Carbohydrate metabolism

1
Carbohydrate metabolism Intermediary Metabolism
Elizabeth F. Neufeld
Suggested reference Champe, Harvey and Ferrier,
Lippincotts Illustrated Reviews Biochemistry,
3rd Edition
2
Kinetic properties of glucose transporters
GLUT-2
Uptake in liver and pancreas b-cells is
proportional to plasma concentration
GLUT-1
GLUT-3
Uptake in brain is independent of plasma
concentration over physiological range
Km concentration at which half maximum rate of
transport occurs (1/2 Vmax)
3
GLUT4 activity is regulated by insulin-dependent
translocation
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is withdrawn, GLUT4 proteins return to their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.
4
Fate of glucose in the liver
Glucose
GLUT2
Glucose
Glucokinase
Glucose-6-P
Glycogen synthesis
Pentose phosphate
Glycolysis
5
Glucokinase vs. Hexokinase
Glucokinase Km 10 mM, not inhibited by glucose
6-phosphate. Present in liver and in pancreas b
cells.
Hexokinase Km 0.2 mM, inhibited by glucose
6-phosphate. Present in most cells.
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Glucokinase vs. Hexokinase

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

• Glucokinase is found in liver and b-cells of
  pancreas

• Glucokinase allows liver to respond to blood
  glucose levels

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

• 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

7
Glucose action in the b-cell
Glucose enters the b-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.
8
Fate of glucose in muscle
Insulin

GLUT4
Glucose
Glucose
Hexokinase
Glucose-6-P
Glycolysis
Glycogen synthesis
9
Glycogen accumulation in muscle
10
Fate of glucose in adipocytes
Lipoproteins
Insulin

Insulin
LPL

GLUT4
Fatty acids
Glucose
Glucose
Hexokinase
Insulin
Glucose-6-P
-
Glycerol-3-P
Triglycerides
11
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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How is metabolism regulated?
High ATP concentrations inhibit catabolic
pathways and stimulate anabolic pathways
13
How is metabolism regulated?
Fast mechanisms, for immediate changes
Substrate concentration Allosteric regulation
(feedback, feed forward) Phosphorylation-dephospho
rylation Signals emanating from hormone action
Slow mechanisms, for long-term changes
Genetic regulation Response to diet and other
environmental variables
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How is metabolism regulated?
long term effects
Rapid effect
Rapid effects
15
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.
16
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
18
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
20
Phosphofructokinase (PFK-1) as a regulator of
glycolysis
PFK-1 activated by Fructose-2,6-bisphosphate
(F-2,6-P2)
F-2,6-P2
Activates PFK-1 by increasing its affinity for
fructose-6-phosphate and diminishing the
inhibitory effect of ATP.
21
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

22
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.

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GLUCOSE
GK
G-6-Pase
G-6-P
Glycolysis
Gluconeogenesis
F-6-P
FBPase 1
PFK 1
F-1,6-P2
P-ENOLPYRUVATE
PEPCK
PK
OXALOACETATE
PYRUVATE
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GLUCOSE
GK
G-6-Pase
G-6-P
Glycolysis
Gluconeogenesis
F-6-P
FBPase 1
PFK 1
F-1,6-P2
Decrease Hepatic Glucose Output
Increase Hepatic Glucose Utilization
P-ENOLPYRUVATE
PEPCK
PK
OXALOACETATE
PYRUVATE
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GLUCOSE
GK
G-6-Pase
G-6-P
Glycolysis
Gluconeogenesis
F-6-P
FBPase 1
PFK 1
F-1,6-P2
Increase Hepatic Glucose Output
Decrease Hepatic Glucose Utilization
P-ENOLPYRUVATE
PEPCK
PK
OXALOACETATE
PYRUVATE
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F
-
6
-
P

/

F
-
1
,
6
-
P

S
U
B
C
Y
C
L
E
2
G-6-P
F-6-P
FBP
ase 2
PFK 2
F-2,6-P2
-

FBP
PFK 1
ase 1
-1,6-P
F
2

PK
27
The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP
ase 2
PFK 2
Fructose-6-P
Fructose-2,6-bis-P
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The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP
ase 2
PFK 2
Fructose-6-P
Fructose-2,6-bis-P
Phosphorylation of PFK2 by PKA promotes
gluconeogenesis
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The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP
ase 2
PFK 2
Fructose-6-P
Fructose-2,6-bis-P
Double mutant, blocks phosphorylation of PFK2 and
phosphatase activity of FBPase2
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The bifunctional enzyme
Fructose-2,6-bis-P
Fructose-6-P
FBP
ase 2
PFK 2
Fructose-2,6-bis-P
Fructose-6-P
Increased PFK1, Increased glycolysis,
Hepatic overexpression of the double mutant
results in a gene expression profile consistent
with the fed state, and protection from Type I
and II diabetes
Fed State
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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 regulated
Converts 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
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Irreversible steps in gluconeogenesis

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

• 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 1,6- Bisphosphatase (FBPase)
fructose-6-phosphate Pi
35
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.

Citrate - AMP - F 2,6-BP
Citrate - AMP F 2,6-BP
Reciprocal controlprevents simultaneous
reactions in same cell.