GLYCOGEN METABOLISM

1. GLYCOGEN METABOLISM Learning objectives: Describe composition and glycosidic bonds in glycogen Describe the biochemical pathway of glycogen synthesis Describe the biochemical pathway of glycogenolysis Discuss regulation of glycogen metabolism

2. Glycogen Glycogen is a branched homopolysaccharide composed of α-D-glucose units bound by α-1,4 and (at branch points) α-1,6 glycosidic bonds. On average, there are branches for every 8-10 glycosyl residues.

3. Glycogen A single molecule can have a molecular mass of up to 108 Da with more than 500,000 glucosyl residues. Glycogen forms intracellular glycogen granules in the cytoplasm.

4. Electron micrograph of a section of a liver cell showing glycogen deposits as accumulations of electron dense particles (arrows).

5. Glycosyl residue attached by an α-1,6 glycosidic bond Glycosyl residue at a non-reducing end Glycosyl units are attached and mobilized from the reducing ends

6. Glycogen is an intracellular storage form of readily available glucose Main stores of glycogen in the human body: Liver - Approximately 100 g or 10% of the fresh weight Muscle - Approximately 400 g or 1-2% of the fresh weight Most other cells have small amounts of glycogen stored

7. LIVER MUSCLE Glycogen Glucose 6-P Glucose Glycogen Glucose 6-P G6Pase GLYCOLYSIS Energy Blood glucose

8. Sources of blood glucose after a meal mM glucose 8 4 Meal Glycogen Gluconeogenesis 8 16 24 2 7 30 Hours Days

9. Glycogen synthesis Glycogenesis Glycogen is synthesized from molecules of α-D-glucose. Synthesis occurs in the cytosol Synthesis requires energy ATP for phosphorylation of glucose UTP for generating an activated form of glucose: UDP-glucose

10. Glycogen synthesis - Glycogenesis Glucose Glucose 6-phosphate Glucose 1-phosphate UDP-glucose Glycogenn+1 Glycogenn+1 with an additional branch ATP ADP Hexokinase/Glucokinase Phosphoglucomutase UDP-glucose pyrophosphorylase Glycogen synthase Branching enzyme UTP PPi Pyrophosphatase 2 Pi + H2O Glycogenn

11. CH2OH CH2OPO32- Glucokinase Hexokinase O O H H H H H H + ATP + ADP OH H OH H OH OH OH OH H OH H OH Glucose Glucose 6-phosphate Same reaction, same enzymes, and same regulation as in glycolysis Irreversible Hexokinase Glucose 6-phosphate (low phosphofructokinase activity) Glucokinase High blood glucose (release from GKRP, High Km) Insulin stimulates gene transcription (only in liver) - + +

12. Phosphoglucomutase OPO32- Ser CH2OPO32- O H H H CH2OPO32- OH H O OH OH H H OH H Ser H OH OH H Glucose 6-phosphate OH OPO32- CH2OH H OH O H H Glucose 1,6-bisphosphate H OH H OH OPO32- OPO32- H OH Ser Glucose 1-phosphate

13. CH2OH O O O O O- O- O- H H H + O- – P - O – P – O – P – O - uridine OH H OH OPO32- H OH UTP Glucose 1-phosphate UDP-glucose pyrophosphorylase CH2OH O H H H O O O- O- O O O- O- OH H O- – P – O – P – O- OH O – P – O – P – O - uridine + H OH Pyrophosphate (PPi) UDP-glucose

14. O O O- O- Pyrophosphatase O- – P – O – P – O- + H2O 2 Pi NB: Irreversible reaction Pyrophosphate (PPi) Glucose 1-phosphate + UTP UDP-glucose + PPi PPi + H2O 2 Pi Glucose 1-phosphate + UTP + H2O UDP-glucose + 2 Pi The irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose

15. CH2OH CH2OH O O H H H H H H O O O- O- OH H OH H + O - R OH O – P – O – P – O - uridine HO α-1,4 H OH H OH UDP-glucose Glycogen (n residues) Glycogen synthase CH2OH CH2 O O H H H H O O O- O- H H OH H + OH H O- – P – O – P – O - uridine O - R O HO α-1,4 α-1,4 H OH H OH UDP Glycogen (n+1 residues)

16. Priming of glycogen synthesis Glycogen synthase can NOT add glucosyl residues to free glucose or to oligosaccharides of less than 8 glucosyl residues Priming is catalyzed by the protein GLYCOGENIN The first glucosyl residue is attached in an O-glycosidic linkage to the hydroxyl group of tyrosine of Glycogenin itself 7 additional residues are attached by glycogenin Glycogenin remains attached to the reducing end of the glycogen molecule

17. Tyr HO 8 UDP-glucose + Glycogenin Glycogenin Tyr O Glycogenin

18. … Cleaveage of α-1,4 bond Non-reducing end “Branching enzyme” Amylo-α(1,4) →α(1,6)-transglucosidase α-1,6 bond Non-reducing ends …

19. Stoichiometry Glucose + ATP + UTP + H2O + Glycogenn→ Glycogenn+1 + ADP + UDP + 2 Pi

20. Degradation of glycogen Glycogenolysis Occurs in cytoplasm Major product is glucose 1-phosphate from breaking α-1,4 bonds Minor product is glucose from breaking α-1,6 bonds Glucose 1-phosphate : Glucose ≈ 10:1

21. Glycogen synthesis - Glycogenesis Glycogenn Glycogenn-1 Glycogen with branch Glucose Glycogen with one less branch Pi Glycogen phosphorylase Glucose 1-phosphate Glucose 6-phosphate Glucose Phosphoglucomutase G6Pase … H2O Pi “Debranching enzyme” H2O Glycolysis

22. CH2OH CH2OH CH2 O O O O O- H H H H H H H H H + O- – P – OH OH H OH H OH H O - R O O HO α-1,4 α-1,4 H OH H OH H OH Phosphate Glycogen with n residues Glycogen phosphorylase CH2OH CH2 CH2OH O O O H H H H H H H H H + OH H OH H OH H O - R O HO OH OPO32- α-1,4 H OH H OH H OH Glucose 1-phosphate Glycogen with n-1 residues

23. Lys N H C OH CH3 2-O3PO-CH2 + NH Pyridoxal phosphate is a coenzyme for the phosphorylase reaction. Pyridoxal phosphate is bound to a nitrogen of a lysyl residue of glycogen phosphorylase The phosphate of pyridoxal phosphate exchanges protons with the phosphate reactant, which allows the reactant to donate a proton to the oxygen atom on carbon 4.

24. CH2OH Phosphoglucomutase O OPO32- H H Ser H OH H OH OPO32- CH2OPO32- H OH O H H Glucose 1-phosphate OH H Ser OH H OH OPO32- H OH CH2OPO32- Glucose 1,6-bisphosphate O H H H OH H OH OH OPO32- Ser H OH Glucose 6-phosphate

25. Glucose-6- phosphatase (G6Pase) CH2OH CH2OPO32- O O H H H H H H + H2O + Pi OH H OH H OH OH OH OH H OH H OH Glucose Glucose 6-phosphate Same reaction as in gluconeogenesis Occurs in endoplasmic reticulum and involves a glucose 6-phosphatase transporter and a catalytic subunit The catalytic subunit is regulated at the level of transcription

26. Glycogen phosphorylase stops when 4 glucosyl units remain on each chain from a branch point a’ b’ c’ α-1,6 bond d’ … a b c d e Oligo-α(1,4)→α(1,4)-glucan transferase (debranching enzyme) Amylo-α(1,6)-glucosidase (debranching enzyme) d’ α-1,6 bond … a’ b’ c’ a b c d e H2O d’ + … a’ b’ c’ a b c d e Glucose

27. Approximate Stoichiometry Glycogenn+11 + 10 Pi + H2O → Glycogenn + 10 Glucose 6-phosphate + Glucose

28. LIVER MUSCLE Glycogen Glucose 6-P Glucose Glycogen Glucose 6-P G6Pase GLYCOLYSIS Energy Blood glucose

29. Regulation of glycogen metabolism Skeletal muscle Glycogen must be broken down to provide ATP for contraction, when the muscle is rapidly contracting, or in anticipation of contractions in stress situations like fear or excitement. In rapidly contracting muscle: Low [ATP], High [AMP] High [Ca++] Stress: High [Epinephrine] Glycogen stores are replenished when muscles are resting. Resting state: Low [AMP], High [ATP]

30. Hormonal regulation of metabolism Hormone Type Secreted by Secreted in response to Insulin Protein Pancreatic beta cells High blood [glucose] Glucagon Polypeptide Pancreatic alpha cells Low blood [glucose] Epinephrine Catecholamine Adrenal medulla Stress (adrenalin) Nervous system Low blood [glucose] Glucocorticoids Steroid hormone Adrenal cortex Stress Low blood [glucose] Glucagon is the most important hormone signaling low blood glucose concentration, while epinephrine and glucocorticoids play secondary roles.

31. Regulation of glycogen metabolism Liver Glycogen must be broken down to provide glucose for maintaining blood glucose in fasting or for providing additional glucose for skeletal muscles in stress situations. Fasting: High [Glucagon] Stress: High [Epinephrine] Glycogen stores must be replenished in the fed state Fed state: High [Insulin] High [Glucose]

32. Muscle Glycogen Glucose 6-phosphate Glycogen Glucose 6-phosphate Rapidly contracting state Stress Resting state and with abundant energy Liver Glycogen Glucose 6-phosphate Glycogen Glucose 6-phosphate Fasting state Stress Fed state

33. Key regulatory enzyme of glycogen breakdown: Glycogen phosphorylase Key regulatory enzyme of glycogen synthesis: Glycogen synthase

34. Glycogen phosphorylase is a dimer of identical subunits. Glycogen phosphorylase can exist in an active R (relaxed) and an inactive T (tense) state. In the T state, the catalytic site is partly blocked

35. Red: active site Yellow: Glycogen binding site Red site: Allosteric site for AMP binding Blue/green sites: Phosphorylation sites

36. Allosteric regulation of glycogen phosphorylase Regulation by energy state. AMP (binding favors the active R state) ATP (binding favors the inactive T state) + - Regulation by feedback inhibition. Glucose 6-phosphate (G6P) G6P concentration increases when G6P is generated faster than it can be further metabolized, e.g. by glycolysis - Regulation by high blood glucose Glucose (Only liver glycogen phosphorylase) In the fed state with a high blood glucose concentration, there is no need for the liver to secrete glucose -

37. Regulation of glycogen phosphorylase by phosphorylation Phosphorylase kinase ATP ADP P Glycogen phosphorylase b Glycogen phosphorylase a P Inactive Active T state R state Pi H2O Protein phosphatase 1 (PP1) Phosphorylation occurs in the fasted or stressed state Dephosphorylation is stimulated in the fed state

38. Phosphorylase kinase is regulated by phosphorylation and Ca++ binding One subunit is the Ca++ -binding calmodulin Ca++ Ca++ Inactive Partly active Fully active Phosphorylation occurs in the fasted or stressed state. Dephosphorylation is stimulated in the fed state. Ca++ binding occurs when the [Ca++] is high, e.g. during rapid muscle contractions

39. Cell membrane

40. cAMP Adenylyl cyclase ATP cAMP + PPi H2O Phosphodiesterase AMP

41. Glucagon receptors and epinephrine receptors are G-protein-coupled receptor GDP Adenylyl cyclase GDP Receptor beta and gamma subunit of G-protein alpha subunit of G-protein GTP When hormone is no longer present, intrinsic GTP hydrolase activity of the G-protein alpha subunit hydrolyzes GTP to GDP, the alpha subunit re-associates with the beta and gamma subunits, and stimulation of adenylyl cyclase ends. cAMP is converted to AMP by phosphodiesterase. Thus, in the absence of hormone, the cAMP concentration rapidly falls.

42. α α α α α α Insulin β β β β β β P P P P Insulin receptor It functions as a tyrosine kinase when insulin is bound P P P P Autophosphorylation P Insulin receptor substrate Activation of protein phosphatases Activation of multiple signaling pathways Activation of protein kinases In general, the protein kinases activated by insulin have opposite biological effects from those activated by glucagon In general, the protein phosphatases activated by insulin dephosphorylate proteins that are phosphorylated by glucagon-stimulated protein kinases, such as PKA

43. Regulation of glycogen synthase Regulation by feed-forward mechanism. Glucose 6-phosphate (G6P) G6P concentration increases at high glucose concentrations when G6P is generated faster than it can be further metabolized + NB: Reciprocal regulation of glycogen synthase and glycogen phosphorylase by glucose 6-phosphate

44. Regulation of glycogen synthase by phosphorylation PKA and Glycogen synthase kinase ATP ADP P P Active Inactive Pi H2O Protein phosphatase 1 (PP1) Phosphorylation occurs in the fasted or stressed state Dephosphorylation is stimulated in the fed state

45. Reciprocal regulation of glycogen phosphorylase and glycogen synthase by phosphorylation Fasting/stress (glucagon/epinephrine) + PKA Phosphorylase kinase Active Glycogen phosphorylase Glycogen synthase + PP1 Fed state (insulin)

46. And it is even more complex.. Scaffolding proteins of different subtypes in liver and muscle can bind the glycogen particle, PP1, glycogen phosphorylase, and glycogen synthase Binding brings participants of glycogen metabolism together. Regulation of PP1 is itself complex with various inhibitors responding to the metabolic state of the organism.