PEPCK
The Regulation of Eukaryotic
Gene Expression
..using the example of PEPCK
PEPCK
• The enzyme is expressed in liver, kidney,
adipose tissue and to a lesser extent in
muscle
• It is a key enzyme in gluconeogenesis
(the synthesis of new glucose, usually
from lactate, pyruvate or alanine) and
glyceroneogenesis (the synthesis of
glycerol, usually from lactate, pyruvate or
alanine)
PEPCK overexpression in
muscle
• The youtube video
• http://www.youtube.com/watch?v=4PXC_mctsgY
• is of a mouse with PEPCK overexpressed in muscle
only.
• This mouse hit the popular press in 2007 and put Case
Western Reserve University in Cleveland Ohio on the
map!
• Earl Sutherland, the discoverer of cAMP also hailed from
Case Western.
• This is an acronym for an enzyme
• PhosphoEnol Pyruvate CarboxyKinase
• This enzyme is ONLY regulated by gene
expression!
• No allosteric activators, covalent
modification etc
• No activation by cAMP, inhibition by
insulin etc
Why choose PEPCK?
• It is an enzyme. Why would this be good?
• It is not post-translationally regulated. Why
would this be good?
• A number of hormones influence gene
expression in different tissues.
The Supermouse….
• Eats 60% more food
than wild type mice
• Weighs 40% less
than wild type mice
• Can run for >4 h until
exhaustion whereas
the control littermates
stop after only 10 min
• Has 2 – 3 fold less
adipose tissue
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PEPCK overexpression in
muscle
• This mouse was leaner than wild type
mice, ran for longer and lived longer!
• They were also more aggressive.
• The overexpression had switched the
muscle fuel usage to fatty acids with little
lactate production.
PEPCK overexpression in fat cells
PEPCK overexpression in
adipose tissue
• A less famous cousin mouse has the
PEPCK enzyme overexpressed in adipose
tissue.
• The results couldn’t be further from
supermouse!
PEPCK overexpression in
adipose tissue
• These mice are obese although
metabolically healthy (as measured by glucose
tolerance and insulin sensitivity) until you put them
on a high fat diet.
• Then you see insulin resistance and
diabetes emerging.
PEPCK overexpression in liver
• Leads to altered glucose tolerance
• Insulin resistance, NIDDM
• Increased gluconeogenesis causes
increased hepatic glucose production
which is released into the blood stream
• This caused increased insulin secretion
but ultimately insulin resistance.
PEPCK Knock out in liver
• Surprisingly these mice can maintain
blood glucose under starvation conditions
• They develop liver steatosis (fatty livers)
probably because of impaired oxidation of
fatty acids
• A total PEPCK knock out in all tissues is
lethal…mice die within days of birth.
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The reaction!
Why the dramatically different
outcome for the mouse when
PEPCK is overexpressed in
different tissues?
CO
H2C
COOH
C
COOH
O
It is after all the same enzyme
catalysing the same reaction.
2
COOH
C
GTP
PO3
CH2
GDP
Phosphoenol
pyruvate
Oxaloacetate
Where does it fit in?
O
Glyceroneogenesis
CHO
H OH
Glucose
H
H
COOH
OH
HO
O
Phosphoenol pyruvate
PEP
H
HO
OH
HO
H
Gluconeogenesis
H
OH
H
OH
GDP
OH
H
C
CO2
O
PO3
CH2
Alanine
H
CH2OH
PEPcarboxykinase
GTP
Glycolysis
CO2
COOH
O
PEP
NADH
NAD+
COOH
Pyruvate
OAA
C
O
HC
LDH
CH3
C
C
H2C
COOH
OH
COOH
Glyceroneogenesis
Fatty acids
H 3C
PPAR
response
element
CO
CH 2OPO 3
HC
HC
OH
LDH
CH3
Pyruvate
Lactate
PPARRE
cAMP
response
element
Glucocorticoid
response
element
IRE
GRE
TRE
CRE
TATA
OH
CH2OPO 3
CH2OPO3
CH 2OH
C
Glycerol 3-P
O
HC
CH2OH
Dihydroxyacetone
phosphate (DHAP)
PEP
O
CH3
PEPCK gene
S-CoA
Triglycerides
NAD+
COOH
Pyruvate Carboxylase
oxaloacetate
OAA
CH3
NADH
COOH
OH
C
O
H
-1000
-400
Insulin
response
element
-300
-100
Thyroid
response
element
Glyceraldehyde
3-P
Promoter and regulatory region
3
PEPCK regulation in liver
• PEPCK activity is highest in liver during
starvation
• Glucocorticoids such as cortisol and
glucagon both activate the expression of
the PEPCK gene in liver
• The glucocorticoids are steroid hormones
whereas glucagon is a peptide hormone
Activating PEPCK activity in liver
during starvation
• Let’s consider the glucocorticoid response first.
• Cortisol is the active glucocorticoid hormone.
• Pharmaceutical analogues are cortisone
(converted to cortisol by a dehydrogenase) and
the synthetic analogues prednisone and
dexamethasone
• Often administered for their immunosuppressive
properties
Activating PEPCK activity in liver
during starvation
Activating PEPCK activity in liver
during starvation
• Cortisol is produced and released by the
adrenal gland….it travels through the
circulation and can pass through the cell
plasma membrane (unlike peptide
hormones)
• Once inside the cell it binds to a cytosolic
receptor in specific cells
• The formation of the cortisol:receptor
complex exposes a nuclear localisation
signal
• The complex moves to the nucleus
• It binds as a dimer to the glucocorticoid
response element (a sequence of DNA
upstream of a number of genes including
PEPCK)
Activating PEPCK activity in liver
during starvation
Activating PEPCK activity in liver
during starvation
• The binding of this complex greatly
enhances the frequency of initiation of the
basal transcription apparatus (RNA pol II
with all the bits).
• Other protein factors (coactivators) also
bind. These factors reside in the nucleus
of liver cells and are known as hepatic
nuclear factors (HNFs).
• It is thought that both the cortisol:receptor
complex and one or more of the HNFs
need to be bound for effective
enhancement.
• This is important for the tissue specific
nature of the PEPCK up-regulation.
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blood
cortisol
PEPCK gene
PPAR
response
element
PPARRE
-1000
cAMP
response
element
Glucocorticoid
response
element
Cortisol
receptor
cytoplasm
IRE
GRE
-400
Insulin
response
element
TRE
CRE
-300
TATA
HNFs
-100
nucleus
Thyroid
response
element
NLS
NLS
NLS
RNA
pol II
TATA
Cortisol binds to its
receptor, exposing
the NLS
Promoter and regulatory region
Differing response to
glucocorticoids in different tissues
• While cortisol up regulates PEPCK
transcription in the liver.
• It down regulates PEPCK in adipose
tissue.
• The same gene (single copy in the
genome) with the same promoter and
regulatory regions! How is this possible?
Activating PEPCK activity in liver
during starvation
• During starvation glucagon is secreted by
the alpha cells of the pancreas (it is
synthesised there)
• Glucagon is a peptide hormone which
cannot cross the plasma membrane
• It binds to a cell surface receptor (a Gcoupled protein receptor)
PEPCK down regulation by
cortisol in adipose tissue
• We are not sure! The accepted logic at
present is that for effective up regulation in
the liver you need both the
cortisol:receptor dimer and some HNFs
bound.
• With different adipocyte specific nuclear
factors you can get the reverse result.
Activating PEPCK activity in liver
during starvation
• The binding of glucagon to this receptor
causes a conformational change,
associations of subunits and ultimately the
activation adenylyl cyclase.
• This causes an increase in cAMP
activates Protein Kinase A moves to the
nucleus phosphorylates transcription
factors (CREBs)
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Activating PEPCK activity in liver
during starvation
PEPCK gene
• The phosphorylated CREBs then bind to
the CRE (cAMP response element) site on
the DNA
• effective enhancement of PEPCK
transcription (amongst other genes you
need up regulated in starvation)
cAMP
response
element
Glucocorticoid
response
element
PPAR
response
element
PPARRE
IRE
GRE
TRE
-300
-400
-1000
Insulin
response
element
CRE
TATA
-100
Thyroid
response
element
Promoter and regulatory region
glucagon
Blood
Liver
cytoplasm
Glucagon
receptor
GDP
Glucagon binds to
receptor
Blood
Adenylyl
cyclase
G protein
Liver
cytoplasm
Adenylyl
cyclase
GTP
R
C
Liver
cytoplasm
Nucleus
R
C
Protein kinase A
Adenylyl
cyclase
Glucagon binds to
receptor
Blood
PEPCK down regulation by Insulin
GTP
R
C
P
P
CREB
CREB
Nucleus
R
C
C
Protein kinase A
Nucleus
GDP
R
ATP
cAMP
What we know…..
R
• Insulin inhibits the basal PEPCK
transcription apparatus
• Insulin antagonizes the induction of
PEPCK expression by glucagon or
glucocorticoids
R
C
CREB
R
P
CREB
C
C
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PEPCK down regulation by Insulin
Summary: Transcriptional
Regulation of PEPCK
• It is thought that intermediates in the
insulin signalling pathway are involved.
• In spite of all we know about insulin we still
don’t know exactly how insulin inhibits the
transcription of PEPCK.
• It would be nice to say that an
intermediate produced by insulin signalling
phosphorylated a transcription factor
which binds to the IRE…. BUT I CAN’T
• Use the liver in starvation as the context
• PEPCK needs to be up-regulated to make
glucose (GLNG) to maintain blood glucose
and thus to supply the brain with fuel
• In adipose tissue it has the role of making
glycerol for the packaging of fatty acids to
triglycerides
Summary: Transcriptional
Regulation of PEPCK
Summary: Transcriptional
Regulation of PEPCK
Cortisol, a steroid hormone, up-regulates
PEPCK
Cortisol can enter the cell (because it is
hydrophobic enough) where it binds to a
cytosolic receptor NLS unmasked enters nucleus dimerises binds to
GRE
Post transcriptional regulation of
PEPCK
• Glucocorticoids and cAMP also stabilise
the PEPCK mRNA in the liver cytoplasm.
• Insulin destabilises it.
• mRNA stability contributes significantly to
the overall up or down regulation of gene
expression.
• PEPCK is normally very unstable.
• mRNA stability is measured by its half life.
• Glucagon, a peptide hormone upregulates PEPCK
• Glucagon can’t enter the cell binds to
G-coupled protein receptor activates
adenylyl cyclase cAMP↑ binds to
Protein kinase A R subunits dissociate
from C subunits C subunits enter
nucleus phosphorylate CREB dimerise and bind to CRE
Why would it be advantageous
for an mRNA sequence like
PEPCK to be unstable?
• If PEPCK is only regulated by gene
expression it is difficult to down regulate
the sequence at the level of synthesis if
the mRNA persists in the cytoplasm.
• This also applies to the Trp operon
enzymes
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PEPCK mRNA stability
cytoplasm
AAAAAAAAA 3’
5’ MeG
Translation
Processed mature mRNA
Nucleus
5’ MeG
AAAAAAAAA 3’
Processing
Transcription
Primary transcript
DNA
• A sequence at the 3’ UTR of PEPCK
mRNA has been identified which
“destabilises” the mRNA.
• If that sequence is inserted into the 3’UTR
of other more stable mRNAs, such as
globin, the half life reduces significantly.
• We are yet to determine how cAMP or
cortisol stabilises this mRNA.
PEPCK gene expression in
adipose tissue
RXR
PPARγ activates the
transcription of genes
involved with
adipogenesis and fat
storage
RXR
TZDs are artificial
ligands for PPARγ.
These are used as
insulin sensitising
agents.
cytoplasm
• Another response element becomes
significant, the PPARRE
• Peroxisomal Proliferator Activator
Receptor (PPAR) Response Element
• There in fact 4 PPARs; one of the ones of
interest to adipocytes is PPARγ, the other
is PPAR δ
• liver has PPARα and PPARγ
PPARγ
Nucleus
PPARγ
RXR
Pharmaceutical applications
TZDs
• A new group of insulin sensitizers, the
thiazolidinediones (TZDs) act on PPARγ.
• The most commonly prescribed are
Rosiglitozone and Piogliterzone
• These are artificial ligands for PPARγ.
• We don’t even know the natural ligand for
PPARγ although the favoured candidates
are fatty acids and their derivatives, in
particular polyunsaturated fatty acids.
cytoplasm
PPARγ
Nucleus
PPARγ
RXR
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Pharmaceutical applications
• They work to sensitize the body to insulin
in an interesting way.
• Insulin resistance is thought, in part to be
brought on by elevated free fatty acids
(FFA) in the serum interfering with insulin
signalling.
• Elevated FFAs are commonly associated
with obesity which gives one of the
putative links between obesity and insulin
resistance.
Fat mice who are metabolically
healthy
Pharmaceutical applications
• Obesity is characterised by lots of large
adipocytes which become leaky, hence
losing weight is one of the most effective
ways of enhancing insulin sensitivity.
• There are some mice that, although fat are
metabolically healthy (remember the
PEPCK mouse)
• They have adipocytes that can contain the
FFAs
Pharmaceutical applications: TZDs
• act to up-regulate PEPCK synthesis in
adipocytes, thus increase
glyceroneogenesis more repackaging
of FFAs in the adipocyte less FFAs in
serum
• Stimulate adipogenesis (differentiation of
new fat cells from fibroblasts) thus
increasing the storage for FFAs and again
lowering FFAs in serum.
Implications of TZD treatment
Obesity: other areas
• The patient may actually put on weight as
adipogenesis is stimulated
• BUT the fat cells will be able to contain the
FFAs and stop the release into the
bloodstream.
• The increase in PEPCK activity will
improve the fat storage in the adipocyte.
• As well as elevated FFAs obese adipose
tissue is often characterised by
macrophage infiltration.
• Obesity is now considered to be a low
grade, chronic inflammatory condition.
• The inflammatory response may account
for the cardiovascular and diabetic
symptoms associated with most sufferers.
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Obesity
• There is a strong link between nutrient
sensing and pathogen sensing in an
organism
• There has been very strong selection for
– strong immune response
– The ability to process and store energy
– In times of chronic nutrient overload the
immune response may become overly
sensitive
For the final exam….
• ELMA will NOT be examined
• Material from the labs after the ELMA will
be examined:
– Beta galactosidase induction (gene
expression)
– Protein purification
Obesity: other areas
• Some recent treatments for type-2
diabetes associated with obesity involve
treating patients with anti-inflammatory
drugs to reduce the inflammatory effects
and so lessen the type 2 diabetic
symptoms.
For the final exam….
The BCHM contribution
• All material covered in my lectures and
Gareth’s lectures will be examined.
• I will place some reading material on the
web and send it to your usyd email
address. This material will also appear in
the exam.
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