DECLARATION OF CONFLICT OF INTEREST
none
Spontaneous cardiac hypertrophy in a
novel mouse model of diabetes: a
mechanistic insight
3rd Year PhD student
Stephen Gibbons
University of Manchester
UK
Diabetes and Heart disease

Diabetes is the fastest growing metabolic disease, with estimations
indicating that by the year 2030 over 380 million people will have
diabetes (WHO statistics).

Adults with diabetes have heart disease
death rates about 2 to 4 times higher
than adults without diabetes.


Animal models of diabetes at present
use either chemical induction or non
human relevant mutations
Requirement for a human relevant
mouse model of diabetes!!
GENA348 mice
•
Novel mouse model
•
Have a SNP mutation giving rise to the MODY2 model
•
MODY2 is caused by a loss-of-function mutation in the gene which encodes the enzyme
glucokinase
WT
Isoleucine
HO
Phenylalanine
GENA348, a human relevant mouse model
Glucose

GENA348
have
a
glucokinase (Gck) mutation

Analogous to that found in
the human form of MODY2
β Cell
GLUCOSE
Glucokinase
Glucose-6-phosphate
Insulin
Blood Glucose
Muscle
GENA348, a human relevant mouse model
Glucose

GENA348
have
a
glucokinase (Gck) mutation

Analogous to that found in
the human form of MODY2

Raises the threshold for
Glucokinase (change in
sensor)
β Cell
GLUCOSE
Glucokinase
Glucose-6-phosphate
Insulin
Hyperglycaemia
Muscle
Glucokinase activity/expression
GCK expression
WT
GCK activity
Glk
WT
HO
50kDa
LIVER
Tubulin
55kDa
HO
Glk
50kDa
HEART
Tubulin
GCK activity and expression are reduced in the GENA348 mice
Toye et al; Diabetes 2004
55ka
Metabolic features of GENA348 mice
Blood glucose levels
25
***
2.5
***
***
2
***
15
insulin (ug/L)
blood glucose (mmol/L)
20
Blood insulin levels
10
5
1.5
1
0.5
0
WT
HO
3 months
WT
HO
6 months
WT
HO
12 months
0
WT
HO
GENA348 diabetic mice develop chronic hyperglycaemia with NORMAL insulin levels
N=5-8
“What are the effects of glucose, independent of
insulin, on the cardiac phenotype”?
HYPERTROPHY?
CARDIAC DYSFUNCTION?
HYPERGLYCEMIA
What effect does hyperglycaemia have on cardiac structure?
Heart
weight normalised
to tibia
length
Left
ventricular
posterior wall
thickness
*
8.5
**
1.58
1.4
7.5
Left ventricular diastolic diameter
**
**
1.3
7
mg/mm
1.2
6.5
mm
mm
1.1
16
0.9
5.5
0.8
5
0.7
4.5
0.6
0.54
Wt
Wt
HO
HO
3m onths
N=10
N=10
3months
Wt
Wt
HO
HO
6m onths
6months
Wt
Wt
HO
HO
12m onths
12months
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
*
WT
HO
12 months
Hyperglycaemia leads to progressive cardiac hypertrophy and dilatation
What effect does hyperglycaemia have at the cellular level?
6 months
12 months
Real time PCR quantification of BNP
WT
cell size (um2) Relative expression BNP
3
**
2.5
HO
2
*
1.5
400
**
350
1
WT
*
HO
**
300
0.5
250
0
200
WT
3 months
6 months
12 months
150
100
50
0
Wt
HO
Wt
Cellular hypertrophy and age
dependant
increases
in HO
BNP levels were evident
6months
12months
What effect does hyperglycaemia have on cardiac function?

Both systolic and diastolic
function were assessed by
echocardiography
Diastolic Function
3.5

Diastolic dysfunction was
evident
Systolic function was
preserved
E:A ratio

*
3
2.5
2
1.5
1
Wt
HO
3 Months

In diabetic patients diastolic
dysfunction
precedes
systolic dysfunction by up to
a decade
*
Wt
HO
6 Months
Wt
HO
12 Months
Summary (1)
3 MONTHS
Blood glucose

Normal structure and
function
6 MONTHS


Cardiac hypertrophy
Diastolic dysfunction
12 MONTHS



Cardiac hypertrophy
Diastolic dysfunction
Dilatation
Cardiac phenotype develops with increasing duration of hyperglycaemia
Molecular mechanism hypothesis
HYPERGLYCEMIA
INCREASED SERUM
AGE CONCENTRATION
AGE: Advanced Glycation End product
RAGE: Receptor for AGE
AGE
RAGE
PI3K
P-Akt
Cardiomyocyte
Hypertrophy
P-GSK3β
Elevated serum levels of Advanced Glycation End products
(AGE)
HYPERGLYCEMIA
INCREASED SERUM
AGE CONCENTRATION
Serum levels of CML
AGE
60
*
[CML] ng/mL
50
40
30
20
10
12
10
0
WT
60% increase
HO
Increased expression of Receptor for Advanced Glycation
End products (RAGE) in heart tissue
3 month
Wt
HO
12 month
6 month
Wt HO
Wt
HYPERGLYCEMIA
HO
RAGE
45kDa
Tubulin
55kDa
INCREASED SERUM
AGE CONCENTRATION
AGE
Relative expression from WT of same age
6
***
5
4
3
*
2
1
0
3 month
6 month
12 month
Increased
RAGE
expression
RAGE
Activation of Akt through phosphorylation
WT
HO
3 months p-Akt
60kDa
6 months p-Akt
60kDa
12 months p-Akt
60kDa
t-Akt
60kDa
HYPERGLYCEMIA
INCREASED SERUM
AGE CONCENTRATION
AGE
PI3K
Rleative Akt phosphorylation to WT of same age
6
Increased
RAGE
expression
*
5
4
RAGE
P-Akt
**
3
2
1
0
3 months
6 months
12 months
Increased
Akt
activation
Deactivation of GSK3β via phosphorylation
WT
HO
INCREASED SERUM
AGE CONCENTRATION
HYPERGLYCEMIA
p-GSK3β
46kDa
t-GSK3β
46kDa
AGE
2
**
Relative expression of p-GSK3B
1.8
PI3K
Increased
RAGE
expression
1.6
1.4
1.2
RAGE
1
P-Akt
0.8
0.6
0.4
0.2
Increased
GSK3β
phosphorylation
0
WT
HO
Cardiomyocyte
Hypertrophy
pGSK3β
Increased
Akt
activation
What are the direct effects of Advanced Glycation End
products (AGEs) on cardiomyocytes?
Control
CML
Plate Neonatal Rat Cardiomyocytes
60kDa
p-Akt
Control
Control media
AGE
Carboxy methylysine (CML)
t-Akt
60kDa
RAGE
45kDa
Tubulin
55kDa
3.5
Phospho Akt

RAGE
Exogenous AGE compounds CAN
activate Akt through phosphorylation
and increase RAGE expression
Relative expression
3
*
control
CML
2.5
2
*
1.5
1
0.5
0
p-Akt
RAGE
What are the direct effects of Advanced Glycation End
products (AGEs) on WT mice?
Osmotic pump
*
*
CML infusion promoted cardiac hypertrophy in
WT mice independently of diabetes
3 month old WT mice
Is PI3K the modulator of Akt?
INCREASED SERUM
AGE CONCENTRATION
HYPERGLYCEMIA
AGE
PI3K
Increased
RAGE
expression
P-Akt
Increased
GSK3β
phosphorylation
Cardiomyocyte
Hypertrophy
Wortmannin
RAGE
pGSK3β
Increased
Akt
activation
Readout:
Is Akt phosphorylation
reduced?
Relative expression of phosph Akt
Does wortmannin prevent CML induced Akt
phosphorylation?
CML
++
++
++
++
-
-
wortmannin
--
--
++
++
-
-
p-Akt
60kDa
60kDa
t-Akt
60kDa
60kDa
4.5
**
4
*
3.5
Wortmannin
prevents CML
induced Akt
phosphorylation
3
2.5
2
1.5
1
0.5
0
control
CML
+wortmainin
Can inhibition of the pathway in vivo prevent cardiac
hypertrophy developing?
Osmotic mini-pump containing
wortmannin
5 month old
GENA348 mice
Analyse cardiac phenotype
Week1
Week 6
**
7.50
**
200
**
6.5 month old
GENA348 mice
**
180
7.00
160
140
120
6.00
LV mass
HW:TL ratio
6.50
5.50
100
80
60
5.00
40
4.50
20
0
4.00
wortmannin
-
WT
+
-
HO
+
wortmannin
-
WT
+
-
HO
+
Wortmannin prevents the development of cardiac hypertrophy in GENA348
diabetic mice
Summary (2)
1.
2.
3.
4.
GENA348 mice have a glucokinase mutation
Develop hyperglycaemia
Do not develop hyperinsulinaemia
Have elevated levels of AGE
INCREASED
AGE CONCENTRATION
AGE
RAGE
3 MONTHS
6 MONTHS
12 MONTHS
P-Akt
WT

Normal heart


Cardiac hypertrophy
Diastolic dysfunction



GENA
Cardiac hypertrophy
Diastolic dysfunction
Dilatation
The cardiac hypertrophy phenotype can be prevented through inhibition of PI3K
Conclusions

GENA348 represents a human relevant model of diabetes and
therefore may provide more clinically valuable information regarding
the cardiovascular complications of diabetes.

GENA348 mice allow the examination of the effects of glucose,
independent of insulin on cardiac structure and function.

AGE:RAGE modulation of the Akt pathway may represent a novel
pathway through which cardiac hypertrophy can be treated in
diabetic patients.
Acknowledgements

Group Head: Professor Ludwig Neyses

Supervisors: Dr Mamas Mamas
Dr Elizabeth Cartwright

Collaborators: Prof. R Cox (Oxford)
Dr. M. Goldsworthy

Lab members:
Riham Abou-leisa
Florence Baudoin-Stanley
Zeinab Hegab
Arfa Maqsood
Tamer Mohamed
Delvac Oceandy
Ros Poulton
Sukhpal Prehar
Celine Schelcher
Mohamed Shaheen
Nicholas Stafford
Min Zi
FP 4-6