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Transcript of Regulation of glycolysis and gluconeogenesis, and the mechanism of anti-diabetic drugs Prof. A. P....
Regulation of glycolysis and gluconeogenesis, and the mechanism of anti-diabetic drugs
Prof. A. P. Halestrap
ReferencesPilkis, 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
De novo synthesis of glucose as opposed to glycogenolysisWhat
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)
Where
NH4+ NH4
+
CO2
CO2
HCO3-
H+
H+ Na+
Na+Glutamine Glutamate 2-Oxoglutarate Glucose
NH3 NH3
NH3 NH3
BLOOD
URINE
Liver proximal tubule epithelial cell
GlucoseGlutamine
Glutaminase Glutamate DH GNG
After exercise, starvation, diabetes, at birth.When
Lactic acid (exercise / Cori cycle)
Substrates
Fructose (from sucrose)
Glycerol and propionate (from odd chain fatty acid -oxidation) are the only components of triglycerides that can be used for glucose production.
Alanine Alanine
Amino acids
2-Oxo acids
Glutamate
Urea
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).
2-Oxoglutarate
Pathway reverse of glycolysis except for three steps with very negative G.
Fructose-1,6-bisphosphatase instead of phosphofructokinase
Glucose-6-phosphatase instead of glucokinase (hexokinase)
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
Glutamate Glutamate
2-oxoglutarate 2-oxoglutarate
Note that pyruvate carboxylation is mitochondrial whereas PEPCK is cytosolic; hence we need oxaloacetate to cross mitochondrial inner membrane.
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).
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)
Pyruvate carboxylase in mitochondria
Cytosol
Regulation can be:Long term (e.g. starvation and diabetes)
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.
Short term (e.g. during and after exercise and other stresses - Cori cycle).
Medium term (birth and acidosis)
Regulation
# By regulator protein. Note also that pyruvate carboxylase is regulated by allosteric effectors and substrate supply
#
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 -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).
Long and medium term regulation
Thyroid hormone response element
Glucocorticoid 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!
GHPEPCK
This involves both substrate supply and hormones.
Short term regulation
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 -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 (1-receptors), opiates, vasopressin and angiotensin work through activation of phospholipase c
Hormone Receptor PLC activation
PIP2
DAG IP3 Ca2+
Protein kinase C Calmodulin-dependent protein kinases
Mitochondrial metabolism
Identification of control points
1. Effects of hormones on the rates of gluconeogenesis from different substrates
Glucagon and Ca-hormones
Glucagon
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 -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
LACPYR
MALPEP
3-PGADHA
G3PF16bisP
G6PGluc
0
50
100
150
200
250
100
Me
tab
oli
te l
ev
el
as
% c
on
tro
l
L-Lactate as substrate
DHA as substrate
Crossover
Crossover
Glucagon produces a crossover at both PEPCK / PK and PF-1-K / Fru-1,6-
Pase
-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
These data show that pyruvate carboxylase is the most rate limiting process
Most rate determining
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 M).
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.
Pathologically, the enzyme is inhibited if tryptophan levels are high. Tryptophan is broken down to quinolinate which chelates Fe2+, an essential cofactor.
COO
COO
Fe2+
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).
[PEP] mM1 2
Act
ivity
PhosphorylationAlanineATP
F16P2
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.
d) Pyruvate carboxylase Exclusively mitochondrial enzyme with Km for
pyruvate of about 200M. 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.
[Acetyl CoA] M
250 500
Act
ivity
Physiological range
Enzyme in mitochondria
Fatty acid oxidation
oxidationFatty acid
[Acetyl-CoA]
Cyclic AMP PKA and
CPT1
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.
Stimulation of
Pyruvatecarboxylase
gluconeogenesis
Ca-sensitivedehydrogenases
NADH
NAD
Hormones
Mitochondrial [Ca ]2+
Matrix
Matrix
K entry into+
[PPi]
Matrix
volume Activation ofrespiration
ATPADP
[ 2-OG]
[Glu]Relieve
inhibition of PEPCK
Sites used for inhibiting GNG
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 1M.
O
R COOH
O
R COSCoACoA
ATP AMP + PPi
Cl CH2(CH2)4CH3(CH2)13-
POCA Tetradecylglycidate
Inhibitors of -oxidation such as hypoglycin (unripe ackee fruit - Jamaican vomiting sickness)
CH2 NH2 CH2 O
Hypoglycin Transamination
CH2 C CH-CH2-CH-COOH CH2 C CH-CH2-C-COOH
Methylene-cyclopropyl-propionic acid
(Pent-4-enoate has a similar effect)
CH2 C CH-CH2-C-S-CoA
CH2 O
Methylene-cyclopropyl-acetyl-CoA
Irreversible inhibitor of butyryl-CoA dehydrogenase
Oxidative decarboxylation
B. Inhibitors of the respiratory chain
The respiratory chain has a high flux control coefficient for gluconeogenesis
50 1000
Rate of GNGRespiratory chain activity
[ATP]
[Respiratory chain inhibitor]
V / JAlthough [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.
0 100 200 300 400Time (min)
0
20
40
60
80
100
Rat
e o
f re
spir
atio
n (
% o
f co
ntr
ol)
Succinate
Glutamate + malate
Incubation with 10mM metformin at 8oC
The diabetic drugs metformin and phenformin (biguanides) act on the respiratory chain.
Metformin
N-C-NH2
NHCH3
CH3
+2
0 2.5 5 7.5 10
[Metformin] (mM)
0
20
40
60
80
100
Res
pir
ato
ry r
ate
as %
of
con
tro
l
0 0.1 0.2 0.3 0.4 [Phenformin] (mM)
K0.5 14.9 ± 1.19 mM
K0.5 0.05 ± 0.0015 mM
Incubation at 8oC with inhibitor for 4 hr
(metformin) or 5 min (phenformin)
Phenformin
N-C-NH2
NH
CH3
CH2
+2
Phenformin
Metformin
0 10 20 30 40 50
[Metformin] (mM)
0
20
40
60
80
100
Res
pir
ato
ry r
ate
as %
of
con
tro
l
0 2 4 6 8 10
[Phenformin] (mM)
K0.5 79.0 ± 3.4 mM
K0.5 2.23 ± 0.18 mM
Metformin inhibits immediately in sub-mitochondrial particles but requires higher concentrations
Cf 15 mM in intact energised mitochondria
Cf 0.05 mM
Metformin
= -180mV
Accumulation
Positive charge allows slow accumulation in mitochondria where they act as weak inhibitors of complex 1.
Uptake is self-limiting: if excessive inhibition occurs drops preventing further accumulation.
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.
N-C-NH2
NHCH3
CH3
+2
50
60
70
80
90
100
110
120
130
Sta
te 3
re
sp
ira
tio
n r
ate
as
% C
on
tro
l
Glutamate / malate Succinate
[Metformin] 50M 100M 50M 100M
(5)*
(4)* (4)**
(5)*
(4)
(4)
(3)
(4)
24 hours
60 hours
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.
0 50 100 150 200
0
150
300
450
600
750
900
Control
Glu
co
se
pro
du
cti
on
(n
mo
le/m
g)
Metformin
Time (min)
2 mM Metformin
5 mM Metformin
45 90 1500
20
40
60
80
100
Pe
rce
nta
ge
in
hib
itio
no
f g
luc
on
eo
ge
ne
sis
5/1
36
/9
Time of incubation (min)
2mM Metformin5mM Metformin
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
Inhibition of respiration
NADH
NAD
ATP
ADP
[Lactate]
[Pyruvate]
Pyruvate
Biguanides
and fatty acid oxidation
[Acetyl-CoA]
carboxylase
Inhibition ofgluconeogenesis
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.
[Triose phosphates]
[2- + 3-PGA]
[PEP]
Pyruvatekinase
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).
AMPKK
(AMPK Kinase)
AMPKAMPK-P
(Active)
Phosphorylation of target proteins
Inhibition of the respiratory chain [AMP] Metformin
Metformin increases the calculated free [AMP] which could account for this but no increase in total [AMP] can be measured.
LKB1 tumour supressor
? Metformin?
Metformin fails to activate AMPK in cells from an LKB1 knockout mouse
Either total [AMP] measurements mask changes in free [AMP] (quite likely) or metformin acts via some unidentified mechanism.
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) 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
?Inhibition of the
respiratory chain
[AMP] [ATP]/[ADP]
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
Ca 2+
OGlucose K+
Pyruvate
Insulin
mitochondrion
[ATP]
Inhibition of potassium efflux
causes depolarisation and calcium entry
sulphonylureasglyburide = glibenclamide
D. Insulin SensitizersThiazolidinediones 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 (PPAR), an orphan member of the nuclear hormone receptor superfamily that is expressed at high levels in adipocytes.
PPAR 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)
Acrp30 is adiponectin PDK4 is PDH kinase 4
Moller, D. E. (2001) New drug targets for type 2 diabetes and the metabolic syndrome. Nature 414: 821-827
Mechanisms of short term regulation of gluconeogenesis
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
Fructose-2,6-bisP
ATP
ADP
Pi
F-2,6-bisPase
Enzyme is 49kDa dimer with both activities on the
same polypeptide
PiInhibited bycitrate and
PEP
Inhibited byF-6-P
(Activates PFK1 and inhibits F-1,6-bisPase)
2. Phosphofructokinase / Fructose-1,6-bisphosphatase step
PFK2Activity switches depending on its
phosphorylation state
Pi
ATP ADPcAMPGlucagon PKA
P
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.
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
Glycogen storage diseases
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.
Glucokinase (GK)
In crude cytosolic extracts of liver F-1P activates GK and F-6P inhibits.
Effect was lost on purification but sensitivity to inhibition by F-6P restored upon addition of an ancillary inhibitory protein (68kDa)
[F-6P] M50 100
GK
Act
ivity
No regulatory protein
With regulatory protein + 200M F-1P
With regulatory protein
Van Schaftingen - the effect correlated with an increase in tissue [Fructose-1-P] and a decrease in [Fructose-6-P].
Note that some individuals have GK deficiency and show early onset and severe Type 2 diabetes.
Regulatory protein resides in the nucleus where GK is also sequestered.
GKR
F-6P
Inactive
R’ R
GK
R’
F-1P
F-1P
R
F-6P
F-6P
Active
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