Carbohydrate Metabolism V & VI

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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 ◦

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Glycogen molecule

UDP-Glucose

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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

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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

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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?

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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

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Regulation of glycogen phosphorylase

Allosteric regulation  Allosteric inhibitors ◦ ATP ◦ Glucose 6-phosphate (insulin leads to ______increased glucose 6-phosphate) ◦ Free glucose (liver only)

 Allosteric activator ◦ 5’ AMP – binds 5’AMP binding site of muscle phosphorylase isoenzyme ◦ Liver phosphorylase isoenzyme does not have 5’AMP binding site ◦ 5’ AMP is a potent signal of the energy state of the muscle cell

Phosphorylase b (inactive) 2 X ADP ATP + 5’AMP

P Phosphorylase a (active)

Glycogen metabolism Differences between liver and muscle

Liver Muscle

Amount of total Percentage of tissue weight Total body content glycogen stored High High

Regulators Hormones: insulin, glucagon, Hormones: insulin, epinephrine, norepinephrine epinephrine, norepinephrine Allosteric regulators: ATP, Allosteric regulators: ATP, Glucose 6-phosphate, Free glucose Glucose 6-phosphate, 5’-AMP

Role of the stored 1. Breakdown of glycogen 1. Breakdown of glycogen into free glucose glycogen into glucose 2. Glucose is released into 2. Glucose is used for the circulation energy in the muscle

Glucose-1-P Glucose-6-P

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