Carbohydrate metabolism
Intermediary Metabolism
Elizabeth F. Neufeld
Suggested reference:
Champe, Harvey and Ferrier, Lippincott’s Illustrated
Reviews – Biochemistry, 3rd Edition
1
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)
2
GLUT4 activity is regulated by insulindependent 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 When insulin signal is withdrawn, GLUT4 proteins return to their
3
intracellular pool. GLUT4 is present in muscle and adipose tissue.
Fate of glucose in the liver
Glucose
GLUT2
Glucose
Glucokinase
Glucose-6-P
Glycogen
synthesis
Pentose
phosphate
Glycolysis
4
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 6phosphate. Present in
most cells.
5
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
6
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 insulincontaining secretory
vesicles. Glucose also
stimulates synthesis of
new insulin.
7
Fate of glucose in muscle
Insulin
+
Glucose
GLUT4
Glucose
Hexokinase
Glucose-6-P
Glycogen
synthesis
Glycolysis
8
Glycogen accumulation in muscle
9
Fate of glucose in adipocytes
Lipoproteins
+
Insulin
+
Glucose
GLUT4
Insulin
LPL
Fatty
Glucose
Hexokinase acids
Glucose-6-P
Glycerol-3-P
Insulin
Triglycerides
10
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
11
How is metabolism regulated?
Movement
Active transport
Signal amplification
Biosynthesis
Oxidation
of fuel
molecules
High ATP concentrations inhibit catabolic pathways and
stimulate anabolic pathways
12
How is metabolism regulated?
Fast mechanisms, for immediate changes
Substrate concentration
Allosteric regulation (feedback, feed forward)
Phosphorylation-dephosphorylation
Signals emanating from hormone action
Slow mechanisms, for long-term changes
Genetic regulation
Response to diet and other environmental variables
13
How is metabolism regulated?
long term
effects
Rapid effect
Rapid effects
14
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.
15
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 2,6bisphosphate levels activate)
16
Control points in glycolysis
Glucose-6-P
-
hexokinase
*
17
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.
18
Phosphofructokinase (PFK-1) as a
regulator of glycolysis
PFK-1
fructose-6-phosphate
fructose-1,6-bisphosphate
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.
19
Phosphofructokinase (PFK-1) as a
regulator of glycolysis
PFK-1 activated by:
Fructose-2,6-bisphosphate (F-2,6-P2)
PFK-2
F-6-P
PFK-1 +
F-1,6-P2
glycolysis
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.
20
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
kinase
ATP
ADP
fructose-2,6-bisphosphate
fructose-6-phosphate
Pi
phosphatase
21
Hormonal control of F-2,6-P2 levels and glycolysis
Hormonal regulation of
bifunctional enzyme
• Glucagon (liver) or
epinephrine (muscle) increase
cAMP levels, activate cAMPdependent protein kinase. In
liver, this leads to decreased
F-2,6-P and inhibits glycolysis.
The effect is opposite in
muscle; epinephrine
stimulates glycolysis.
Phosphorylation of PFK2 by
• Insulin decreases cAMP,
protein kinase activates
increases F-2,6-P stimulates
its phosphatase activity on
glycolysis.
F2,6P in liver.
22
GLUCOSE
GK
G-6-Pase
G-6-P
F-6-P
Gluconeogenesis
FBPase 1
Glycolysis
PFK 1
F-1,6-P2
P-ENOLPYRUVATE
PEPCK
OXALOACETATE
PK
PYRUVATE
23
GLUCOSE
GK
G-6-Pase
G-6-P
F-6-P
Gluconeogenesis
FBPase 1
Glycolysis
PFK 1
F-1,6-P2
Decrease
Hepatic Glucose Output
Increase
Hepatic Glucose Utilization
P-ENOLPYRUVATE
PEPCK
OXALOACETATE
PK
PYRUVATE
24
GLUCOSE
GK
G-6-Pase
G-6-P
F-6-P
Gluconeogenesis
FBPase 1
Glycolysis
PFK 1
F-1,6-P2
Increase
Hepatic Glucose Output
Decrease
Hepatic Glucose Utilization
P-ENOLPYRUVATE
PEPCK
OXALOACETATE
PK
PYRUVATE
25
F-6-P / F-1,6-P 2 SUBCYCLE
G-6-P
F-6-P
FBP ase 2
PFK 2
F-2,6-P2
-
+
PFK 1
FBPase 1
F-1,6-P 2
+
PK
26
The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP ase 2
Fructose-6-P
PFK 2
P
Fructose-2,6-bis-P
27
The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP ase 2
Fructose-6-P
PFK 2
P
Fructose-2,6-bis-P
Phosphorylation of PFK2 by PKA
promotes gluconeogenesis
28
The bifunctional enzyme
Fructose-6-P
Fructose-2,6-bis-P
FBP ase 2
Fructose-6-P
PFK 2
Fructose-2,6-bis-P
Double mutant, blocks phosphorylation
of PFK2 and phosphatase activity of FBPase2
29
The bifunctional enzyme
Fructose-2,6-bis-P
Fructose-6-P
FBP ase 2
PFK 2
Fructose-6-P
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
Fructose-2,6-bis-P
Increased PFK1,
Increased glycolysis,
Fed State
30
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%)
31
Gluconeogenesis takes energy and is regulated
Converts pyruvate to
glucose
Glucose-6-P
-
hexokinase
Glucose 6-phosphatase
Gluconeogenesis is not
simply the reverse of
glycolysis; it utilizes
unique enzymes
(pyruvate carboxylase,
PEPCK, fructose-1,6bisphosphatase, and
glucose-6-phosphatase)
for irreversible
reactions.
6 ATP equivalents are
consumed in
synthesizing 1 glucose
from pyruvate in this
pathway
32
Irreversible steps in gluconeogenesis
• First step by a gluconeogenic-specific
enzyme occurs in the mitochondria
Pyruvate
pyruvatecarboxylase oxaloacetate
• 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
33
Irreversible steps in gluconeogenesis (continued)
• Fructose 1,6-bisphosphate + H2O
Fructose 1,6Bisphosphatase
(FBPase)
fructose-6-phosphate + Pi
• 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!
1) G-6-P transporter
2) Ca-binding stabilizing
protein (SP)
3) G-6-Pase
4) Glucose transporter
5) Pi transporter
34
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
+ Citrate
Citrate AMP +
F 2,6-BP +
6-phosphofructo
-1-kinase
Fructose
1,6-bisphosphatase
- AMP
- F 2,6-BP
Fructose 1,6-bisphosphate
Reciprocal control—prevents simultaneous reactions in same cell.
35