Chapter 6 Carbohydrate Metabolism

Jia-Qing Zhang

张嘉晴

Biochemistry department Medical college Jinan university Mar. 2007

1

What’s metabolism?

2

Metabolism…..

What is Life?

What are the properties of life?

Movement Reproduction of one’s kid Metabolism

3

Lipid metabolism Carbohydrate metabolism Protein metabolism

4

5

metabolism

Carbohydrate metabolism

Metabolism of lipid Catabolism of protein

6

Carbohydrate Metabolism

7

Section 1 Introduction

Carbohydrates are the major source of carbon atoms and energy for living organisms.

8

Carbohydratesf of the diet Starch Sugar Lactose cellulose

9

Starch Sugar Cellulose

10

Glucose , the hydrolyzed product of most starch, will be focused in this chapter.

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Glucose transport

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The fate of absorbed glucose

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Section 2 Anaerobic degradation of glucose

Glycolysis Glucos e Pyruvate or lactate AT P cytosol

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2.1 Basic process of glycolysis

Glucos e Phase 1 Pyruvat e Phase 2 Lactate

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Phase1 Pyruvate formation from glucose Reaction1 Glucose Glucose-6 Phosphate Hexokinase

16

CH 2 OH

O OH

Hexokinase CH 2 OPO 3

O OH 17

Hexokinases

Hexokinases is a key enzyme in glycolysis and have 4 isoenzymes , isoenzyme 4 present in liver, and named glucokinase. 1 2 3 4 Hexokinases in all extrahepatic cells Glucokinase present in liver

18

Hexokinase has a low Km 0.1mol/L, high affinity for glucose.

Hepatic glucokinase has high Km > 10mol/L, a low affinity for glucose

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Glucose-6-

Phosphate Reaction 2: Glucose-6 Phosphate Fructose-6 Phosphate Phosphohexose isomerase

20

CH 2 OPO

O

Phosphohexose isomerase

OH

3

CH 2

OPO 3

O CH 2 OH OH 21

Reaction 3: Fructose-6 Phosphate Fructose-1,6 Phosphate

CH 2

OPO 3

O CH 2

Phosphofructokinase

OH CH 2

OPO 3

O CH 2

OPO 3

OH OH 22

Phosphofructokinase Phosphofructokinase

23

helpful

Reaction 4:

Fructose-1,6 Phosphate CH 2 OPO 3 C=O CH 2 OH Aldolase Glyceraldehyde 3 Phosphate

+

Dihydroxyacetone Phosphate(DHAP) CHO H-C-OH CH 2 OPO 3

24

Aldolase

25

Reaction 5:

Glyceraldehy de 3-Phosphate

+

Dihydroxyace tone Phosphate 2

×

Glyceraldehyde 3-Phosphate Triose Phosphate Isomerase

26

Triose Phosphate Isomerase

27

Reaction 6:

Glyceraldehyde 3-Phosphate CHO H-C-OH CH 2 OPO 3 1,3 Bisphosphoglycerate Glyceraldehyde 3-Phosphate Dehydrogenase O C ~ OPO 3 High energy H-C-OH CH 2 OPO 3

28

Glyceraldehyde 3-Phosphate Dehydrogenase

29

Reaction 7:

Substrate level phosphorylation 1,3 Bisphosph oglycerate 3 Phosphoglycerate O C ~ OPO 3 H-C-OH CH 2 OPO 3 Phosphoglycerate Kinase COO H-C-OH CH 2 OPO 3

30

Phosphoglycerate Kinase

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Reaction 8:

3 Phosphoglycerate 2 Phosphoglycerate COO H-C-OH CH 2 OPO 3 Phosphoglycerate Mutase COO H-C OPO 3 CH 2 OH

32

Mutase

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Reaction 9:

2-Phosphoglycerate COO H-C OPO 3 CH 2 OH Enolase Phosphoenolpyruvate COO C~ OPO3 PEP CH 2 High energy

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Enolase

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Reaction 10:

Phosphoenolpyruvate COO C~ OPO3 PEP CH 2 Pyruvate Kinase Pyruvate COO C=O CH 3

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Pyruvate Kinase

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Glucose O2 pyruvate no O2 CO2 + H2O lactate

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Conversion of pyruvate to lactate

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Conversion of pyruvate to lactate

NADH + H + NAD + Pyruvate Lactate Lactate dehydrogenase(LDH) COO NADH + H + HO-C-H COO C=O CH 3 L-lactate NAD + CH 3 Pyruvate

40

How many ATP are produced in above process?

2? 4?

Net ATP in glycolysis is 2

41

The features of the glycolysis pathway

    Major anaerobic pathway in all cells NAD + is the major oxidant Requires PO 4 Generates 2 ATP’s per glucose oxidized   End product is lactate (mammals) Connects with Krebs cycle via pyruvate 42

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2.2 Regulation of Glycolysis

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6-phosphofructokinase-1

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6-phosphofructokinase-1(PFK-1)

Allosteric enzyme

negative allosteric effectors

Citrate , ATP

Positive allosteric effectors

AMP, fructose1,6-bisphosphate, fructose2,6-bisphosphate

 Response to changes in energy state of the cell (ATP and AMP) 48

49

effector of PFK-1.

Fructose-2,6-bisphosphate formed by phosphorylation of Fructose--6-PO 4 catalyzed by PFK-II.

50

Regulation of Pyruvate Kinase

51

52



Allosteric enzyme

Inhibited by ATP. alanine Activated by fructose 1,6 bisphosphate



Regulated by phosphorylation and dephosphorylation PO4 enzyme

53

Regulation of Hexokinase Allosteric enzyme Inhibitor: Glucose-6-phosphate except for glucokinase

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The Energy Story of Glycolysis Glucose + 2ADP + 2P i + 2NAD + 2 Pyruvate + 2ATP + 2NADH + 2H + + 2H 2 O Overall ANAEROBIC (no O 2 ) 2Pyruvate + 2NADH Lactate + 2NAD + Overall AEROBIC(O 2 ) 2NADH 5 ATPs Oxidative phosphorylation

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The Significance of Glycolysis

  Glycolysis is the emergency energy yielding pathway----ineffient Main way to produce ATP in some tissues

red blood cells, retina, testis, skin, medulla of kidney

 In clinical practice

acidosis

56

Section 3 Aerobic Oxidation of Glucose

1.

2.

3.

Oxidation of glucose to pyruvate in cytosol Oxidation of pyruvate to acetylCoA in mitochondria Tricarboxylic acid cycle and oxidative phosphorylation

57

Oxidation of pyruvate to acetylCoA

Pyruvate + CoA Pyruvate dehydrogenase complex Acetyl CoA + CO 2 mitochondria This reaction is irreversible.

58

Pyruvate dehydrogenase complex Comprises of 3 kinds of enzyme and 5 cofactors: E1: pyruvate dehydrogenase E2:dihydrolipoyl transacetylase E3:dihydrolipoyl dehydrogenase Cofactors: Thiamine pyrophosphate(TPP), FAD, NAD, CoA and lipoic acid.

59

 60

Pyruvate Dehydrogenase Complex ..

Acetyl-CoA C-CH 3 O HS-CoA ..

acetyl CH 3 -C ..

O CH 3 -C TPP OH E 1 E 2 E 3 hydroxyethyl Pyruvate Dehydrogenase Dihydrolipoyl Transacetylase NAD + FAD ..

H 2 NADH ..

Dihydrolipoyl dehydrogenase

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Tricarboxylic Acid Cycle

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All Mean the Same

63

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CH 3 C ~S-CoA O

CARBON BALANCE

4 4 4 Oxaloacetate Malate Fumarate Citrate 6 2 carbons in 2 carbons out

TCA cycle

Isocitrate 6 a -ketoglutarate

CO 2

5 4 Succinate

8 reactions

Succinyl-CoA 4

CO 2

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Reaction 1.

Oxaloacetate + Acetyl CoA Citrate Synthase Citrate

+

Coenzyme A

66

COO C=O CH 2 COO Oxaloacetate (OAA) CH 3 -C~SCoA O

Citrate Synthase

HS-CoA COO OOC -CH 2 C-OH CH 2 COO CH 2 COO HO-C-COO CH 2 COO Acetyl-CoA Citric Acid or Citrate

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Reaction 2 Citrate Aconitase Isocitrate

69

Isocitrate Formation CH 2 COO HO-C-COO -H 2 O H-C-COO H CH 2 COO C-COO +H 2 O H C-COO CH 2 COO H-C-COO HO-C-COO H Citrate cis-Aconitate Isocitrate

Aconitase

70

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Reaction 3 Isocitrate

a

-Ketoglutarate + Carbon Dioxide Isocitrate Dehydrogenase

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CH 2 COO H-C-COO HO-C-COO H NAD + Isocitrate CO 2 NADH + H + COO CH 2 CH 2 C=O COO -

a

-Ketoglutarate

Isocitrate Dehydrogenase

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Reaction 4

a

-Ketoglutarate + CoA Succinyl CoA + Carbon Dioxide

a

-Ketoglutarate Dehydrogenase

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COO CH 2 CH 2 C=O COO NAD + FAD Lipoic acid HS-CoA TPP COO CH 2 CH 2 C~SCoA O CO 2

a

-Ketoglutarate Succinyl-CoA

a

-Ketoglutarate dehydrogenase Complex

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ketoglutarate

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Reation 5 Succinyl CoA Succinate + CoA Succinyl CoA Synthetase

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Thioester bond energy conserved as GTP COO CH 2 CH 2 C~SCoA P i + GDP GTP HS-CoA O Succinyl-CoA COO CH 2 CH 2 COO Succinate

Succinyl-CoA Synthetase

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Reaction 6 Succinate Fumarate Succinate Dehydrogenase

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Reaction 7 Fumarate Fumarase Malate

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Reaction 8 Malate Oxaloacetate Malate Dehydrogenase

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C C FAD COOH COOH FADH 2 H 2 O NAD + H COOH C H COOH C OH C COOH H C COOH Succinate Fumarate Malate NADH + H + COOH C=O C COOH Oxaloacetate

87

CH 3 C ~S-CoA O

CARBON BALANCE

4 4 4 Oxaloacetate Malate Fumarate Citrate 6 2 carbons in 2 carbons out

3 NADH 1 FADH2

Isocitrate 6 a -ketoglutarate

CO 2

5 4 Succinate

GTP

Succinyl-CoA 4

CO 2

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ATP Generated in the Aerobic Oxidation of Glucose

 There are two ways for producing ATP

Substrate level phosphorylation

Succinyl CoA to succinate

Oxidative phosphorylation

89



3.2 ATP Generated in the Aerobic Oxidation of Glucose

In aerobic oxidation of glucose

Gycolysis: 2 NADH and 2ATP produced by substrate level phosphorylation Production of acetylCoA: 2 NADH TCA cycle: 2

×

3NADH ,2

×

1 FAD and 2GTP

Stoichiometry: 2.5 ATP per NADH 1.5 ATP per FADH

Table 6-1

32 ATP are produced for one glucose

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Features: Acetyl-CoA enters forming citrate 3 NADH, 1 FADH 2 , and 1 GTP are formed Oxaloacetate returns to form citrate

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3.3 the regulation of aerobic oxidation of glucose The regulation of pyruvate dehydrogenase complex The regulation of tricarboxylic acid cycle

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Regulation of Pyruvate Dehydrogenase complex Pyruvate + HS-CoA + NAD +



Acetyl-CoA + NADH + H + Activators: Inhibitors High NADH means that the cell is experiencing a surplus of oxidative substrates and should not produce more. Carbon flow should be redirected towards synthesis. High Acetyl-CoA means that carbon flow into the Krebs cycle is abundant and should be shut down and rechanneled towards biosynthesis

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Mechanism:

1. allosteric regulation NADH and acetyl-CoA TPP 2. Covalent Modification E-1 subunits of PDH complex is subject to phosphorylation FAD H PO 4 = Active E 1 -OH ATP Epinephrine Glucagon 1 2 Insulin 3

PDH phosphatase

H 2 O E 1 -O PO 3 Inactive

PDH kinase

ADP Cyclic-AMP protein kinase ATP

94

Regulation of the Citric Acid Cycle Key enzymes : 1. Citrate synthase 2. Isocitrate dehydrogenase 3. α-ketoglutarate dehydrogenase complex Modulators: The ratios of [NADH]/[NAD] and [ATP]/[ADP], high ratios inhibit Additonally, Ca 2+ is an activitor Succinyl CoA is a inhibitor summary of TCA

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Pentose Phosphate Pathway

96

PENTOSE PHOSPHATE Pathway

Take Home: The PENTOSE PHOSPHATE pathway is basically used for the synthesis of NADPH and D-ribose. It plays only a minor role (compared to GLYCOLYSIS ) in degradation for ATP energy.

97

The primary functions of this pathway are: 1. To generate NADPH, 2.

To provide the cell with ribose-5-phosphate.

98

NADPH differs from NADH physiologically : 1)its primary use is in the synthesis of metabolic intermediates (NADPH as reductant provides the electrons to reduce them), 2) NADH is used to generate ATP

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Basic Process

  Found in cytosol Two phases Oxidative phase nonreversible Nonoxidative phase reversible 100

101

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The significance of PPP 1) Ribose 5- phosphate: Ribose 5- phosphate is the starting pointing for the synthesis of the nucleotides and nucleic acids.

103

2) NADPH: a. NADPH is very important ”reducing power”for synthesis of fatty acids and cholesterol, and the synthesis of amino acids via glutamate dehydrogenase.

b. In erythrocytes, NADPH is the coenzyme of glutathione reductase to keep the normal level of reduced glutathione Additonally, NADPH serves as the coenzyme of mixed funtion oxidases.

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Glycogen Formation and Degradation

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Location of glycogen Glycogen is the storage form of glucose in animals and humans Glycogen is synthesized and stored mainly in the liver and the muscles

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Features: The structure of glycogen consists of long polymer chains of glucose units connected by an alpha glucosidic bonds. All of the monomer units are alpha-D-glucose,



93% of glucose units are joined by a-1,4-glucosidic bond



7% of glucosyl residues are joined by a-1,6-glucosidic bonds



Fig.6-11

107

108

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Main chains: branch point every 3 units on average.

Branch: 5-12 glucosyl residues. Two properties of this structure: 1) High solubility.

many terminals 4 hydroxyl groups

2) More reactive points for synthesis and degradation of glycogen.

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Glycogen Formation (glycogenesis)

Occurs in cytosol of liver and skeletal muscle Dived into 3 phases:



ACTIVATION OF D-GLUCOSE



GLYCOSYL TRANSFER



BRANCHING

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Glucose-6 Phosphate Glucokinase(liver Hexokinase(muscle) phosphoglucomutase Glucose-1-phosphate

112

ACTIVATION: UDP-GLUCOSE G-1-P + UTP UDP-GLUCOSE + PPi

UDP-Glucose pyrophosphorylase

2 Pi

O H CH 2 OH O HO OH H H OH O O P O O O P O O H O N CH 2 O Uridine diphosphate (UDP) Glucose HO N OH 113

O H CH 2 OH O HO OH H H OH H HO N O O P O O O P O O O CH 2 O N OH

NEW GLYCOSYL TRANSFER UDPG

H CH 2 OH O HO OH H H OH O H CH 2 OH O OH H H OH O

NON-REDUCING END

H CH 2 OH O HO OH H H OH O H CH 2 OH O OH H H OH O H CH 2 OH O OH H H OH O 114

115

BRANCHING

Cleave Glycogenin

 a1.,4->1,6-glucantransferase 116

GLYCOGEN SYNTHESIS ENZYMES

   UDP-glucose pyrophosphorylase  forms UDP-glucose Glycogen Synthase  major polymerizing enzyme a1.,4->1,6-glucantransferase 117

Glycogen Degradation ( Glycogenolysis )

Glycogenolysis is not the reverse of glycogenesis

118

Glycogen Synthesis

Glycogen Degradati o n

Synthesis

Glucose-6-PO 4 Glucose-1-PO 4 glucose UDP-Glucose

119

Phosphorylase and Debranching Enzyme Highly branched core

Phosphorylase Phosphorylase Phosphorylase

G-1-p Glycogen Debranching enzyme1 Limit Branch Debranching enzyme2 + D-glucose

Debranching enzyme: a tandem enzyme Oligo α1,4 α 1,4 glucantransferase Transfer a trisaccharide unit glucosidase Hydorlyze a 1,6 branch point

121

Glycogen Breakdown

PO 4 Glycogen

Phosphorylase and Debranching Enzyme

Glucose-1-Phosphate

Phosphoglucomutase

Glucose-6-Phosphate Glucose Glycolysis Take home: Glycogen contributes glucose to glycolysis and to blood glucose (Liver)

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The regulation of glycogensis and glycogenolysis

123

Regulatory site of glycogenesis and glycogenolysis :

•

Phosphorylase

•

Glycogen synthase

124

Phosphorylase

Phosphorylase G-1-p

125

Inactive b Glucagon,epinephrine PKA protein kinase A Adenylate cyclase cAMP

Phosphoryl ase b kinase Phosphoryl ase b kinase

inactive Active a

Phosphorylase

126

127

Glycogen synthase

128

Glycogen Glycogen synthase

+ +

129

active a Glucagon,epinephrine Adenylate cyclase PKA protein kinase A cAMP inactive b

Glycogen synthase

130

synthase inactive b Glucagon,epinephrine PKA protein kinase A Adenylyl cyclase cAMP phosphorylase b Phosphorylating inhibitor-1 Active Protein phosphatase-1

131

Active inactive

132

Allosteric regulation: Phosphorylase: Activitor: AMP Inhibitor: ATP, glucose-6-phosphate Glycogen synthase: Activitor: ATP, Glucose-6-phosphate

133

TAKE HOME: DEGRADATION What activates glycogen degradation inactivates glycogen synthesis.

SYNTHESIS

What activates glycogen synthesis inactivates glycogen degradation

134

The Significance of Glycogenesis and Glycogenolysis

 Liver 

maintain blood glucose concentration

Skeletal muscle

fuel reserve for synthesis of ATP

135

Glycogen Storage Diseases

 Deficiency of

glucose 6-phosphatase liver phosphorylase liver phosphorylase kinase branching enzyme debranching enzyme muscle phosphorylase Table 6-2

136

Gluconeogenesis

Gluconeogenesis: The process of transformation of non-carbohydrates to glucose or glycogen

glucogenic amino acids lactate glycerol organic acids liver, kidney

Glucose Glycogen

137

138

139

140

Phosphatase PO 4 Blood Glucose Glucose Kinase H 2 O G6P F6P Kinase F1,6bisP DHAP Gly-3-P 1,3 bisPGA Kinase 3PGA L-lactate 2PGA PEP Kinase Pyruvate Ribose 5-PO 4 Glycogen OAA PO 4 H 2 O Phosphatase

141

3 irreversible reactions PEP Pyruvate F-6-PO4 F1,6-bisPO4 Glucose Glucose-6-PO4



G o’ = 61.9 kJ per mol



G o’ = 17.2 kJ per mol



G o’ = 20.9 kJ per mol Take home: Gluconeogenesis feature enzymes that bypass 3 irreversible KINASE steps

142

Reaction1 Glucose Hexokinase Glucose-6 Phosphate

143

Reaction 3 Fructose-6 Phosphate Phosphofructokinase Fructose-1,6 Phosphate

144

Reaction 10:

Phosphoenolpyruvate Pyruvate

145

3 reactions need to bypass: Pruvate phosphoenolpyruvate Fructose 1,6-bisphosphate fructose 6-phosphate Glucose 6-phosphate glucose

146

The conversion of pyruvate to phosphoenolpyruvate(PEP) Pyruvate CO 2 Pyruvate carboxylase mitochondria oxaloacetate

147

malate oxaloacetate aspartate cytosol oxaloacetate PEP malate aspartate mitochondria

148

Mitochondria or cytosol GTP GDP oxaloacetate CO 2 PEP Phosphoenolpyruvate carboxykinase

149

The conversion of Fructose 1,6-bisphosphate to Fructose 6-phosphate Fructose 1,6 bisphosphate Fructose 6-phosphate Fructose 1,6-bisphosphatase

150

The conversion of glucose 6-phosphate to Glucose glucose 6-phosphate Glucose Glucose 6-phosphatase

151

Substrate cycle The interconversion of two substrates catalyzed by different enzymes for singly direction reactions is called substrate cycle.

Glucose glucose-6-phosphote

152

153

Significance

:

Primarily in the liver (80%); kidney (20%) Maintains blood glucose levels The anabolic arm of the Cori cycle

154

Cori Cycle

155

Cori cycle is a pathway in carbohydrate metabolism that links the anaerobic glycolysis in muscle tissue to gluconeogenesis in liver.

156

Liver is a major anabolic organ L-lactate D-glucose Blood Lactate THE CORI CYCLE Blood Glucose L-lactate D-glucose Muscle is a major catabolic tissue

157

Significance of cori cycle:

•

avoid the loss of lactate and accumulation of lactate in blood to low blood pH and acidosis.

•

6 ATP are sonsumed per 2 lactate to glucose

158

Regulation of gluconeogenesis

There are 2 important regulatory points: Fructose 1,6 bisphosphate Fructose 6-phosphate + P Fructose 1,6-bisphosphatase i

159

Fructose 1,6-bisphosphatase Inhibitor: Fructose 2,6-bisphosphate and AMP Activitor: Citrate

160

161

To summarize, when the concentration of glucose in the cell is high, the concentration of fructose 2,6 bisphosphate is elevated. This leads to a stimulation of glycolysis . Conversely, when the concentration of glucose is low, the concentration of fructose 2,6-bisphosphate is decreased. This leads to a stimulation of gluconeogenesis. Gluconeogenesis predominates under starvation conditions.

162

Pyruvate + CO 2 ATP + H 2 O .

+ oxaloacetate + ADP + P pyruvate carboxylase + 2 H + i Pyruvate carboxylase is allosterically activated by

acetyl CoA

163

  

The Significance of Gluconeogenesis

Replenishment of glucose and maintaining normal blood sugar level Replenishment of liver glycogen

“three carbon” compounds

Regulation of Acid-Base Balance  Clearing the products 

lactate, glycerol

Glucogenic amino acids to glucose 164

 

Blood Sugar and Its Regulation

Blood sugar level

3.89-6.11mmol/l

Sources of blood sugar---income

digestion and absorption of glucose from dietary gluconeogenesis glycogen other saccharides

Outcome: aerobic oxidation Glycogen PPP Lipids and amino acids

165

Regulation of Blood Glucose Concentration

 Insulin 

decreasing blood sugar levels

Glucagon, epinephrine glucocorticoid

increasing blood sugar levels

166

Insulin

The unique hormone responsible for decreasing blood sugar level and promoting glycogen formation, fat, and proteins simultaneously.

167

168

169

The effects of insulin:

Effects on membrane actively transport.

Effects on glucose utilization Effects on gluconeogenesis.

170

Glucagon

171

Epinephrine

Stimulates glucogen degradation and gluconeogenesis

172

Glucocorticoids

Inhibit the utilization of glucose Stimulate gluconeogenesis by stimulating protein degradation to liberate amino acids

173

Review questions

174

Glucagon

175

•

Epinephrine

•

glucocorticoids

176