Transcript Karbohidrat Metabolizması

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
Gluconeogenesis
What
De novo synthesis of glucose as opposed to glycogenolysis
Where
Liver and proximal convoluted tubules of the kidney (late in
starvation - pH regulation in acidosis involves conversion of
glutamine to ammonia (excreted) and 2-oxoglutarate which
forms glucose by gluconeogenesis)
Glutamine
CO2
Liver proximal tubule epithelial cell
CO2
HCO3
When
-
Glucose
BLOOD
Glutaminase
Glutamate DH
GNG
Glutamine
Glutamate
2-Oxoglutarate
Glucose
H
+ Na+
NH3
NH3
+
+
Na
H
NH3
NH3
NH4+
NH4+
URINE
After exercise, starvation, diabetes, at birth.
Substrates
Lactic acid (exercise / Cori cycle)
Some amino acids and especially alanine and glutamine
(alanine cycle and glutamine cycle used to transfer amino
groups from muscle to liver for urea synthesis).
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.
Urea
Amino acids
Glutamate
2-Oxoglutarate
Alanine
2-Oxo acids
Alanine
Pathway reverse of glycolysis except for
three steps with very negative DG.
Glucose-6-phosphatase
instead of glucokinase
(hexokinase)
Fructose-1,6bisphosphatase instead
of phosphofructokinase
Gluconeogenesis
needs NADH
Gluconeogenesis
needs ATP
Pyruvate carboxylase plus
phosphoenolpyruvate carboxykinase
(PEPCK) instead of pyruvate kinase.
HCO3-
Uses 2 ATPs to reverse a
glycolytic step that makes 1 ATP
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).
Glutamate
2-oxoglutarate
Glutamate
2-oxoglutarate
Cytosol
Pyruvate carboxylase in
mitochondria
Regulation
Regulation can be:
Long term
(e.g. starvation and diabetes)
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.
#
# By regulator protein.
Note also that pyruvate carboxylase is regulated by allosteric effectors and substrate
supply
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).
Glucocorticoid response element
Thyroid hormone response element
cAMP response element
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
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.
Stress hormone including adrenaline (a1-receptors), opiates, vasopressin and
angiotensin work through activation of phospholipase c
Hormone
Receptor
PLC
activation
DAG
Protein kinase C
PIP2
Mitochondrial metabolism
IP3
Ca2+
Calmodulin-dependent protein kinases
Identification of control points
1. Effects of hormones on the rates of gluconeogenesis from different substrates
Glucagon
Glucagon and
Ca-hormones
2. Futile cycle measurements
Futile cycling only occurs to a significant extent in the fed state and is insignificant in
the starved state.
Glucagon inhibits futile-cycling at both PEPCK / PK and PF-1-K / Fru-1,6-Pase
whilst Ca-mobilising hormones (e.g.vasopression and a-adrenergic agonists) only
inhibit futile-cycling at PEPCK / PK and to a lesser extent than glucagon.
3. Crossover plots
Glucagon induced changes in metabolite concentration
Metabolite level as % control
250
L-Lactate as substrate
200
Crossover
Crossover
150
100
DHA as substrate
50
0
LAC
MAL
PYR
3-PGA
PEP
G3P
G6P
DHA
F16bisP
Gluc
Glucagon produces
a crossover at both
PEPCK / PK and
PF-1-K / Fru-1,6Pase
a-adrenergic
agonists only
produce a
crossover at
PEPCK / PK step
4. Flux control coefficient measurements
Flux control coefficient x 100
[L-Lactate] 5mM
0.5mM
5mM
0.5mM
Most rate
determining
These data show that pyruvate carboxylase is the most rate limiting process
And that regulation by glucagon at both PEPCK / PK and PF-1-K / Fru-1,6-P2ase
Pyruvate
transport
Mechanisms of short term regulation of gluconeogenesis
1. 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 2oxoglutarate, a competitive inhibitor. Glucagon and Ca-mobilising hormones
decrease the concentration of 2-oxoglutarate by a Ca-mediated activation of
2-oxoglutarate dehydrogenase.
Pathologically, the enzyme is inhibited if tryptophan levels are high. Tryptophan
is broken down to quinolinate which chelates Fe2+, an essential cofactor.
COO
Fe2+
COO
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 calmodulindependent protein kinase has a similar
but less potent inhibitory effect and
accounts for some of the effects of Camobilising hormones on
gluconeogenesis.
F16P2
Phosphorylation
Alanine
ATP
For glucagons in the fed state, there is a
strong correlation between phosphorylation
/ inhibition of PK and stimulation of
gluconeogenesis.
1
[PEP] mM
2
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.
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.
PC is critically dependent on acetyl-CoA which acts as an allosteric activator over
the physiological range of concentrations, and this provides a regulatory link
pyruvate carboxylation to fatty acid oxidation.
Enzyme in
mitochondria
Fatty acid
oxidation
Physiological
range
250
[Acetyl CoA] mM
500
Hormones
Mitochondrial [Ca 2+]
Matrix
[PPi]
Cyclic
AMP
PKA
and
CPT1
K+ entry into
Matrix
Ca-sensitive
dehydrogenases
NADH
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.
[ 2-OG]
NAD
Matrix
volume
Fatty acid
oxidation
Sites used for
inhibiting GNG
Relieve
inhibition of
PEPCK
[Glu]
Activation of
respiration
Pyruvate
carboxylase
ATP
ADP
[Acetyl-CoA]
Stimulation of
gluconeogenesis
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.
R
COOH
R
CoA
O
ATP
POCA
Cl
AMP + PPi
COSCoA
O
Tetradecylglycidate
CH2(CH2)4
CH3(CH2)13-
Inhibitors of b-oxidation such as hypoglycin (unripe ackee fruit - Jamaican
vomiting sickness)
CH2
CH2 C
NH2
CH2
CH-CH2-CH-COOH
Hypoglycin
CH2 C
Transamination
O
CH-CH2-C-COOH
Methylene-cyclopropylpropionic acid
Oxidative decarboxylation
CH2
CH2 C
O
CH-CH2-C-S-CoA
Methylene-cyclopropyl-acetyl-CoA
Irreversible inhibitor of butyryl-CoA dehydrogenase
(Pent-4-enoate has a similar effect)
B. Inhibitors of the respiratory chain
The respiratory chain has a high flux control coefficient for gluconeogenesis
V/J
[ATP]
Although [ATP] changes little
the calculated ATP/ADP ratio
drops a lot and calculated free
[AMP] increases
Rate of GNG
Respiratory chain activity
0
50
100
[Respiratory chain inhibitor]
Thus could mild inhibitors of the respiratory chain are potential antidiabetic 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.
The diabetic drugs metformin and phenformin (biguanides) act on the respiratory chain.
Phenformin
Metformin
CH3
CH3
NH2+
NH2+
CH
2 N-C-NH
2
CH3
N-C-NH2
0
[Metformin] (mM)
2.5
5
7.5
10
Incubation with 10mM
metformin at 8oC
Respiratory rate as % of control
100
80
Metformin
60
K0.5 14.9 ± 1.19 mM
Incubation at 8oC with
inhibitor for 4 hr
(metformin) or 5 min
(phenformin)
40
20
Phenformin
K0.5 0.05 ± 0.0015 mM
0
0
0.1
0.2
0.3
[Phenformin] (mM)
0.4
Metformin inhibits immediately in sub-mitochondrial particles but
requires higher concentrations
0
[Metformin] (mM)
10
20
30
40
50
CH3
100
Respiratory rate as % of control
CH3
NH2+
Metformin
N-C-NH2
Accumulation
Dy = -180mV
80
60
K0.5 79.0 ± 3.4 mM
Cf 15 mM in intact
energised mitochondria
 Positive charge allows slow accumulation
in mitochondria where they act as weak
inhibitors of complex 1.
 Uptake is self-limiting: if excessive
inhibition occurs Dy drops preventing
further accumulation.
40
20
K0.5 2.23 ± 0.18 mM
Cf 0.05 mM
0
0
2
4
6
8
[Phenformin] (mM)
10
 Phenformin is much more potent than
Metformin because it is more
hydrophobic and enter the mitochondria
more rapidly. It has a much higher risk of
causing the rare side-effect of severe
lactic acidosis.
Sta te 3 re sp iratio n ra te as % Co n tro l
Prolonged exposure allows metformin to inhibit the respiratory
chain at therapeutic doses
130
Glutamate / malate
(4) Succinate
(4)
120
24 hours
110
60 hours
(3)
(4)
100
(5)*
Hepatoma cell incubated with
metformin for the time shown
and then mitochondrial
respiration measured in
permeabilised cells.
90
80
(4)*
(4)**
(5)*
70
60
50
[Metformin] 50m M
100 m M
50 m M
100 m M
Time dependent inhibition of gluconeogenesis in rat liver
cells by metformin
Direct effects of metformin on GNG via changes in
ATP/ADP ratio and NADH/NAD+ ratio
Biguanides
Inhibition of respiration
and fatty acid oxidation
[Triose phosphates]
[Lactate]
[Pyruvate]
NADH
NAD
[Acetyl-CoA]
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.
ATP
ADP
[2- + 3-PGA]
[PEP]
Pyruvate
kinase
Inhibition of
gluconeogenesis
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 antidiabetic effects. (AMPK inhibitor blocks effects but not very specific).
Activation of AMPK is through an indirect mechanism - (no effect on isolated AMPK).
Metformin increases the calculated free [AMP] which could account for this but no
increase in total [AMP] can be measured.
Either total [AMP] measurements mask changes in free [AMP] (quite likely) or
metformin acts via some unidentified mechanism.
Metformin fails to activate AMPK in cells
from an LKB1 knockout mouse
AMPKK
(AMPK Kinase)
AMPK
?
LKB1 tumour
supressor
AMPK-P
(Active)
 [AMP]
?
Metformin
Phosphorylation of
target proteins
Inhibition of the
respiratory chain
Metformin
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 Hardie’s group
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) upregulation (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.
Inhibition of the
SREBP-1c (Sterol Response
respiratory chain
?
Element Protein)– an important
insulin stimulated transcription factor
 [AMP]
 [ATP]/[ADP]
implicated in the pathogenesis of
insulin resistance
AMPK may also phosphorylate IRS-1 leading to increased insulin sensitivity
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.
Sulphonylureas stimulate insulin secretion
Glucose
O
Inhibition of
potassium efflux
causes depolarisation
and calcium entry
+
K
Ca
2+
Pyruvate
[ATP]
mitochondrion
sulphonylureas
glyburide = glibenclamide
Insulin
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
18;409(6818):307-12)
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
Mechanisms of short term regulation of gluconeogenesis
2. Phosphofructokinase / Fructose-1,6-bisphosphatase 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
Inhibited by ATP
citrate and
PEP
PFK2
ADP
Enzyme is 49kDa dimer
with both activities on the
same polypeptide
Activity switches
depending on its
phosphorylation state
Pi
Inhibited by
F-6-P
F-2,6-bisPase
Fructose-2,6-bisP
(Activates PFK1 and inhibits F-1,6-bisPase)
Pi
P
ATP
ADP
PKA
Glucagon
cAMP
Glucagon [F-2,6-bisP] hence stimulating F-1,6-bisPase and inhibiting PFK1.
Calmodulin-dependent protein kinase does not phosphorylate the enzyme,
accounting for the lack of effect of Ca-mobilising hormones on this step.
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.
Glycogen storage diseases
Deficiency of G-6-Pase causes glycogen storage disease (Von
Gierke’s 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
Glucokinase (GK)
Repressed in starvation and diabetes.
Short term regulation by fructose which stimulates the conversion of glucose to glucose6-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].
GK Activity
No regulatory protein
With regulatory protein + 200mM F-1P
Effect was lost on purification but
sensitivity to inhibition by F-6P
restored upon addition of an
ancillary inhibitory protein (68kDa)
With regulatory protein
50
[F-6P] mM
In crude cytosolic extracts of liver
F-1P activates GK and F-6P
inhibits.
100
F-1P
F-6P
F-6P
F-1P
R’
R’
R
R
GK
Active GK is released from the regulatory protein in
response to F-1P or glucose (by some ill-defined
mechanism,) and translocated to the cytosol
Active
F-6P
Regulatory protein resides in the
nucleus where GK is also sequestered.
R
GK
Inactive
Note that some individuals have GK deficiency and show early onset
and severe Type 2 diabetes.