Gilles Mithieux-Fabienne Rajas

Inserm u.855/Université Lyon 1

October 2nd, 2010, Milan

MRAR

Evolution of GSD1a with time in a liver-specific model

of the pathology

Dysregulation of glucose homeostasis

GSD1a

•Prevalence: 1:100 000•Rare disease

•No endogenous glucose production

•LOW glucose blood

Type 2 diabetes

•Prevalence: 5-8%•Epidemic

•Endogenous glucose

production ++++

•HIGH glucose blood

Mirror metabolic diseases

-G6PT : transport subunit (ubiquitous)

Endogenous glucose production and Glucose-6 Phosphatase

G6P

T

Glucose -6 P

Glucose-6 P Glucose + Pi

Cytosol

Endoplasmic reticulum

G6PCG

6PT

GSD1a

GSD1b

Glycogenolysis Gluconeogenesis

-G6PC: catalytic subunit (liver, kidney and intestine)

G6Pase complex

EGP Liver

Kidney Intestine

•Hypoglycemia

•Liver disease:

-Hepatomegaly with accumulation of glycogen

-Liver steatosis

-Liver adenomas (or carcinomas)

•Kidney disease:

- Nephromegaly with accumulation of glycogen

- Renal failure

•Intestinal disease (under-estimated):

-digestion dysfunction-Diarrheas

Phenotype of GSD1a

GSD 1a livernormal liver

Mouse models of GSD1a

in the Liver and/or Intestineand/or Kidney

+ invalidation inducible by tamoxifen

Lethal without injection of glucose every 8 hours

(Mutel et al., J Hepatol, 2010)

(Chou et al, 1999)

Total invalidation of g6pc :

Tissue-specific invalidation of g6pc :

-To know if expression of G6Pase at one site is sufficient to compensate the lack of expression at the others

-To study the mechanisms of the deficiency organ by organ-Liver gene therapy

glucose

Targeted invalidation of G6Pase in liver of mice

B6.G6pclox/lox

Injection of tamoxifen(1mg/day for 5 days)

at 6-8 weeks

L.G6pc-/-

Liver-specific Cre

B6.SACreERT2/+

x =

B6.SACre ERT2/+

G6pclox/lox

*

**0

20

40

60

80

-/- +/- +/+G

6Pas

e ac

tivi

ty (

U/g

pro

t)

J.Hepatol. in press

Metabolic effects of hepatic G6pc deletion with time

A B

0

50

100

150

200

Glu

cose

(m

g/d

L)

10 d 1 mth 6 mth 18 mth

0

10

20

30

40

50

0 3 6 9 12 15 18

Bo

dy

we

igh

t (g

)

Time after gene deletion (month)

C

D E F

0

1

2

3

4

TG

(g

/L)

10 d 1 mth 6 mth 18 mth

**

* **

0

1

2

3

Ch

ole

ste

rol (

g/L

)

10 d 1 mth 6 mth 18 mth

** ** **

**

0

10

20

30

40

Uric

aci

d (

mg

/L)

10 d 1 mth 6 mth 18 mth

*

*

0

2

4

6

La

ctic

aci

d (

mM

)

10 d 1 mth 6 mth 18 mth

** **

Improvement of plasmatic parameters with time, except for cholesterol

LG6pc-/- mice Control mice

L.G6pc-/- mice develop hepatomegaly and steatosis

0

20

40

60

80

10 d 1 mth 18 mth

TG

co

nte

nt

(m

g/g

of

live

r)

**

**

0

20

40

60

80

10 d 1 mth 18 mthG

lyco

ge

n c

on

ten

t (

mg

/g o

f liv

er)

**

**

**

0

1

2

3

4

10 d 1 mth 18 mth

Liv

er

we

igh

t (g

)

**

****

Liver disease:

• Accumulation of glycogen in the liver of LG6pc-/- mice: HEPATOMEGALY

•Triglyceride storage in the liver of LG6pc-/- mice: STEATOSIS

•Development of hepatic adenomas with time

LG6pc-/- mice Control miceControlL-G6pc-/-

Late appearance of hepatocellular adenoma in L.G6pc-/- mice

Follow up of mouse liver by MRI from 3 months to 18 months

In collaboration with O. Beuf & F. Pilleul, Creatis, LyonMRI 7.0 Tesla

12 months 18 months

Liver of a LG6pc-/- mouse

Muttel et al, J.Hepatol. in press

Late appearance of hepacellular adenoma in L.G6pc-/- mice

Livers of LG6pc-/- miceControl liver

Time after G6pc deletion % mice with lesions Size of lesions

<6 months 0 0

9 months 15 1 mm

12 months 30 1-2 mm

15 months 40-50 1-3 mm

18 months 100 1-10 mm

Histology study

Liver adenomas (80%) Liver pre-carcinomas (20%)

AdAd

Development of adenoma in the liver of Lg6pc-/- mice

•Adenoma with steatosis and large hepatocytes •Adenoma without steatosis - small hepatocytes

Loss of tissue organizationAbsence of portal space

Necrosis and inflammationGlycogenic nuclei

• Viable, permitting to study the liver pathology along the mouse life

• Liver disease characteristic of GSD1a:

-Hepatomegaly

-Liver steatosis

-Liver adenomas…and pre-carcinomas

• No apparent kidney disease • No apparent intestinal disease

Excellent model to gene therapy

LG6pc-/- mice exhibit all the hepatic symptoms of the human pathology

In conclusion

Fabienne RajasArmelle Penhoat

Valérie Large Amandine Stein

Elodie Mutel Aya Abdul-WahedSylvie Casteras Anne Stefanutti

Isabelle Houberdon Gilles Mithieux

UMR Inserm u.855/UCBL, Lyon

Thanks to

Olivier Beuf Frank Pilleul

Niri RamamonjisoaSophie CavassilaHélène Ratiney

Créatis, Lyon

MRI