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Carbohydrate metabolismIntermediary Metabolism

Elizabeth F. Neufeld

Suggested reference:

Champe, Harvey and Ferrier, Lippincott’s Illustrated Reviews – Biochemistry, 3rd Edition

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Kinetic properties of glucose transporters

Uptake in liver and pancreas -cells is proportional to plasma concentration

GLUT-2

GLUT-3

GLUT-1

Uptake in brain is independent of plasma concentration over physiological range

Km = concentration at which half maximum rate of transport occurs (1/2 Vmax)

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Intracellular pool of GLUT4 in membranous vesicles translocate to the cell membrane when insulin binds to its receptor. The presence of more receptors increases the Vmax for glucose uptake (does not affect Km). When insulin signal is withdrawn, GLUT4 proteins return to their intracellular pool. GLUT4 is present in muscle and adipose tissue.

GLUT4 activity is regulated by insulin-dependent translocation

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Fate of glucose in the liver

GLUT2

Glucose

Glucose

Glucose-6-P

Glucokinase

Glycogensynthesis

Pentose phosphate

Glycolysis

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Glucokinase vs. Hexokinase

Glucokinase: Km = 10 mM, not inhibited by glucose 6-phosphate. Present in liver and in pancreas cells. Hexokinase: Km= 0.2 mM, inhibited by glucose 6-phosphate. Present in most cells.

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Glucokinase vs. Hexokinase

• Glucokinase is found in liver and -cells of pancreas

• Glucokinase allows liver to respond to blood glucose levels

• At low glucose levels, very little taken up by liver, so is spared for other tissues.

• Not inhibited by glucose 6-phosphate, allowing accumulation in liver for storage as glycogen

• It has a high Km, so it does not become saturated till very high levels of glucose are reached

• Hexokinase has low Km and therefore can efficiently use low levels of glucose. But is quickly saturated.

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Glucose action in the -cell

Glucose enters the -cell as blood glucose concentration rises. Glycolysis to generate ATP closes K+ channels in the cell membrane, stopping outward transport, and opening Ca+ channels. Inward flux of Ca+ causes exocytosis of insulin-containing secretory vesicles. Glucose also stimulates synthesis of new insulin.

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Fate of glucose in muscle

GLUT4Glucose Glucose

Glucose-6-P

Hexokinase

Glycogensynthesis

Glycolysis

Insulin

+

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Glycogen accumulation in muscle

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Fate of glucose in adipocytes

GLUT4Glucose Glucose

Insulin

+

Glucose-6-P

Hexokinase

LPL

Insulin+

Glycerol-3-P

Triglycerides

Fatty acids

Insulin

-

Lipoproteins

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How is metabolism regulated?Two broad classes of pathways

• Catabolic – break down molecules to generate energy• Anabolic - require energy for synthesis of molecules

The two pathways are kept distinct by regulatory mechanisms and/or sequestration in different cell compartments.

Pathways contain recurring enzymatic mechanisms• Oxidation-reduction reactions• Isomerization reactions• Group transfer reactions• Hydrolytic reactions• Addition or removal of functional groups

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MovementActive transport

Signal amplificationBiosynthesis

Oxidation of fuel

molecules

High ATP concentrations inhibit catabolic pathways and stimulate anabolic pathways

How is metabolism regulated?

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How is metabolism regulated?

Fast mechanisms, for immediate changes

Substrate concentrationAllosteric regulation (feedback, feed forward)Phosphorylation-dephosphorylationSignals emanating from hormone action

Slow mechanisms, for long-term changes

Genetic regulationResponse to diet and other environmental variables

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long term effects

How is metabolism regulated?

Rapid effect

Rapid effects

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Overview of glucose metabolic pathways

• Glycolysis: from G6P to pyruvate• Gluconeogenesis: from oxaloacetate to G6P • Glycogen synthesis: from G6P to glycogen• Glycogenolysis: from glycogen to G6P• TCA cycle

The pathways must be carefully regulated to keep pathways going in opposite directions from proceeding simultaneously.

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Regulation of glycolysis

• Glycolytic flux is controlled by need for ATP and/or for intermediates formed by the pathway (e.g., for fatty acid synthesis).• Control occurs at sites of irreversible reactions

• Phosphofructokinase- major control point; first enzyme “unique” to glycolysis• Hexokinase or glucokinase• Pyruvate kinase

•Phosphofructokinase responds to changes in:• Energy state of the cell (high ATP levels inhibit)• H+ concentration (high lactate levels inhibit)• Availability of alternate fuels such as fatty acids, ketone bodies (high citrate levels inhibit)• Insulin/glucagon ratio in blood (high fructose 2,6-bisphosphate levels activate)

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Control points in glycolysis

hexokinaseGlucose-6-P -

*

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Why is phosphofructokinase, rather than hexokinase, the

key control point of glycolysis?

Because glucose-6-phosphate is not only an intermediate in glycolysis. It is also involved in glycogen synthesis and the pentose phosphate pathway.

PFK catalyzes the first unique and irreversible reaction in glycolysis.

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Phosphofructokinase (PFK-1) as a regulator of glycolysis

fructose-6-phosphate fructose-1,6-bisphosphatePFK-1

PFK allosterically inhibited by:

• High ATP lower affinity for fructose-6-phosphate by binding to a regulatory site distinct from catalytic site.• High H+ reduced activity to prevent excessive lactic acid formation and drop in blood pH (acidosis).• Citrate prevents glycolysis by accumulation of this citric acid cycle intermediate to signal ample biosynthetic precursors and availability of fatty acids or ketone bodies for oxidation.

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Phosphofructokinase (PFK-1) as a regulator of glycolysis

PFK-1 activated by:Fructose-2,6-bisphosphate (F-2,6-P2)

F-6-P

F-1,6-P2

F-2,6-P2

glycolysis

+

PFK-2

PFK-1

Activates PFK-1 by increasing its affinity for fructose-6-phosphate and diminishing the inhibitory effect of ATP.

F-2,6-P2

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Phosphofructokinase-2 (PFK-2) is also a phosphatase (bifunctional

enzyme)Bifunctional enzyme has two activities:• 6-phosphofructo-2-kinase activity, decreased by phosphorylation • Fructose-2,6-bisphosphatase activity, increased by phosphorylation

fructose-6-phosphate fructose-2,6-bisphosphate

phosphatase

kinase

ATP ADP

Pi

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Hormonal control of F-2,6-P2 levels and glycolysis

Hormonal regulation of bifunctional enzyme

• Glucagon (liver) or epinephrine (muscle) increase cAMP levels, activate cAMP-dependent protein kinase. In liver, this leads to decreased F-2,6-P and inhibits glycolysis. The effect is opposite in muscle; epinephrine stimulates glycolysis.

• Insulin decreases cAMP, increases F-2,6-P stimulates glycolysis.

Phosphorylation of PFK2 by protein kinase activates its phosphatase activity on F2,6P in liver.

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GLUCOSE

G-6-Pase GK

G-6-P

F-6-P

P-ENOLPYRUVATE

PEPCKPK

PYRUVATEOXALOACETATE

FBPase 1 PFK 1

F-1,6-P2

GlycolysisGluconeogenesis

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GLUCOSE

G-6-Pase GK

G-6-P

F-6-P

P-ENOLPYRUVATE

PEPCKPK

PYRUVATEOXALOACETATE

FBPase 1 PFK 1

F-1,6-P2

GlycolysisGluconeogenesis

IncreaseHepatic Glucose Utilization

DecreaseHepatic Glucose Output

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GLUCOSE

G-6-Pase GK

G-6-P

F-6-P

P-ENOLPYRUVATE

PEPCKPK

PYRUVATEOXALOACETATE

FBPase 1 PFK 1

F-1,6-P2

GlycolysisGluconeogenesis

DecreaseHepatic Glucose Utilization

IncreaseHepatic Glucose Output

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+

F-6-P / F-1,6-P2 SUBCYCLE

FBPase 1 PFK 1

F-1,6-P2

FBPase 2 PFK 2

PK

-

+

F-6-P

G-6-P

F-2,6-P2

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The bifunctional enzyme

FBPase 2 PFK 2

Fructose-6-P

Fructose-2,6-bis-P

Fructose-2,6-bis-P

Fructose-6-P P

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The bifunctional enzyme

FBPase 2 PFK 2

Fructose-6-P

Fructose-2,6-bis-P

Fructose-2,6-bis-P

Fructose-6-P P

Phosphorylation of PFK2 by PKApromotes gluconeogenesis

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The bifunctional enzyme

FBPase 2 PFK 2

Fructose-6-P

Fructose-2,6-bis-P

Fructose-2,6-bis-P

Fructose-6-P

Double mutant, blocks phosphorylationof PFK2 and phosphatase activity of FBPase2

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The bifunctional enzyme

FBPase 2 PFK 2

Fructose-6-P

Fructose-2,6-bis-P

Fructose-2,6-bis-P

Fructose-6-P

Increased PFK1,Increased glycolysis,

Fed State

Hepatic overexpressionof the double mutant results in a gene expression profileconsistent with the fed state,and protection fromType I and II diabetes

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Gluconeogenesis• Mechanism to maintain adequate glucose levels in tissues, especially in brain (brain uses 120 g of the 160g of glucose needed daily). Erythrocytes also require glucose.

• Occurs exclusively in liver (90%) and kidney (10%)

• Glucose is synthesized from non-carbohydrate precursors derived from muscle, adipose tissue: pyruvate and lactate (60%), amino acids (20%), glycerol (20%)

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Gluconeogenesis takes energy and is regulatedConverts pyruvate to glucose

Gluconeogenesis is not simply the reverse of glycolysis; it utilizes unique enzymes (pyruvate carboxylase, PEPCK, fructose-1,6-bisphosphatase, and glucose-6-phosphatase) for irreversible reactions.

6 ATP equivalents are consumed in synthesizing 1 glucose from pyruvate in this pathway

hexokinaseGlucose-6-P - Glucose 6-phosphatase

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Irreversible steps in gluconeogenesis

• First step by a gluconeogenic-specific enzyme occurs in the mitochondria

pyruvate oxaloacetate

Pyruvate carboxylase

• Once oxaloacetate is produced, it is reduced to malate so that it can be transported to the cytosol. In the cytosol, oxaloacetate is subsequently dexcarboxylated/phosphorylated by PEPCK (phosphoenolpyruvate carboxykinase), a second enzyme unique to gluconeogenesis.

The resulting phosphoenol pyruvate is metabolized by glycolysis enzymes in reverse, until the next irreversible step

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Irreversible steps in gluconeogenesis (continued)

• Fructose 1,6-bisphosphate + H2O

fructose-6-phosphate + Pi

Fructose 1,6-Bisphosphatase

(FBPase)

• In liver, glucose-6-phosphate can be dephosphorylated to glucose, which is released and transported to other tissues. This reaction occurs in the lumen of the endoplasmic reticulum.

Requires 5 proteins!

2) Ca-binding stabilizing protein (SP)

1) G-6-P transporter

3) G-6-Pase4) Glucose transporter5) Pi transporter

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Gluconeogenesis and Glycolysis are reciprocally regulated

• Fructose 1,6-bisphosphatase is main regulatory step in gluconeogenesis.

• Corresponding step in glycolysis is 6-phosphofructo-1-kinase (PFK-1).

• These two enzymes are regulated in a reciprocal manner by several metabolites.

Fructose-6-phosphate

Fructose 1,6-bisphosphate

6-phosphofructo-1-kinase

Fructose 1,6-bisphosphatase

+ Citrate

- AMP

- F 2,6-BP

Citrate -

AMP +

F 2,6-BP +

Reciprocal control—prevents simultaneous reactions in same cell.