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 10 8 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. Glycogen Glucose 6-P Glucose Blood glucose Glycogen Glucose 6-P G6Pase GLYCOLYSIS LIVER MUSCLE

8. Sources of blood glucose after a meal Meal Glycogen Gluconeogenesis 8 16 24 2 7 30 Hours Days mM glucose 8484

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

11. O H OH OH H CH 2 OH H OH HH + ATP+ ADP Glucokinase Hexokinase Same reaction, same enzymes, and same regulation as in glycolysis Irreversible Glucose Glucose 6-phosphate O H OH OH H CH 2 OPO 3 2- H OH HH Hexokinase Glucose 6-phosphate (low phosphofructokinase activity) Glucokinase High blood glucose (release from GKRP, High K m ) Insulin stimulates gene transcription (only in liver) - + +

12. O H OH OH H CH 2 OPO 3 2- H OH HH O H OH OH H CH 2 OH H OH HH OPO 3 2- O H OH OH H CH 2 OPO 3 2- H OH HH OPO 3 2- Glucose 6-phosphate Glucose 1-phosphate Glucose 1,6-bisphosphate OH Ser OPO 3 2- Ser OPO 3 2- Ser Phosphoglucomutase

13. O H OH OH H CH 2 OH H OH HH OPO 3 2- Glucose 1-phosphate O H OH OH H CH 2 OH H OH HH O – P – O – P – O - uridine UDP-glucose O O - + O - – P - O – P – O – P – O - uridine O O O O - O - O - UTP O - – P – O – P – O - O O - Pyrophosphate (PPi) + UDP-glucose pyrophosphorylase

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

15. O H OH OH H CH 2 OH H OH HH O – P – O – P – O - uridine UDP-glucose O O - + O H OH OH H CH 2 OH H HH HO O - R α-1,4 Glycogen (n residues) O H OH OH H CH 2 H HH O H OH OH H CH 2 OH H HH O α-1,4 O - R α-1,4 HO O - – P – O – P – O - uridine O O - + Glycogen (n+1 residues) UDP Glycogen synthase

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 Glycogenin Tyr O Glycogenin 8 UDP-glucose + Glycogenin

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

19. Stoichiometry Glucose + ATP + UTP + H 2 O + Glycogen n → Glycogen n+1 + ADP + UDP + 2 P i

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 n Glycogen n-1 Glycogen with branch Glucose Glycogen with one less branch Pi Glycogen synthesis - Glycogenesis Glucose 1-phosphate Glucose 6-phosphate Glucose Phosphoglucomutase G6Pase H2OH2O Glycolysis Glycogen phosphorylase … “Debranching enzyme” H2OH2O Pi

22. O H OH OH H CH 2 OH H HH O H OH OH H CH 2 H HH O H OH OH H CH 2 OH H HH O O α-1,4 O - R HO O - – P – OH OO-OO- + + O H OH OH H CH 2 OH H OH HH OPO 3 2- Glucose 1-phosphate Glycogen with n-1 residues O H OH OH H CH 2 H HH O H OH OH H CH 2 OH H HH O α-1,4 O - R HOHO Phosphate Glycogen with n residues Glycogen phosphorylase

23. NHNH C N H Lys OH CH 3 2- O 3 PO-CH 2 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. O H OH OH H CH 2 OH H OH HH OPO 3 2- O H OH OH H CH 2 OPO 3 2- H OH HH OPO 3 2- Glucose 1-phosphate Glucose 1,6-bisphosphate OH Ser OPO 3 2- Ser OPO 3 2- Ser Phosphoglucomutase O H OH OH H CH 2 OPO 3 2- H OH HH Glucose 6-phosphate

25. O H OH OH H CH 2 OPO 3 2- H OH HH Glucose 6-phosphate O H OH OH H CH 2 OH H OH HH Glucose + H 2 O + P i Glucose-6- phosphatase (G6Pase) 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. … α-1,6 bond Glycogen phosphorylase stops when 4 glucosyl units remain on each chain from a branch point bc a’ b’ a c’ d’ de … bcade b’c’a’ α-1,6 bond d’ Oligo-α(1,4)→α(1,4)-glucan transferase (debranching enzyme) Amylo-α(1,6)-glucosidase (debranching enzyme) … bcade b’c’a’ d’ + Glucose H2OH2O

27. Approximate Stoichiometry Glycogen n+11 + 10 P i + H 2 O → Glycogen n + 10 Glucose 6-phosphate + Glucose

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

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 HormoneTypeSecreted by Secreted in response to InsulinProteinPancreatic beta cellsHigh blood [glucose] GlucagonPolypeptidePancreatic alpha cellsLow blood [glucose] EpinephrineCatecholamineAdrenal medullaStress (adrenalin)Nervous systemLow blood [glucose] GlucocorticoidsSteroid hormoneAdrenal cortexStress 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 Glycogen Glucose 6-phosphate Glycogen Glucose 6-phosphate Rapidly contracting state Stress Resting state and with abundant energy Fasting state Stress Fed state Liver

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. Regulation by energy state. - + AMP (binding favors the active R state) ATP (binding favors the inactive T state) Allosteric regulation of glycogen phosphorylase 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 P P Inactive Active T state R state Glycogen phosphorylase b Glycogen phosphorylase a ATPADP Phosphorylase kinase Protein phosphatase 1 (PP1) H2OH2O PiPi Phosphorylation occurs in the fasted or stressed state Dephosphorylation is stimulated in the fed state

38. 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 Phosphorylase kinase is regulated by phosphorylation and Ca ++ binding One subunit is the Ca ++ -binding calmodulin Ca ++ Inactive Partly active Fully active

39. Cell membrane

40. ATP cAMP + PP i Adenylyl cyclase cAMP H2OH2O Phosphodiesterase AMP

41. Receptor Adenylyl cyclase GDP GTP beta and gamma subunit of G-protein alpha subunit of G-protein Glucagon receptors and epinephrine receptors are G-protein-coupled receptor GDP 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. β β αα Insulin Insulin receptor It functions as a tyrosine kinase when insulin is bound β β αα P P P P Insulin receptor substrate Autophosphorylation β β αα P P P P P Activation of multiple signaling pathways Activation of protein phosphatases Activation of protein kinases, the protein kinases activated by insulin have opposite biological effects In general, the protein kinases activated by insulin have opposite biological effects from those activated by glucagon, the protein phosphatases activated by insulin dephosphorylate proteins 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 P P Active Inactive ATPADP PKA and Glycogen synthase kinase Protein phosphatase 1 (PP1) H2OH2O PiPi 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 Fed state (insulin) PP1 + + Phosphorylase kinase Glycogen phosphorylase Glycogen synthase Active

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.