeding + glucagon, fasting -

9. Regulation of activity of Glucokinase (GK ) regulatory protein (RP) fructose glucose glucose + GK active GK-RP inactive glucose 6-phosphate + - fructose 6-phosphate RP ATP ADP PFK1 fructose 1,6-bisphosphate FK fructose Fructose 1-phosphate

10. Glucokinase – glucose sensor function - blood glucose - +  cell glucokinase hepatocyte glucokinase +  cell Insulin secretion + +

11. Glucose -6-phosphatase system SP=stabilizing protein T1= glucose 6-phosphate transporter T2= phosphate transporter T3= glucose transporter

12. Glucose -6-phosphatase system • Liver, kidney, pancreatic beta cells,gall bladder, testis, spleen, intestines • Km : 2 mM • G-6-P (0.2 mM) the flux though this step is proportional to i.c. G-6-P • At 5 mM level GK activity is balanced by opposing activity of G-6-P phosphatase • G-6-P phosphataseis activeted in starvation and diabetes • Gene transcription occurs similarly as PEPCK (insulin inhibits) • Induced after birth

14. Regulation of glycolysis (PFK1) and gluconeogenesis (Fructose1,6-Bisphosphatase) • The opposite effectsof adenine nucleotides on • Phosphofructokinase (Glycolysis) • Fructose-1,6-bisphosphatase (Gluconeogenesis) • ensures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP. • When ATP is high it is more useful to the cell to store glucose as glycogen. • When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose.

15. Phosphofructokinase 1 • Allosteric inhibitors: ATP, citrate, fatty acids • Activators: AMP, F-2,6-P • Irreversible, main place of regulation „committed step” • Citrate increases the inhibitory effect of ATP • F-2,6-P –inhibition of the inhibitory effect of ATP T – R transition Tetramer structure (370 kD) – sigmoidal curve M:muscle, P:platelet, L:liver isoenzymes

16. PFK1 is inhibited by ATP PFK activity At high concentration, ATP binds at a low-affinity regulatory site, promoting the tense conformation. Sigmoidal dependence of reaction rate on [fructose-6-phosphate] is observed at high [ATP ]

17. Phosphofructokinase, the rate-limiting step ofGlycolysis, is allosterically inhibited by ATP. • At high concentration, ATP binds at a low-affinity regulatory site, promoting the tense conformation. • Sigmoidal dependence of reaction rate on [fructose-6-phosphate is observed at high [ATP ] • PFK activity in the presence of the globally controlled allosteric regulator fructose-2,6-bisphosphate is similar to that at low ATP. • Fructose-2,6-bisphosphate promotes the relaxed state, activating Phosphofructokinase even at high [ATP]. • Thus activation by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supersedes local control by [ATP].

18. The effect of F2,6BP on F1,6BP phosphatase

19. ATP ADP Pi H2O F 2,6-bP PFK1 – F1,6bPase co-ordinated regulation fructose 6-phosphate tandem enzyme insulin glucagon ATP ADP Pi H2O F1,6- bPase GLUCONEOGENESIS GLICOLYSYS PFK1 AMP, ADP ATP, citrate FA, H+ fructose 1,6-bisphosphate Reciprocal control

20. Fructose 2,6 –bisphosphate is not a glycolyses intermediate Phosphorylated form:phosphatase Dephosphorylated form:kinase

21. PFK2/FBPase2

23. Pyruvate kinase Tissue-specific isoenzymes. PK-L (in liver) is regulated allosterically Feedforward activation by F-1,6 BP +, alanin, ATP -. and hormonally (cAMP dependent phosphorylation = inactivation) PK-M (in skeletal muscle) is not regulated.

24. dephosphorylation phosphorylation Regulation of pyruvate kinase in liver fructose 1,6-bisphosphate feed-forward activation phosphoenolpyruvate INSULIN ADP ATP PK-L GLUCAGON ATP alanine pyruvate

25. Pyruvate Carboxylase (pyruvate oxaloacetate) is allosterically activated by acetyl CoA. [Oxaloacetate] tends to be limiting for Krebs cycle. When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.

26. PEPCK • Not an allosteric enzyme • Induced by glucagon

27. Coordinated Regulation of Gluconeogenesisand GlycolysisSUMMARY • Regulation of enzyme quantity • Fasting: glucagon, cortisol • induces gluconeogenic enzymes • represses glycolytic enzymes • liver making glucose • Feeding: insulin • induces glycolytic enzymes • represses gluconeogenic enzymes • liver using glucose

28. Coordinated Regulation of Glycolysis and Gluconeogenesis • Allosteric Effects • Pyruvate kinase vs Pyruvate carboxylase • PK - Inhibited by ATP and alanine • PC - Activated by acetyl CoA • PFK-1 vs FBPase-1 • PFK-1 activated by AMP and & F2,6P2 • FBPase-1 inhibited by AMP & F2,6P2

29. Coordinated Regulation of Gluconeogenesis and Glycolysis • Short-term Hormonal Effects • Glucagon, Insulin • cAMP & F2,6P2 • PFK-2 & FBPase-2 • A Bifunctional enzyme • cAMP • Inactivates PFK-2 • Activates FBPase-2 • Decreases F2,6P2 • Reduces activation of PFK-1 • Reduces inhibition of FBPase-1 • Low blood sugar results in • High gluconeogenesis • Low glycolysis

30. glycolysis and gluconeogenesis Summary • The gluconeogenesis pathway issimilar to the reverse of glycolysisbut differs at critical sites. • control of these opposing pathways is reciprocal so that physiologicalconditions favoring one disfavor theother and vice versa • General principles of metaboliccontrol -- a) pathways are not simplereversals of each other andb) under reciprocal control

31. Digestion of carbohydrate

32. GI Tract Functions • •Digestion -breakdown of complex macromolecules to di-& monomeric molecules. • •Absorption-fuels traverse GI track to cells & tissues of the body. • •Fuel sources. • –Carbohydrates. • –Lipids. • –Proteins.

33. Digestion of carbohydrates • Dietary carbohydrate from which humans gain energy enter the body in complex forms, such as disaccharides and the polymers starch (amylose and amylopectin) and glycogen. • The polymer cellulose is also consumed but not digested. • The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers to simpler, soluble forms that can be transported across the intestinal wall and delivered to the tissues.

34. The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and contains lingual amylase that begins the digestion of carbohydrates. The action of lingual amylase is limited to the area of the mouth and the esophagus; it is virtually inactivated by the much stronger acid pH of the stomach. Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific gastric proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as chyme, moves to the small intestine.

36. Amylase • The α-amylases are calciummetalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. • In animals, it is a major digestive enzyme and its optimum pH is 6.7-7.0. • In human physiology, both the salivary and pancreatic amylases are α-Amylases.

37. Sucrase • Sucrase is the name given to a number of enzymes that catalyse the hydrolysis of sucrose to fructose and glucose • Sucrose intolerance (also known as Congenital Sucrase-Isomaltase Deficiency (CSID) or Sucrase-isomaltase deficiency) occurs when sucrase is not secreted in the small intestine. With sucrose intolerance, the result of consuming sucrose is excess gas production and often diarrhea and malabsorption. • Sucrase is secreted by the tips of the villi of the epithelium in the small intestine. Its levels are reduced in response to villi-blunting events such as celiac sprue and the inflammation associated with the disorder. The levels increase in Pregnancy/Lactation and Diabetes as the villi hypertrophy.

38. Lactase • Lactase (LCT), a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharidelactose into constituent galactose and glucosemonomers. • In humans, lactase is present predominantly along the brush bordermembrane of the differentiated enterocytes lining the villi of the small intestine. • Lactase is essential for digestive hydrolysis of lactose in milk. Deficiency of the enzyme causes lactose intolerance.

39. Carbohydrate Digestion & Absorption • Carrier mechanisms for monosaccharides(glucose, fructose and galactose). • –Na+-independent facilitated diffusion. • Fructose transport. • In conjunction with glucose transporter (GLUT-5). • –Located on serosalside of enterocytemembrane. • –Moves glucose into capillaries. • –Fructose moves down its concentration gradient.

41. Glut5 5

42. Sodium-dependent glucose cotransporters • Sodium-dependent glucose cotransporters are a family of glucose transporter found in the intestinal mucosa of the small intestine (SGLT1) and the proximal tubule of the nephron (SGLT2 and SGLT1). They contribute to renal glucose reabsorption. • These proteins use the energy from a downhill sodium gradient to transport glucose across the apical membrane against an uphill glucose gradient. Therefore, these co-transporters are an example of secondary active transport. Both SGLT1 and SGLT2 are known as symporters since both sodium and glucose are transported in the same direction across the membrane.

43. Passive transport - GLUTs • Facilitated diffusion of glucose through the cellular membrane is catalyzed by glucose carriers (protein symbol GLUT, gene symbol SLC2 for Solute Carrier Family 2) that belong to a superfamily of transport facilitators (major facilitator superfamily). • Molecule movement by such transporter proteins occurs by facilitated diffusion. This makes them energy independent.

44. GLUT transporters Plasma membrane carriers of glucose. Catalyze facilitated diffusion (passive, bi-directional trp.) 12 transmembrane helices. More than 5 isoforms with different function and characteristics.

45. GLUT transporters

46. GLUT transporters GLUT1 and GLUT3 high affinity (KM≈ 1 mM) Expressed in every cell except hepatocytes (liver) and pancreatic β-cells. Ensures steady glucose uptake in RBC, CNS, kidney medulla, testis (glucose-dependent cells). Blood- brain, blood placenta- barrier GLUT2 low affinity (KM ≈ 15 mM) Expressed in hepatocytesand pancreatic β-cells (glucose sensor cells). Makes glucose uptake proportional with blood glucose concentration. GLUT4 Intermediate affinity (KM ≈ 5 mM) Insulin-dependent expression in skeletal muscle and adipocytes (facultative glucose consuming cells). Adjusts glucose consumption to availability. GLUT5 Expressed in intestinal epithelial cells and kidney tubular epithelial cells. Participates in glucose absorption and re-absorption.