9/13/2019 Carbohydrate Metabolism V & VI - Regulation of Glycolysis & Gluconeogenesis - - Glycogen Metabolism - FScN4621W Food Science and Nutrition University of Minnesota Unit V Regulation of Glycolysis & Gluconeogenesis 1 9/13/2019 Regulation of Glycolysis and Gluconeogenesis Regulation of Enzyme Activities Key Reactions in Glycolysis and Gluconeogenesis glycolysis Glucokinase Hexokinase Glucose glucose 6-phosphate Glucose 6-phosphatase gluconeogenesis Enzymes are regulated at both activity and gene expression levels 2 9/13/2019 Hexokinase/Glucokinase Hexokinase I ◦ Predominate in Skeletal Muscle ◦ Low Km/high affinity for glucose ◦ Activity is coordinated with GLUT4 ◦ Allosterically inhibited by its product glucose 6-phosphate ◦ Hexokinase activity controls glucose uptake and phosphorylation Hexokinase/Glucokinase Glucokinase (Hexokinase IV) ◦ Predominate in Liver and Pancreas ◦ High Km/low affinity for glucose ◦ Activity is coordinated with GLUT2 ◦ Glucokinase - GLUT2 system is very active when blood glucose is high ◦ NOT inhibited by glucose 6-phosphate ◦ Indirectly inhibited by fructose 6-phosphate (Fru-6-P) ◦ Activated by fructose 1-phosphate (Fru-1-P) ◦ Glucokinase regulatory protein is involved in the regulation by Fru-6-P and Fru-1-P 3 9/13/2019 Regulation of Glucokinase Activity Fructose Fru-1-P Fructosekinase Glucose Fru-6-P Fructose Fru -1-P GKRP nucleus + • Regulation of GK by substrates - - Fructose - Fru-1-P + - Fru-6-P - + - Glucose + + • Fructose-Fru-1-P/glucose stimulate Fru-6-P the disassociation of GK from GKRP cytosol • Fru-6-P promotes the binding of GK to GKRP, thereby inhibiting the Insulin disassociation of GK from GKRP GLUT2 • Regulation of GK by hormone Glu - Insulin + • Translocation of GK from nucleus to cytosol GK: glucokinase GKRP: glucokinase regulatory protein Liver + Stimulate or - Inhibit the disassociation of GK from GKRP Glucose 6-Phosphatase Located in Endoplasmic Reticulum (ER) Linked to glucokinase, forming a Substrate Cycle that controls glycolysis and gluconeogenesis INSULIN increases GK activity and decreases glucose 6-phosphatase activity Glucose Glucose Glucose + Pi + Fru-6-P INSULIN G6Pase INSULIN _ GK Glucose Glucose G6P G6P ER Lactate Glycogen GK GKRP N G6P: glucose-6-phosphate 4 9/13/2019 Key Reactions in Glycolysis and Gluconeogenesis glycolysis 6-Phosphofructo-1-kinase Fructose 6-phosphate Fructose 1,6-bisphosphate Fructose 1,6-biphosphatase gluconeogenesis Regulation of 6-Phosphofructo-1-kinase and Fructose 1,6-BisPhosphatase Insulin Glucagon Bifunctional enzyme 6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase Fructose 2,6-bisphosphate (F-2,6-BisP) 6-Phosphofructo-1-kinase Key enzyme in glycolysis Fructose 1,6-BisPhosphatase Key enzyme in GNG Coordinately control the rate of glycolysis and gluconeogenesis 5 9/13/2019 Fructose-2,6-Bisphosphate Fructose 2,6-bisphosphate (Fru-2,6-P2) is a metabolite, and is synthesized and broken down by the bifunctional enzyme, phosphofructokinase 2/fructose-2,6- bisphosphatase (PFK-2/FBPase-2). NOT directly involved in the glycolytic pathway F-2,6-BisP is increased by carbohydrate feeding or insulin administration glycolysis In the fasted state, when glucagon and epinephrine levels are high, F-2,6-BisP levels are low, and F-1,6-BisPase activity is increased gluconeogenesis Bifunctional Enzyme 6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase H H Fructose 6-phosphate Ser Fructose 6-phosphate ATP Pi synthesis 6PF-2kinase F2,6-Pase breakdown ADP H2O Insulin and glucagon regulate Bif via phosphorylation/dephosphorylation Fructose 2, 6-bisphosphate Fructose 2, 6-bisphosphate 6 9/13/2019 Bifunctional Enzyme 6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase Ser + + Breakdown of Synthesis of F2,6-Pase Fru 2, 6-bisphosphate 6PF-2kinase Fru 2, 6-bisphosphate Insulin and glucagon regulate Bif via phosphorylation/dephosphorylation Insulin Glucagon + - - + 6PF-2kinaseF2,6-Pase 6PF-2kinase F2,6-Pase Fructose 2,6-bisphosphate Fructose 2,6-bisphosphate (F-2,6-BisP) (F-2,6-BisP) A. Insulin Fed State glycolysis gluconeogenesis Fasted State 7 9/13/2019 Key Reactions in Glycolysis and Gluconeogenesis glycolysis Pyruvate kinase Phospoenolpyruvate pyruvate 2 1 Phosphoenolpyruvate oxaloacetate Pyruvate carboxykinase carboxylase gluconeogenesis Pyruvate Kinase (PK) Insulin Fructose 1,6-bisphosphate + Pyruvate Kinase - Glucagon Alanine 8 9/13/2019 Pyruvate Carboxylase and PEPCK Pyruvate carboxylase ◦ Positively regulated by acetyl CoA, which signals the need for more oxaloacetate PEPCK ◦ The rate determining enzyme in gluconeogenesis ◦ Activity is NOT regulated by allosteric or covalent modifiers ◦ It is changed at the gene expression level Unit VI Glycogen Metabolism ◦ Glycogenesis – glycogen biosynthesis ◦ Glycogenolysis – glycogen breakdown ◦ Regulation of glycogen metabolism 9 9/13/2019 Glycogen Glycogen – major storage carbohydrate in animals Where does it occurs? ◦ _______ and _________ The role of glycogen ◦ Liver - Release glucose to maintain blood glucose between meals or during starvation ◦ Muscle - Provide glucose for glycolysis within the muscle for muscle energy use Glycogen metabolism Glycogenesis (Glycogen synthesis) ◦ Enzymes – UDPGlc pyrophosphorylase, glycogen synthase, branching enzyme ◦ UTP (uridine diphosphate) ◦ Glycogenin Glycogenolysis (Glycogen breakdown) ◦ Enzymes - Glycogen phosphorylase, glucan transferase, debranching enzyme ◦ 10 9/13/2019 Glycogen molecule UDP-Glucose 11 9/13/2019 Formation of UDP-Glucose Glucose G-6-P G-1-P + UTP UDPGluc pyrophosphorylase UDP-Glucose Glycogenin A 37 Da protein serving as the primer molecule for glycogen synthesis First binding glucose from UDP-glucose (UDPGlc) to tyrosine residue of glycogenin Further glucose is attached to the existing glucose or glucose chain via 1-4 linkage from UDPGlc Form a short chain polysaccharide – glycogen primer with 8 glucose molecules Glycogen synthase takes over extending the chain Branching enzyme – branched chain polysaccharides 12 9/13/2019 Pathways of glycogenesis and glycogenolysis Biosynthesis Breakdown Branching Enzyme UDP Insulin Pi - - + Glycogen Glycogen cAMP Synthase Phosphorylase + - Glucagon Glucan Epinephrine Transferase UDP Debranching Enzyme UDPGlc - pyrophosphorylase UTP G-6-P Refer to Harper’s Glucokinase Glucose-6-Phosphatase Glucose Pathways of glycogenesis and Fed glycogenolysis Insulin Glucagon Biosynthesis Branching Enzyme UDP Insulin Pi - - + Glycogen Glycogen cAMP Synthase Phosphorylase + - Glucagon Glucan Epinephrine Transferase UDP Debranching Enzyme UDPGlc - pyrophosphorylase UTP G-6-P Refer to Harper’s Glucokinase Glucose-6-Phosphatase Glucose 13 9/13/2019 Pathways of glycogenesis and Fasting glycogenolysis Insulin Glucagon Breakdown Branching Enzyme UDP Insulin Pi - - + Glycogen Glycogen cAMP Synthase Phosphorylase + - Glucagon Glucan Epinephrine Transferase UDP Debranching Enzyme UDPGlc - pyrophosphorylase UTP G-6-P Refer to Harper’s Glucokinase Glucose-6-Phosphatase Glucose Regulation of glycogen metabolism cAMP-dependent regulation Hormone ◦ Insulin Insulin ◦ Glucagon (liver only) Cyclic Nucleotide ◦ Epinephrine - Phosphodiesterase Key enzyme Glycogen + + Glycogen cAMP Phosphorylase ◦ Glycogen synthase Synthase ◦ Glycogen + phosphorylase Glucagon Epinephrine Intracellular second message ◦ cAMP Which pathway does insulin or glucagon/ epinephrine promote? 14 9/13/2019 Regulation of glycogen metabolism cAMP-dependent regulation Hormone ◦ Insulin ◦ Glucagon (liver only) Insulin ◦ Epinephrine - Key enzyme Cyclic Nucleotide (catalyzes the formation of cAMP) ◦ Glycogen synthase Phosphodiesterase ◦ Glycogen AMP phosphorylase Glycogen + - Glycogen cAMP Intracellular second Synthase Phosphorylase message ◦ cAMP Which pathway does insulin or glucagon/ epinephrine promote? • High cAMP levels promote glycogen phosphorylase activity, but inhibit glycogen synthase activity • Low cAMP levels promote glycogen synthase activity, but reduce glycogen phosphorylase activity Regulation of glycogen synthase and phosphorylase activity Phosphorylation – dephosphorylation Glycogen synthase and phosphorylase exist in both phosphorylated and dephosphorylated states The effect of phosphorylation on the activity of both enzymes is opposite Glycogen synthase ◦ Glycogen synthase b - phosphorylated: INACTIVE ◦ Glycogen synthase a – dephosphorylated: ACTIVE Glycogen phosphorylase ◦ Glycogen phosphorylase a – phosphorylated: ACTIVE ◦ Glycogen phosphorylase b – dephosphorylated: INACTIVE 15 9/13/2019 Regulation of glycogen phosphorylase Phosphorylation – dephosphorylation Regulators ◦ Hormones – insulin, glucagon, epinephrine, norepinephrine ◦ cAMP concentration – effector of hormonal action ◦ cAMP-depedent protein kinase – PKA (protein kinase A) Phosphorylase b (inactive) Hormones Glucagon (liver only) cAMP PKA Epinephrine Norepinephrine P Phosphorylase a (active) Glycogen degradation Hormonal regulation of glycogen metabolism Fed state Fasted state What hormones are What hormones are secreted? secreted? Glucagon insulin ◦ ◦ Epinephrine How glycogen synthase is How glycogen synthase is regulated? regulated? ◦ increased by insulin ◦ decreased due to decreased insulin How glycogen How glycogen phosphorylase is phosphorylase is regulated? regulated? decreased due to increased insulin ◦ ◦ increased by glucagon & decreased glucagon/epinephrine & epinephrine What happens to glycogen What happens to glycogen metabolism? metabolism? ◦ glycogen stored ◦ glycogen degradation 16 9/13/2019 Regulation of glycogen phosphorylase