Glucose and Glycogen Regulation PDF

Glucose and Glycogen Regulation Marc A. Ilies, Ph. D. Lehninger - Chapter 15 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas...

Glucose and Glycogen Regulation Marc A. Ilies, Ph. D. Lehninger - Chapter 15 [email protected]; lab 517, office 517A (Tu, Fr 3-5) For questions, comments please use the discussion tool in Canvas ©MAIlies2024 1  Glucose dynamics in living organisms - the amount of glucose in blood (glycemia) is tightly regulated and is the result of several processes in which glucose is produced or consumed : - excess glucose is stored as glycogen (glucose is a potent osmolyte, it cannot be stored in large amounts) from which it can be liberated when needed, in order to maintain normal glycemia 2  Glycogen Degradation - glycogen: branched polymer of glucose (1→4 and 1→6 linkages) - in vertebrates glycogen found in liver (up to 10% weight) and skeletal muscles (up to 1-2% weight) - glucose residues are removed from the non-reducing end, via phosphorolysis: 3 (note that sometimes glycogen can be also hydrolyzed with H2O, with maltase – slide 6)  Glycogen Degradation: debranching - mechanism: Phosphorolysis - outcome: G-1P Debranching 1 Debranching 2 - outcome: Glucose 4  Glycogen degradation and regulation of glucose level - glucose-1-phosphate is converted to glucose-6-phosphate: phosphoglucomutase - glucose-6-phosphate can undergo glycolysis, the pentose pathway, or it can regenerate glucose (gluconeogenesis) via glucose-6-phosphatase (see 14-24); last process segregated into ER of liver cells, kidney cells: Glucose-6 phosphatase H2O Pi - muscle and adipose tissue cells do not have glucose 6-phosphatase, 5 gluconeogenesis is done mainly in the liver Glycogen Storage Diseases - a small amount of glycogen is degraded on a continuous basis by an enzyme known as acid maltase or (14)-glucosidase. Why this happens is not fully understood; - About 1:40,000 newborns exhibit α(14)-glucosidase deficiency, translating into Pompe Disease. - In April of 2006 the FDA approved a recombinant form of this enzyme sold as Myozyme . Movie about the story: https://www.yo utube.com/watch?v=PQiB0ElK84Q -deficiency in muscle glycogen phosphorylase: McArdle Syndome (myopathy: exercise intolerance, early fatigue, painful muscle cramps, myoglobinuria) 6  Glycogen Storage Diseases - Glucose 6-Phosphatase deficiency: Von Gierke Disease - most common glycogen storage disease 7  Glycogen Synthesis - abundant glucose (after meals) enters liver and muscle cells where is phosphorylated by hexokinase to glucose 6-phosphate (trapped inside cells) phosphoglucomutase pyrophosphorylase 8 Glycogen Synthesis - chain elongation: 9  Glycogen Synthesis - branching: - a glycogen branching enzyme will cut the linear chain after another three sugar units (will cut an α1→4 linkage) and will reattach it to position 6 of the reference monosaccharide (will form an α1→6 linkage); both non-reducing ends can be further elongated and (re)branched 10  Glycogen Synthesis - Priming is ensured by protein glycogenin (a glucosyltransferase), which attaches first 6 glucose units on itself (on Tyr-194 OH group): 11  Regulation of metabolic pathways - cells and organisms maintain a dynamic steady state: v1 v2 A→S→P when v1 = v2 , [S] = constant (homeostasis) - factors determining enzyme activity 12  Covalent modifications of enzymes - occur within seconds or minutes of a regulatory signal (extracellular signal) - most common: phosphorylation/dephosphorylation; alters the electrostatics: Kinases add phosphates Phosphatases remove them. Effects: - conformational changes (vmax, KM) - active site modification - interaction with other proteins promoted/altered 13 Assessment of individual enzyme contribution to a pathway - the contribution of each enzyme to metabolic flux through a pathway can be determined experimentally: 14 Coordinated regulation of glycolysis and gluconeogenesis - achieved at the level of the three irreversible (exergonic) steps: - G values for liver in kJ/mol: -32.9 -26.4 - if both pathways are left unregulated: ATP + F-6P → ADP + F-1,6-BP F-1,6-BP + H2O → F-6P + Pi -24.4 -8.6 ATP + H2O → ADP + Pi + heat (PFK) (FBPase) (futile cycle) All reactions highlighted are exergonic ! -26.4 -22.6 15  Regulation of glycolysis at hexokinase level - hexokinase - 4 isozymes; hexokinase I in muscles, hexokinase IV (glucokinase) in liver - isozymes I-III have low Km, low Vmax and are inhibited by glucose 6- phosphate; take care of glucose phosphorylation in muscles and other tissues - liver isozyme IV (glucokinase) has special kinetic properties as compared with muscle isozyme – higher Km, higher Vmax, not inhibited by glucose 6-phosphate; when blood glucose increase, its activity increases too: 16  Hexokinase isozyme regulation Hexokinase (isozymes I-III) Glucokinase (isozyme IV) Distribution Most Tissues Liver and  cells Km Low 0.1mmol/L (2mg%) High 10mmol/L ( 200mg %) Vmax Low High Inhibition by G-6-P Yes No - GK functions when glucose levels are high - after meals - the high Vmax allows for efficient removal of glucose and the high Km prevents the enzyme from becoming saturated - GK is not inhibited by its substrate G-6-P so it can continuously process glucose, bringing its level in blood to normal 17  Regulation of glycolysis at PFK level - PFK-1 is allosterically regulated by ATP: (enhanced further by citrate – link with citric acid cycle) 18  Regulation of glycolysis at pyruvate kinase level - enzyme allosterically inhibited by ATP, acetyl-CoA, fatty acids (all signs of energy abundance), and alanine; it is activated by F1,6BP; - the enzyme in the liver is additionally regulated by glucagon, through the activation of a cAMP- dependent protein kinase (PKA), which phosphorylates the enzyme, inactivating it (liver switched on 19 glucose production when glucose level is low); a protein phosphatase (PP) can restore the activity Coordinated regulation of gluconeogenesis - first enzyme in each pathway is regulated allosterically by acetyl-CoA = fate of pyruvate is determined by the energy requirements of the cell 20  Coordinated regulation of glycolysis and gluconeogenesis - ATP and citrate inhibit glycolysis; ADP, AMP activate glycolysis and inhibit gluconeogenesis: - another important regulatory role is played at this level by fructose-2,6-bisphosphate 21 Fructose 2,6-bisphosphate: allosteric regulator of PFK-1 and FBPase-1 PFK-1 (and glycolysis) FBPase-1 (and gluconeogenesis) Not to be confused with fructose 1,6-bisphosphate intermediate in glycolysis 22 Coordinated regulation of glycolysis and gluconeogenesis - fructose 2,6-bisphosphate activates glycolysis and inhibits gluconeogenesis 23 PFK-2/FBPase-2 – protein with double enzymatic activity - F26BP synthesized/hydrolyzed by a protein with dual activity: (in the same protein) - activity controlled by hormones insulin and glucagon: AKA protein kinase A (PKA) 24 (see also 14-8) Coordinated regulation at the level of F6P/F1,6bisP: overview Gluconeogenesis glucose & glycogen Fructose-6-P Pi Phosphoprotein phosphatase PFK- 2 Stimulates PFK- 2 PFK- 2 (inactive) (active) OPO3 - Insulin OH FBPase-2 FBPase-2 (active) (inactive) FBPase-2 c-AMP dependent (previous two slides protein kinase combined) ADP Stimulate ATP Glucagon Pi c-AMP ATP FBPase-1 Fructose-2,6-P PFK-1 Inhibits Stimulates ADP H 2O Fructose-1,6-P pyruvate Glycolysis 25  Coordinated regulation of glycogen synthesis and breakdown: Control of glycogen breakdown - breakdown of glycogen achieved by phosphorylase; enzyme is present in two forms: phosphorylase a (catalytically active) and phosphorylase b (less active): Cascade-like activation, see next slide 26  Stimulation of glucose generation in muscles and liver via glycogen phosphorolysis - in hepatocytes: only glucagon - in muscles: HO OH CH3 HO CH CH2 N H Epinephrine (Adrenaline) a catecholamine - fight or flight response hormone: - bronchodilation - increased heart rate and stroke - raised blood pressure - Ca2+ and AMP: upregulate 27 glycogenolysis Glycogen phosphorylase in liver can act as a glucose sensor - in response to high glucose, phosphorylase a exposes its phosphates to the action of insulin sensitive phosphatase and thus decreases the breakdown of glycogen by conversion of active phosphorylase a to inactive form b: - Note that phosphorylase is active when phosphorylated (and has phosphate as substrate…!) 28 Coordinated regulation of glycogen synthesis and breakdown: Control of glycogen synthesis - Glycogen synthesis achieved by glycogen synthase, which can exist in two forms: glycogen synthase a (active) and glycogen synthase b (phosphorylated, inactive): Priming with Casein Kinase II - activation by PP1, inactivation by GSK3 after priming by casein kinase II: Note that PP1 is the same enzyme that inactivates glycogen phosphorylase! 29  Control of glycogen synthesis from blood glucose in myocytes - in muscles, besides activation of glycogen synthase, insulin induces relocation of GLUT4 transporter to the plasma membrane and activates hexokinase: Most important in increasing the flux towards glycogen 30  Carbohydrate regulation in liver cells (overview) - antagonistic effects by insulin and glucagon: (review previous slides for all details) 31 Hormones such as insulin act simultaneously at several pathways and at different levels (on different enzymes) to produce the desired overall effect 32  Differences in carbohydrate metabolism in liver and muscles - muscles do not contribute to blood glucose levels (lack phosphatase); stimulated by epinephrine - liver cells stimulated to produce glucose by both glucagon and epinephrine; note the opposite effect of epinephrine for glycolysis in liver and muscles! Phosphatase (lacking in muscles) 33  Effects of Insulin Inhibits adenyl cyclase Inhibits cAMP-dependent protein kinase (PKA) Glycogen synthesis Glycogen degradation is favored is inhibited Stimulates Glycogen Inhibits Phosphorylase Synthase kinase Stimulates Phosphoprotein phosphatase 34 Effects of Glucagon, epinephrine and insulin on carbohydrate metabolism Glucagon Epinephrine Insulin Source Pancreatic Adrenal Pancreatic  cells medulla  cells Primary Target Liver Muscle>Liver Muscle, liver, adipose tissue Effects on: [cyclic-AMP]    [Fructose-2,6,-bisphosphate]   ( in muscle)  Gluconeogenesis    Glycogen breakdown    35 36  Goals and Objectives Upon completion of this series of lectures at minimum you should be able to answer the following: ► How are organisms maintaining the glucose level constant and which are the main pathways involved? ►Which enzymes are involved in glycogen degradation, what are their particularities and products, what diseases can be observed in case of their deficeincy? ►Which are the main steps, intermediates and enzymes involved in glycogen synthesis, what are their particularities? ►Which are the common ways to regulate metabolic pathways in vivo, what common enzyme chemical modification are used (with examples), what are the consequences in their kinetic parameters, and how can we assess them? ►At which steps the coordinated regulation of glycolysis/gluconeogenesis occurs, what enzymes, intermediates, hormones and secondary messengers are involved and what is their specific effect/activity/mechanism of action? ► At which steps the coordinated regulation of glycogenolysis/glycogenoneogenesis occurs, what enzymes, intermediates, hormones and secondary messengers are involved and what is their specific effect/activity/mechanism of action? How is carbohydrate synthesis and dynamics integrated? ►Which are the main effects of glucagon, insulin, epinephrine, which are their primary targets as a function of the tissue, secondary messengers involved, and 37 what is their individual effect on these entities?  Drugs and diseases ► Diseases: Pompe disease, McArdle Syndrome, Von Gierke disease, Diabetes, Obesity ► Drugs: Myozyme, Epinephrine (adrenaline) ► Hormones: Insulin, glucagon, epinephrine (adrenaline) 38