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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
http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=pZTSAH5UonOqsM&tbnid=-eWRZrqVOp_piM:&ved=0CAUQjRw&url=http://www.lsce.ipsl.fr/&ei=qlHIUeKzEKmL0AWhjoGQDg&bvm=bv.48293060,d.ZG4&psig=AFQjCNHSIp8d_hkxZNvMtDHNm8V_R8aD0w&ust=1372168997632535http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=pZTSAH5UonOqsM&tbnid=-eWRZrqVOp_piM:&ved=0CAUQjRw&url=http://www.lsce.ipsl.fr/&ei=qlHIUeKzEKmL0AWhjoGQDg&bvm=bv.48293060,d.ZG4&psig=AFQjCNHSIp8d_hkxZNvMtDHNm8V_R8aD0w&ust=1372168997632535
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
The causes of hepatic steatosis
Supply
LipolysisLipogenesis de novoFood
STEATOSIS
Adipose tissue
Lipolysis
FattyAcids
Glucose
Food
chylomicrons
VLDL
Lipogenesis
esterification
TG
-oxydation
Secretion
Use
-oxydationSecretion
The causes of hepatic steatosis
Supply
LipolysisLipogenesis de novoFood
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
http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=of4zcjZnnPg5EM&tbnid=DHQWa8ptBsrXuM:&ved=0CAUQjRw&url=http://pages.usherbrooke.ca/bcm-514-bl/3a.html&ei=B8rJUYOjGsTE0QXX5oGACg&bvm=bv.48293060,d.d2k&psig=AFQjCNECswCAHwE4CqtOcRwqknUvsXQ9PQ&ust=1372265346028994http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=of4zcjZnnPg5EM&tbnid=DHQWa8ptBsrXuM:&ved=0CAUQjRw&url=http://pages.usherbrooke.ca/bcm-514-bl/3a.html&ei=B8rJUYOjGsTE0QXX5oGACg&bvm=bv.48293060,d.d2k&psig=AFQjCNECswCAHwE4CqtOcRwqknUvsXQ9PQ&ust=1372265346028994
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
O-GlcNAcylation levels and FAS expressionin physiopathological models
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
O-GlcNAcylated protein levels and FAS expression
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
O-GlcNAcylation levels and FAS expressionin physiopathological models
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
O-GlcNAcylated protein levels and FAS expression
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
http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=tjBWw60Z94C7aM&tbnid=-wSPQokxrjhSTM:&ved=0CAUQjRw&url=http://www.tebu-bio.com/index.php?module=tech-info&id_cms=488&type_cms=3&ei=uInIUYntDM6A0AWAoIGAAg&bvm=bv.48293060,d.ZG4&psig=AFQjCNFGwYJivWt2WxjOEizz-n4bUsk0dg&ust=1372183335039591http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=tjBWw60Z94C7aM&tbnid=-wSPQokxrjhSTM:&ved=0CAUQjRw&url=http://www.tebu-bio.com/index.php?module=tech-info&id_cms=488&type_cms=3&ei=uInIUYntDM6A0AWAoIGAAg&bvm=bv.48293060,d.ZG4&psig=AFQjCNFGwYJivWt2WxjOEizz-n4bUsk0dg&ust=1372183335039591
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
http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=N2ak8O02Ok1F7M&tbnid=Zdl_lDXV5oLIGM:&ved=0CAUQjRw&url=http://www.functionalglycomics.org/fg/update/2011/110609/full/fg.2011.24.shtml&ei=Qou4UZ6mPOOy0QXP24DoCA&bvm=bv.47810305,d.ZG4&psig=AFQjCNEWq7CrI80XCsk00FEzuVWm4Aqgtg&ust=1371135156374233http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=of4zcjZnnPg5EM&tbnid=DHQWa8ptBsrXuM:&ved=0CAUQjRw&url=http://pages.usherbrooke.ca/bcm-514-bl/3a.html&ei=B8rJUYOjGsTE0QXX5oGACg&bvm=bv.48293060,d.d2k&psig=AFQjCNECswCAHwE4CqtOcRwqknUvsXQ9PQ&ust=1372265346028994http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=of4zcjZnnPg5EM&tbnid=DHQWa8ptBsrXuM:&ved=0CAUQjRw&url=http://pages.usherbrooke.ca/bcm-514-bl/3a.html&ei=B8rJUYOjGsTE0QXX5oGACg&bvm=bv.48293060,d.d2k&psig=AFQjCNECswCAHwE4CqtOcRwqknUvsXQ9PQ&ust=1372265346028994
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)