Karbohidrat Metabolizmasi

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Regulation of glycolysis and gluconeogenesis, and
the mechanism of anti-diabetic drugs
Prof. A. P. Halestrap
References Pilkis, S. J. Granner, D. K.
(1992). Molecular physiology of the regulation
of hepatic gluconeogenesis and glycolysis. Annu.
Rev. Physiol. 54 885-909. Van Schaftingen, E.
(1993). Glycolysis Revisited. Diabetologia 36,
581-588. Moller, D. E. (2001) New drug targets
for type 2 diabetes and the metabolic syndrome.
Nature 414 821-827 Rutter, G. A. Xavier, G.
D., and Leclerc, I. Roles of 5'-AMP-activated
protein kinase (AMPK) in mammalian glucose
homoeostasis. Biochemical Journal. 2003 3751-16
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Gluconeogenesis
De novo synthesis of glucose as opposed to
glycogenolysis
What
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Substrates
Lactic acid (exercise / Cori cycle)
Fructose (from sucrose) Glycerol and propionate
(from odd chain fatty acid b-oxidation) are the
only components of triglycerides that can be used
for glucose production.
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Pathway reverse of glycolysis except for three
steps with very negative DG.
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Pyruvate carboxylase plus phosphoenolpyruvate
carboxykinase (PEPCK) instead of pyruvate kinase.
HCO3-
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Note that pyruvate carboxylation is mitochondrial
whereas PEPCK is cytosolic hence we need
oxaloacetate to cross mitochondrial inner
membrane.
For most substrates oxaloacetate crosses as
malate and effectively transfers NADH from the
mitochondria (where it is abundant from fatty
acid oxidation and citric acid cycle activity) to
the cytosol (Route 2)
Where L-lactate is the substrate this occurs as
aspartate since lactate conversion to pyruvate
produces NADH to drive glycolysis backwards
(Route 1 in diagram).
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Cytosol
Pyruvate carboxylase in mitochondria
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Regulation can be Long term (e.g. starvation
and diabetes)
Regulation
Medium term (birth and acidosis)
Short term (e.g. during and after exercise
and other stresses - Cori cycle).
Long and medium term regulation involve changes
in gene expression whilst short term regulation
involves a change in enzyme activity or substrate
supply.
Note that both long and short term regulation
involves the those enzymes that can participate
in futile cycles.
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By regulator protein. Note also that pyruvate
carboxylase is regulated by allosteric effectors
and substrate supply
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Long and medium term regulation
Primarily mediated through an increased
glucagon/insulin ratio causing induction of
gluconeogenic enzymes (especially PEPCK, but also
other key GNG enzymes in Table 1) with permissive
effect of glucocorticoids such as cortisol.
Glycolytic enzymes such as GK and PK are
repressed.
Starvation and Diabetes both induce a large
decrease in glucagon / insulin ratio and cause a
5-10 fold increase in PEPCK in liver and 2-3 fold
increase in kidney. In kidney PEPCK induction
also occurs in response to acidosis.
In the liver it can be shown that PEPCK protein
synthesis induced by glucagon follows a rise in
cyclic AMP and mRNAPEPCK synthesis. After 20 min
mRNA increased 5-fold After 90 min 9-fold)
mRNA degradation is not affected (addition of
a-amanitin to block RNA synthesis promotes the
same rate of PEPCK degradation in controls and
glucagon- treated livers).
The mechanism involves a range of regulatory
elements in the PEPCK promoter including cAMP,
gluocorticoid and thyroid hormone response
elements. (Other promoters have similar
regulatory elements).
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Note the immense increase in PEPCK activity seen
at birth are also brought about by large changes
in glucagons/insulin ratios. Transgenic mice in
which the PEPCK promoter is linked to the growth
hormone gene greatly enhances the production of
growth hormone at birth, leading to very large
mice that grow at twice normal rate!
GH
PEPCK
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Short term regulation
This involves both substrate supply and hormones.

Note that alcohol reduces gluconeogenesis by
increasing NADH/NAD and hence decreasing
oxaloacetate.
Stimulation by glucagon and other hormones that
increase cyclic AMP (adrenaline via b -receptors
in some species) regulate enzyme activity through
the activation of protein kinase A.
These effects are antagonised by insulin which
lowers cyclic AMP.
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Identification of control points
1. Effects of hormones on the rates of
gluconeogenesis from different substrates
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2. Futile cycle measurements
a-adrenergic agonists only produce a crossover at
PEPCK / PK step
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4. Flux control coefficient measurements
Flux control coefficient x 100
L-Lactate 5mM 0.5mM 5mM
0.5mM
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Pyruvate transport
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• Mechanisms of short term regulation of
  gluconeogenesis
• Pyruvate to phosphoenolpyruvate step
• a) PEPCK Short term regulation is primarily
  through the supply of oxaloacetate whose
  cytosolic concentrations are less than the
  enzymes Km (about 9 mM).

There may also be regulation through changes in
the concentration of 2-oxoglutarate, a
competitive inhibitor. Glucagon and
Ca-mobilising hormones decrease the concentration
of 2-oxoglutarate by a Ca-mediated activation of
2-oxoglutarate dehydrogenase.
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b)    Pyruvate kinase The liver isoform of PK
is a key regulator of gluconeogenesis in the FED
state. It is inhibited by protein kinase A
mediated phosphorylation , which decreases the
substrate affinity of the enzyme. (The kidney M2
isoform can also be regulated in this way).
Phosphorylation by calmodulin-dependent protein
kinase has a similar but less potent inhibitory
effect and accounts for some of the effects of
Ca-mobilising hormones on gluconeogenesis.
For glucagons in the fed state, there is a strong
correlation between phosphorylation / inhibition
of PK and stimulation of gluconeogenesis.
At the levels of glucagon present in the starved
state PK is already almost totally inhibited and
thus does not play a role in the regulation of
gluconeogenesis under these conditions.
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d) Pyruvate carboxylase Exclusively
mitochondrial enzyme with Km for pyruvate of
about 200mM. This is in the physiological range
and regulation through substrate supply is
important.
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PC is inhibited by glutamate and by increases in
the ADP/ATP ratio. These provide a mechanism by
which glucagon and Ca-mobilising hormones can
stimulate pyruvate carboxylase.
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Hypoglycaemic agents and antidiabetic drugs
A. Inhibitors of fatty acid oxidation
Inhibitors of carnitine palmitoyl transferase 1,
especially cyclo-oxirane derivatives which are
activated by fatty-acyl CoA synthetase to their
CoA derivative which inhibits CPT1 with Ki values
of less than 1mM.
CoA
ATP
AMP PPi
POCA
Tetradecylglycidate
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Inhibitors of b-oxidation such as hypoglycin
(unripe ackee fruit - Jamaican vomiting sickness)
CH2 NH2 CH2 O
CH2 C CH-CH2-C-COOH
CH2 C CH-CH2-CH-COOH
Methylene-cyclopropyl-propionic acid
Hypoglycin Transamination
 
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B. Inhibitors of the respiratory chain
The respiratory chain has a high flux control
coefficient for gluconeogenesis
Although ATP changes little the calculated
ATP/ADP ratio drops a lot and calculated free
AMP increases
Thus could mild inhibitors of the respiratory
chain are potential anti-diabetic agents? The
surprising answer is yes and the most commonly
prescribed antidiabetic drug, metformin, probably
works this way.
Owen, M. R. Doran, E., and Halestrap, A. P.
Evidence that metformin exerts its anti-diabetic
effects through inhibition of complex 1 of the
mitochondrial respiratory chain. Biochemical
Journal. 2000 348607-614.
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The diabetic drugs metformin and phenformin
(biguanides) act on the respiratory chain.
Metformin


Incubation with 10mM metformin at 8oC
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Metformin inhibits immediately in
sub-mitochondrial particles but requires higher
concentrations
Cf 15 mM in intact energised mitochondria
Cf 0.05 mM
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Prolonged exposure allows metformin to inhibit
the respiratory chain at therapeutic doses
Hepatoma cell incubated with metformin for the
time shown and then mitochondrial respiration
measured in permeabilised cells.
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Time dependent inhibition of gluconeogenesis in
rat liver cells by metformin
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Direct effects of metformin on GNG via changes in
ATP/ADP ratio and NADH/NAD ratio
Biguanides
Inhibition of respiration
Lactate
ATP
NADH
ADP
NAD
Pyruvate
Pyruvate
carboxylase
The evidence for the proposed mechanism of action
comes from measurements of metabolite levels in
hepatocytes and whole animals treated with
metformin, and from studies on isolated
mitochondria.
Inhibition of
gluconeogenesis
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Recent data from several labs has shown that
metformin treatment activates AMP dependent
protein kinase (AMPK, and that this may play a
key role in its anti-diabetic effects. (AMPK
inhibitor blocks effects but not very specific).
Activation of AMPK is through an indirect
mechanism - (no effect on isolated AMPK).
Zhou, G et al. (2001) Role of AMP-activated
protein kinase in mechanism of metformin action J
Clin. Invest. 108 1167-1174. Also papers from
Grahame Hardies group
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AMPK activation can account for effects on
metformin on gene transcription (down regulation
of fatty acid oxidation and gluconeogenesis
genes) and glucose transporter (GLUT-4)
up-regulation (expression and translocation) in
muscle. Inhibition of acetyl-CoA carboxylase in
liver also occurs by this mechanism and may help
explain the decrease in plasma free fatty acids
and triglycerides.
SREBP-1c (Sterol Response Element Protein) an
important insulin stimulated transcription factor
implicated in the pathogenesis of insulin
resistance
?
AMPK may also phosphorylate IRS-1 leading to
increased insulin sensitivity
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Problems with the AMPK activation theory
Some of the enzyme activities modulated through
changed gene expression (e.g. fatty acid
synthetase and liver pyruvate kinase) or direct
phosphorylation (acetyl CoA carboxylase) are in
the opposite direction to insulin.
Many experiments have been performed at
concentration of metformin and phenformin far in
excess of those used to treat Diabetes
Note that the liver is exposed to much higher
Metformin than other tissues (except the gut)
since it receives the drug from the gut via the
portal blood supply. This may be why ingestion
of metformin is without major side-effects on
tissues such as the heart and brain that are
highly dependent on an active respiratory chain.
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Sulphonylureas stimulate insulin secretion
Inhibition of potassium efflux causes
depolarisation and calcium entry

Glucose
Insulin
K
O
2
Ca
Pyruvate
ATP
mitochondrion
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D. Insulin Sensitizers Thiazolidinediones such as
ciglitazone act as insulin sensitizers, reducing
the peripheral insulin resistance that occurs in
type 2 diabetes. They are agonists of the
peroxisome proliferatory-activated receptor g
(PPARg), an orphan member of the nuclear hormone
receptor superfamily that is expressed at high
levels in adipocytes.
PPARg is a central regulator of adipocyte gene
expression and differentiation one of whose
effects is to decrease Resistin secretion.
Resistin works in opposition to leptin and
increases insulin resistance (Nature 2001 Jan
18409(6818)307-12)
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Moller, D. E. (2001) New drug targets for type 2
diabetes and the metabolic syndrome. Nature 414
821-827
Acrp30 is adiponectin
PDK4 is PDH kinase 4
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Mechanisms of short term regulation of
gluconeogenesis
2. Phosphofructokinase / Fructose-1,6-bisphosphat
ase step
Key regulation is by fructose 2,6-bisphosphate
(F-2,6-bisPase). Activates , phosphofructokinase
1 (PFK1) and inhibits fructose-1,6-bisphosphatase
F-1,6-bisPase.
Fructose-6-P
ATP
Inhibited by
Inhibited by
Pi
Pi
Enzyme is 49kDa dimer with both activities on the
same polypeptide
F-6-P
citrate and
PEP
F-2,6-bisPase
PFK2
Activity switches depending on its
phosphorylation state
ADP

Fructose-2,6-bisP
(Activates PFK1 and inhibits F-1,6-bisPase)
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3.     Glucose-6-phosphatase / glucokinase
Glucose-6-phosphatase (G-6-Pase) is a microsomal
enzyme that is induced in starvation and diabetes
but for which there is no good evidence for
short-term regulation.

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Deficiency of G-6-Pase causes glycogen storage
disease (Von Gierkes Disease) since the
elevation of G-6-P in the liver inhibits glycogen
phosphorylase leading to massive glycogen
accumulation in the liver (which is
enlarged).Mutations in any of the G-6-Pase
constituent proteins have been shown to produce
the disease.Patients also show severe
hypoglycaemia after a short fast because they
cannot mobilize their liver glycogen which
represents the first source of blood glucose on
starvation
Glycogen storage diseases
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Glucokinase (GK)
Repressed in starvation and diabetes. Short term
regulation by fructose which stimulates the
conversion of glucose to glucose-6-P in isolated
hepatocytes by about 2-4 fold in a reversible
fashion.
Van Schaftingen - the effect correlated with an
increase in tissue Fructose-1-P and a decrease
in Fructose-6-P.
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Regulatory protein resides in the nucleus where
GK is also sequestered.
Inactive
Note that some individuals have GK deficiency and
show early onset and severe Type 2 diabetes.