regulation of hepatic fatty acid synthase properties by o ... · dr anne sophie vercoutter edouart...

Regulation of hepatic Fatty Acid Synthaseproperties by O-GlcNAcylation in vivo and ex vivo

S/T

Baldini Steffi, Anne-Marie Mir, Marlène Mortuaire, Céline Guinez and Tony Lefebvre

CNRS-UMR 8576, Unit of Structural and Functional Glycobiology, FRABio FR3688, Lille 1 University, 59655 Villeneuve d’Ascq, France

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Control of blood glucose by the liver

A l’état nourriFasted

pancreas

+

glucagon

glycogenolysisgluconeogenesis

adipocytes

Fatty Acid

β-oxydation

glucose

gluco-dependent organs

Hepatic glucose production

In the fed state

pancreas

+

insulin

glucose

Glucose absorption

glycogenogenesisglycolysis

lipogenesis

VLDL

adipocytes

Hepatic glucose uptake

Fasted

pancreas

+

glucagon

glycogenolysisgluconeogenesis

adipocytes

Fatty Acid

β-oxydation

glucose

gluco-dependent organs

Hepatic glucose production

Control of blood glucose by the liver

Dysregulation of glucido-lipidic metabolismand hepatic steatosis

15% no obese65% obèse (IMC 30-40)

3% no obese20% obese

1/3

10 à 29%

CHC4 à 27%

The fat represents at least 5 to 10% of the liver weight

The causes of hepatic steatosis

Supply

LipolysisLipogenesis de novoFood

STEATOSIS

Adipose tissue

Lipolysis

FattyAcids

Glucose

Food

chylomicrons

VLDL

Lipogenesis

esterification

TG


Supply


STEATOSIS

Adipose tissue

Lipolysis

FattyAcids

Glucose

Food

chylomicrons

VLDL

Lipogenesis

esterification

TG

-oxydation

Secretion

Use

-oxydationSecretion


Supply


STEATOSIS

Adipose tissue

Lipolysis

FattyAcids

Glucose

Food

chylomicrons

VLDL

Lipogenesis

esterification

TG

-oxydation

Secretion

Use

-oxydationSecretion

26%

59%

14%

Donnelly et al., 2005Hepatic steatosis : Fatty acids of triglycerides come from59% of the lipolyse26% of the lipogenesis14 % of the food

Fatty Acid Synthase

• 2 subunits• 7 activities• liver, adipose tissue, mammary gland

Overall reaction : acetylCoA + 7 malonylCoA + 14 NADPH,H+ palmitoylCoA (C16:0) + 7CO2 + 14 NADP++ 7CoA

Lipogenesis and Fatty Acid Synthase

β-ketoacyl-synthaseMalonyl/acetyl

transferase

Thioesterase

β-ketoacyl-reductase

Glucose

Glucose

Glucose-6-P

Pyruvate Acetyl-CoA Citrate

Pyruvate

Citrate Acetyl-CoA Malonyl-CoAACC

Acyl-CoA

Triglycerides

FAS

CYTOSOL

GLUT

MITOCHONDRIA

Lipogenesis

β-hydroxyacyldehydratase

Enoyl reductase

ACP

ACC : AcetylCoA Carboxylase

glucose

G6P

ChREBP

PEP

L-PK

Gly

coly

sis

Pyruvate citrate FAACC, FAS, SCD-1

Lipogenesis

++

Hepatocytes

+

glucose insulin

insulin+

SREBP-1c

+

At the transcriptional level by the transcription factors,ChREBP and SREBP-1c

Regulation of FAS

At the post translational level by phosphorylation

Functions of O-GlcNAcylation

… Involved in many cellular processes

Signaling

Cell cycle

DevelopmentApoptosis

Proteins degradation

TranscriptionTranslation Cell trafficking

Cellular architecture

… dynamism disturbed in certain diseases

Type 2 diabetes

Neurological diseases

Cardiovascular diseases

Cancers

… Hundreds proteins bearing O-GlcNAc have been identified

Enzymes of metabolism

Kinases/phosphatases

Proteins of cytoskeleton Transcription FactorsProteins of stress

Proteins of pore nuclear Ribosomals proteinsProteasome OGT/OGA

http://images.google.fr/imgres?imgurl=www.kizefo.de/pics/gallery_pict/sonst/apoptosis.jpg&imgrefurl=http://www.kizefo.de/other.html&h=287&w=200&prev=/images?q=apoptosis&svnum=10&hl=fr&lr=&ie=UTF-8&oe=UTF-8&sa=Nhttp://images.google.fr/imgres?imgurl=www.kizefo.de/pics/gallery_pict/sonst/apoptosis.jpg&imgrefurl=http://www.kizefo.de/other.html&h=287&w=200&prev=/images?q=apoptosis&svnum=10&hl=fr&lr=&ie=UTF-8&oe=UTF-8&sa=Nhttp://images.google.fr/imgres?imgurl=dicty.cmb.nwu.edu/chisholm/NUskeleton.jpg&imgrefurl=http://dicty.cmb.nwu.edu/chisholm/NWU cytoskeleton.htm&h=200&w=200&prev=/images?q=cytoskeleton&start=20&svnum=10&hl=fr&lr=&ie=UTF-8&oe=UTF-8&sa=Nhttp://images.google.fr/imgres?imgurl=dicty.cmb.nwu.edu/chisholm/NUskeleton.jpg&imgrefurl=http://dicty.cmb.nwu.edu/chisholm/NWU cytoskeleton.htm&h=200&w=200&prev=/images?q=cytoskeleton&start=20&svnum=10&hl=fr&lr=&ie=UTF-8&oe=UTF-8&sa=Nhttp://www.biw.kuleuven.be/dtp/cmpg/pgprb_images/Proteasome26S.pnghttp://www.biw.kuleuven.be/dtp/cmpg/pgprb_images/Proteasome26S.pnghttp://www.uni-heidelberg.de/zentral/bzh/transport.jpghttp://www.uni-heidelberg.de/zentral/bzh/transport.jpghttp://www.scienceclarified.com/images/uesc_04_img0230.jpghttp://www.scienceclarified.com/images/uesc_04_img0230.jpghttp://folk.uio.no/jamoskau/Lectures/ERN3120 090505_files/slide0078_image062.gifhttp://folk.uio.no/jamoskau/Lectures/ERN3120 090505_files/slide0078_image062.gif

O-N-acetylglucosaminylation (O-GlcNAcylation)

Glc

Glc G6P F6P GlcNH26P GlcNAc6P GlcNAc1PUDP-GlcNAc

GFAT

Gln Glu AcetylCoA

HS-CoA

GlcNH26PAcT

UTP PPi

UDP-GlcNAcPPase

The Hexosamine Biosynthesis Pathway (HBP)

O-GlcNAcylation

S/T S/T

OH

UDP-GlcNAcUDP

OGT (O-GlcNAc transferase)

OGA (O-GlcNAcase)

GlcNAc H2O

Kinase

Phosphatase

ATP ADP

H2O Pi

S/T

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Hypothesis

• FAS expression and O-GlcNAcylation level depend on glucose concentrations

Relation between O-GlcNAcylation, expression and activity of FAS

Relations between FAS O-GlcNAcylation and nutritional conditions

FAS activity

Do O-GlcNAcylation levels interfere with:

FAS expression

FAS stabilization

Is FAS O-GlcNAcylated ?

Fasted

HCHOFasted

Group1 : C57Bl6 Fasted

Group2 : C57Bl6 RefedC57BL6

Ob/ob

3h 15h 3h

Day

Night15h

Fasted

HCHOFasted

Group3 : ob/ob Fasted

Group4 : ob/ob Refed

Model 1 : Use of mice C57BL6 wild type or ob/ob

O-GlcNAcylation levels and FAS expressionin physiopathological models

FAS ARNm

C57

Ob/ob

C57

Ob/ob

Quantification

Fasted Refed Fasted Refed

C5

7B

L6

ob

/ob

Fasted

170-130-100-

70-

55-

C5

7B

L6

Refed

WB: FAS

WB: O-GlcNAc

55-

35-WB: GAPDH

170-

0

0.5

1

1.5

2

2.5

3

3.5

4 ***

***

O-G

lcN

Ac/

GA

PDH

0

0.5

1

1.5

2

2.5

3 **

FA

S/G

APD

H

Rel

ativ

e m

RN

Ale

vel

FA

S /

cycl

op

hil

in

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

*

***

Fasted Refed

C57BL6

ob/ob

O-GlcNAcylated protein levels and FAS expression


Model 2 : Use of mice C57BL6 fed a chow diet or fed a High CarbohydrateDiet.

12 weeks

Chow Diet (CD)

High Carbohydrate Diet (HCD)

75% carbohydrate, 22%

protein, 3% fat

65% carbohydrate, 24%

protein, 11% fat

Fasted

HCHOFasted

Group1 : CD Fasted

Group2 : CD Refed

Fasted

HCHOFasted

Group3 : HCD Fasted

Group4 : HCD Refed

FAS ARNm

CD

HC

D

Fasted

170-130-100-

70-

55-

55-

35-

CD

WB: FAS

WB: O-GlcNAc

WB: GAPDH

170-

Fasted Refed

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Rel

ativ

e m

RN

Ale

vel

FA

S /

cycl

op

hil

in

CD

HCD

NS

***CD

HCD

CD

HCD

Quantification

0

1

2

3

4

5

6

7

CD

HCD

NS

***

O-GlcNAc/GAPDH

0

1

2

3

4

5

6

7

8

9

CD

HCD

****

FAS/GAPDH

Refed

Fasted RefedFasted Refed


Interaction FAS/OGT

WB: FAS

WB: OGT

WB: FAS

WB: OGT

CoIP

OGT

Input

C57

Fasted Refed

IgG

ob C57

170-

130-

100-

170-

ob

130-

100-

WB: FAS

WB: OGT

WB: FAS

WB: OGT

CoIP

OGT

Input

CD

Fasted Refed

IgG

HCD CD

170-

130-

100-

170-

130-

100-

FAS and OGT are partners of interaction

click chemistry

FAS O-GlcNAcylation

CD HC

D

Fasted Refed

CD

Input

- + GlcNAc 0.5M-+- +

WGA beads

WB: FAS170-

170-

Use of lectins : Wheat Germ Agglutinin

C5

7B

L6

ob

/ob

Fasted Refed

C5

7B

L6

Input

- + GlcNAc 0.5M-+- +

WGA beads

WB: FAS170-

170-

Y289L GalT1

UDP

N3UDP

N3

Avidin-bead

biotin

UDP-GalNAz

GalNAz / biotin alkyn- +

WB: Cristallin

WB: Avidin-HRP

Input

Avidin beads

20-

20-

click chemistry

GalNAz / biotin alkyne- +

Input

Avidin beads

CD

Fasted Refed

HCD CD

WB: FASC

D HC

D

Fasted Refed

CD

Input

- + GlcNAc 0.5M-+- +

WGA beads

WB: FAS170-

170-

C5

7B

L6

ob

/ob

Fasted Refed

C5

7B

L6

Input

- + GlcNAc 0.5M-+- +

WGA beads

WB: FAS170-

170-

Use of lectins : Wheat Germ Agglutinin

FAS O-GlcNAcylation

Model 3 : Use of mice C57BL6 presenting an inhibition of OGA

Fasted

HCHOFasted

Group1 : Fasted

Group2 : Refed

Day 0 Day 14

Daily injection of PBS/ThiametG

20 mg/ kg/ day


WB : O-GlcNAc

WB : FAS

WB : GAPDH

130

100

70

50

4040

35

PBS ThiametG Refed PBS

Fasted

0

5

10

15

20

25

30

Rel

ativ

e m

RN

Ale

vel

FA

S /

cycl

op

hil

in

PBS

ThiametG

NS

***

Fasted Refed

Quantification

0

2

4

6

8

10

12

14

PBS

Thiamet-G

Fasted Refed

***

**

O-GlcNAc/GAPDH

0

2

4

6

8

10

12

PBS

Thiamet-G

*

***

FAS/GAPDH

Fasted Refed

FAS ARNm


Ex vivo Cancer human hepatocytes cell line : HepG2 Mouse primary hepatocytes cultures

Glc

Ser/Thr Ser/Thr O-GlcNAcOGT

OGA

GFATGlc G6P F6P GlcNH26P GlcNAc6P GlcNAc1P

UDP-GlcNAc

Gln Glu

Gln GlcNH2

ThiametGNButGT

Fru

Expression of FAS

Mouse primary hepatocytes culture

Liver mice

HepG2

Glucosaminidase

S/T

O-GlcNAc

S/T

0 1 2 6 14 240 1 2 6 14 24

CHX CHX + NButGT

WB : O-GlcNAc

WB : FAS

WB : GAPDH

55

70

100

130

35

Time (h)

270

Refed

55-

70-

100-

130-

35-

kDa

Fasted

- -+ + Glucosaminidase

WB : FAS

WB : O-GlcNAc

WB : GAPDH

270-

PROTEIN

translationCHX

S/T

O-GlcNAc

S/T

NButGT OGA

Role of O-GlcNAcylation on FAS stabilisation

Kinetic with cycloheximide

Treatment with β-N-acetylglucosaminidase

C5

7B

L6

ob

/ob

Fasted RefedC

57

BL

6

Input

- + GlcNAc 0.5M-+- +

WGA beads

170-

170-

CD

HC

D

Fasted Refed

CD

Input

- + GlcNAc 0.5M-+- +

WGA beads

170-

170-

FAS O-GlcNAcylation

µm

ole

s o

f N

AP

DH

oxyd

ized

.

min

-1.m

g-1

of

liver

0

2

4

6

8

10

12***

C57BL6

ob/ob

Fasted Refed

***

µm

ole

s o

f N

AP

DH

oxyd

ized

.

min

-1.m

g-1

of

liver

Fasted Refed

CD

HCD

***

0

50

100

150

200

Fasted Refed

*

µm

ole

s o

f N

AP

DH

oxyd

ized

.

min

-1.m

g-1

of

liver

PBSThiametG

FAS activity

WB: FAS WB: FAS

Role of O-GlcNAcylation on FAS activity

Conclusions

FAS interacts with OGT and it’s O-GlcNAcylated

Roles of the O-GlcNAcylation :

Increase of global O-GlcNAcylation levels is correlated with an increaseof FAS expression through a reduction of its degradation.

FAS activity is in correlation with its O-GlcNAcylation

FAS regulation :

by transcription factors

Glucose pyruvate Fatty acid

Glycolysis Lipogenesis

PK ACC, FAS

ChREBP SREBP-1c

by O-GlcNAcylation

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Perspectives

O-GlcNAcylationStabilisation

PhosphorylationUbiquitinylation

DegradationS/T S/T

+- - -+ MG132

WB : O-GlcNAc

WB : FAS

WB : GAPDH

G5

GlcNH2 G25iø

130-100-

70-55-40-

35-

170-

40-

WB : ub130-100-

170-

Relation O-GlcNAcylation/ubiquitinylation of FAS

The MG132 can restore FAS expression at G5 similar to the condition G25. FAS seems to be degraded by the proteasome in the liver

• Perform siRNA to silent OGT

• Evaluate FAS ubiquitinylation in functionof O-GlcNAcylation levels

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Pr Annick PierceDr Ikram El YazidiDr Anne Sophie Vercoutter EdouartDr Agata SteenackersDr Nao YamakawaMoyira Aquino-gilMaité LeturcqAnne-Marie MirMarlène MortuaireJeanne Vermuse

Team of Pr Tony Lefebvre

Collaborations

Céline Guinez (Unité Environnement Périnatal et santé, V d’Ascq) Catherine Postic (Institut de Cochin, Paris)Isabelle Hainault (Centre des Cordeliers, Paris)David Vocadlo (Simon Fraiser University, Burnaby)

regulation of hepatic fatty acid synthase properties by o ... · dr anne sophie vercoutter edouart...

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