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Regulation of hepatic Fatty Acid Synthase properties 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 Control of blood glucose by the liver Fasted A l’état nourri gluco-dependent organs glucose glycogenolysis gluconeogenesis β-oxydation Fatty Acid + glucagon adipocytes pancreas Hepatic glucose production Control of blood glucose by the liver Fasted In the fed state gluco-dependent organs Glucose absorption glucose glucose glycogenolysis gluconeogenesis β-oxydation Fatty Acid + glucagon adipocytes glycogenogenesis glycolysis lipogenesis VLDL + insulin adipocytes pancreas Hepatic glucose production pancreas Hepatic glucose uptake Dysregulation of glucido-lipidic metabolism and hepatic steatosis The fat represents at least 5 to 10% of the liver weight 15% no obese 65% obèse (IMC 30-40) 1/3 3% no obese 20% obese 10 à 29% 4 à 27% CHC The causes of hepatic steatosis Glucose Food chylomicrons VLDL Lipogenesis TG Fatty Acids esterification Lipolysis Adipose tissue Supply Lipolysis Lipogenesis de novo Food STEATOSIS The causes of hepatic steatosis Glucose Food Lipogenesis TG chylomicrons -oxydation Fatty Acids esterification VLDL Lipolysis Secretion Adipose tissue Supply Lipolysis Lipogenesis de novo Food STEATOSIS Use -oxydation Secretion The causes of hepatic steatosis Glucose Food Lipogenesis 14% 26% TG chylomicrons -oxydation Fatty Acids esterification VLDL 59% Lipolysis Secretion Adipose tissue Supply Lipolysis Lipogenesis de novo Food STEATOSIS Use -oxydation Secretion Donnelly et al., 2005 Hepatic steatosis : Fatty acids of triglycerides come from 59% of the lipolyse 26% of the lipogenesis 14 % of the food Lipogenesis and Fatty Acid Synthase Lipogenesis Glucose ACC : AcetylCoA Carboxylase GLUT Glucose CYTOSOL Citrate Acetyl-CoA Glucose-6-P Pyruvate Pyruvate ACC Malonyl-CoA FAS Acyl-CoA MITOCHONDRIA Acetyl-CoA Citrate Fatty Acid Synthase Malonyl/acetyl transferase Triglycerides β-ketoacyl-synthase β-hydroxyacyl dehydratase Enoyl reductase β-ketoacyl-reductase • 2 subunits • 7 activities • liver, adipose tissue, mammary gland ACP Thioesterase Overall reaction : acetylCoA + 7 malonylCoA + 14 NADPH,H+ palmitoylCoA (C16:0) + 7CO2 + 14 NADP++ 7CoA Regulation of FAS At the transcriptional level by the transcription factors,ChREBP and SREBP-1c insulin glucose glucose + ChREBP SREBP-1c insulin + Glycolysis G6P PEP L-PK + Pyruvate + citrate + ACC, FAS, SCD-1 FA Lipogenesis Hepatocytes At the post translational level by phosphorylation Functions of O-GlcNAcylation … Hundreds proteins bearing O-GlcNAc have been identified Enzymes of metabolism Kinases/phosphatases Proteins of stress Transcription Factors Proteins of cytoskeleton Proteasome Proteins of pore nuclear Ribosomals proteins … Involved in many cellular processes Translation Cell cycle Apoptosis Cell trafficking Transcription Signaling Development Proteins degradation Cellular architecture … dynamism disturbed in certain diseases Neurological diseases Type 2 diabetes Cancers Cardiovascular diseases OGT/OGA O-N-acetylglucosaminylation (O-GlcNAcylation) O-GlcNAcylation UDP UDP-GlcNAc ATP ADP OH OGT (O-GlcNAc transferase) S/T Kinase S/T OGA (O-GlcNAcase) H2 O S/T Phosphatase Pi GlcNAc H2O The Hexosamine Biosynthesis Pathway (HBP) Glc Glc G6P F6P GFAT Gln Glu GlcNH26P GlcNH26P AcT Acetyl CoA GlcNAc6P HSCoA UDP-GlcNAc PPase GlcNAc1P UTP PPi UDPGlcNAc Hypothesis • FAS expression and O-GlcNAcylation level depend on glucose concentrations Relation between O-GlcNAcylation, expression and activity of FAS Is FAS O-GlcNAcylated ? Relations between FAS O-GlcNAcylation and nutritional conditions Do O-GlcNAcylation levels interfere with: FAS expression FAS activity FAS stabilization O-GlcNAcylation levels and FAS expression in physiopathological models Model 1 : Use of mice C57BL6 wild type or ob/ob Day 3h 15h 3h Fasted C57BL6 Fasted HCHO Fasted Ob/ob Fasted HCHO 15h Night Group1 : C57Bl6 Fasted Group2 : C57Bl6 Refed Group3 : ob/ob Fasted Group4 : ob/ob Refed O-GlcNAcylated protein levels and FAS expression C57BL6 Refed ob/ob C57BL6 Fasted 17013010070- WB: O-GlcNAc 55WB: FAS 17055- *** 3 C57 *** Ob/ob 3.5 3 FAS/GAPDH O-GlcNAc/GAPDH 4 WB: GAPDH 35- 2.5 2 1.5 1 * FAS ARNm * C57 Ob/ob 2.5 2 C57BL6 *** 0.45 ob/ob 0.35 1.5 1 0.5 0.5 0.5 0.4 Relative mRNA level FAS / cyclophilin Quantification 0.3 0.25 0.2 0.15 * 0.1 0.05 0 0 Fasted Refed 0 Fasted Refed Fasted Refed O-GlcNAcylation levels and FAS expression in physiopathological models Model 2 : Use of mice C57BL6 fed a chow diet or fed a High Carbohydrate Diet. 12 weeks Fasted Chow Diet (CD) 65% carbohydrate, 24% protein, 11% fat Fasted Fasted High Carbohydrate Diet (HCD) 75% carbohydrate, 22% protein, 3% fat HCHO Fasted HCHO Group1 : CD Fasted Group2 : CD Refed Group3 : HCD Fasted Group4 : HCD Refed O-GlcNAcylated protein levels and FAS expression Refed CD CD HCD Fasted 1701301007055- WB: O-GlcNAc WB: FAS 17055- *** 9 CD 6 * *** 8 HCD 0.07 CD 7 NS 6 4 3 CD5 CD HCD 4 HCD 3 2 2 *** CD HCD 0.06 HCD FAS/GAPDH O-GlcNAc/GAPDH 5 FAS ARNm Relative mRNA level FAS / cyclophilin Quantification 7 WB: GAPDH 35- 0.05 0.04 0.03 NS 0.02 0.01 1 1 0 Fasted Refed 0 0 Fasted Refed Fasted Refed Interaction FAS/OGT Fasted ob C57 ob Refed Fasted C57 CD HCD CD WB: FAS 170- WB: FAS Input 130100- 170130- WB: OGT 100IgG IgG 170130100- Input WB: OGT WB: FAS WB: FAS WB: OGT Refed CoIP OGT 170130100- FAS and OGT are partners of interaction CoIP OGT WB: OGT FAS O-GlcNAcylation Use of lectins : Wheat Germ Agglutinin - + + - HCD Refed CD C57BL6 ob/ob C57BL6 - Fasted - + GlcNAc 0.5M + - + CD Refed Fasted - + GlcNAc 0.5M Input Input 170- WB: FAS WGA beads 170- 170- 170- WB: FAS WGA beads click chemistry UDP-GalNAz UDP biotin N3 UDP Y289L GalT1 N3 Avidinbead FAS O-GlcNAcylation Use of lectins : Wheat Germ Agglutinin + - + - HCD Refed CD C57BL6 ob/ob C57BL6 - Fasted - + GlcNAc 0.5M + - + CD Refed Fasted - GlcNAc 0.5M Input Input 170- + WB: FAS WGA beads 170- WB: FAS WGA beads 170- 170- click chemistry 20- + + GalNAz / biotin alkyne GalNAz / biotin alkyn Fasted Refed CD HCD CD Input WB: Cristallin 20- Input Avidin beads WB: Avidin-HRP WB: FAS Avidin beads O-GlcNAcylation levels and FAS expression in physiopathological models Model 3 : Use of mice C57BL6 presenting an inhibition of OGA Day 0 Daily injection of PBS/ThiametG 20 mg/ kg/ day Day 14 Fasted Fasted HCHO Group1 : Fasted Group2 : Refed O-GlcNAcylated protein levels and FAS expression Fasted ThiametG PBS Refed PBS WB : FAS 130 100 WB : O-GlcNAc 70 50 40 40 WB : GAPDH 35 Quantification 14 FAS ARNm 30 12 ** *** 12 6 Relative mRNA level FAS / cyclophilin FAS/GAPDH O-GlcNAc/GAPDH 10 8 8 6 PBS *** ThiametG 25 10 *** PBS * Thiamet-G 4 4 20 15PBS Thiamet-G 10 NS 5 2 2 0 0 0 Fasted Refed Fasted Refed Fasted Refed Ex vivo Cancer human hepatocytes cell line : HepG2 Mouse primary hepatocytes cultures Glc Fru Gln GlcNH2 Gln Glu Glc G6P F6P GFAT GlcNH26P GlcNAc6P Ser/Thr ThiametG NButGT Mouse primary hepatocytes culture Expression of FAS GlcNAc1P UDP-GlcNAc OGT OGA Ser/Thr O-GlcNAc Role of O-GlcNAcylation on FAS stabilisation HepG2 Kinetic with cycloheximide CHX + NButGT CHX O-GlcNAc Time (h) 0 S/T CHX translation NButGT OGA S/T 1 2 6 14 0 24 1 2 6 14 24 270 WB : FAS 130 100 70 WB : O-GlcNAc 55 PROTEIN WB : GAPDH 35 Liver mice Treatment with β-N-acetylglucosaminidase O-GlcNAc S/T kDa 270- Fasted Refed - - + + Glucosaminidase WB : FAS 130- Glucosaminidase 100- WB : O-GlcNAc 70- S/T 5535- WB : GAPDH Role of O-GlcNAcylation on FAS activity FAS O-GlcNAcylation Refed - + GlcNAc 0.5M CD C57BL6 + Refed HCD - + Fasted CD - ob/ob C57BL6 Fasted - + - + - + Input 170- GlcNAc 0.5M Input 170WGA beads 170- WGA beads 170- WB: FAS WB: FAS *** PBS ThiametG 200 *** 10 8 6 4 2 *** µmoles of NAPDH oxydized. min-1.mg-1 of liver 12 CD HCD C57BL6 ob/ob µmoles of NAPDH oxydized. min-1.mg-1 of liver µmoles of NAPDH oxydized. min-1.mg-1 of liver FAS activity 0 Fasted Refed Fasted Refed * 150 100 50 0 Fasted Refed Conclusions FAS regulation : by transcription factors Glucose Glycolysis PK pyruvate ChREBP Lipogenesis ACC, FAS Fatty acid SREBP-1c by O-GlcNAcylation FAS interacts with OGT and it’s O-GlcNAcylated Roles of the O-GlcNAcylation : Increase of global O-GlcNAcylation levels is correlated with an increase of FAS expression through a reduction of its degradation. FAS activity is in correlation with its O-GlcNAcylation Perspectives Relation O-GlcNAcylation/ubiquitinylation of FAS O-GlcNAcylation Stabilisation S/T Phosphorylation Ubiquitinylation Degradation S/T G5 ø 130100705540170130100- 1704035- GlcNH2 + - + G25i - MG132 WB : O-GlcNAc • Perform siRNA to silent OGT WB : ub • Evaluate FAS ubiquitinylation in function of O-GlcNAcylation levels WB : FAS WB : GAPDH The MG132 can restore FAS expression at G5 similar to the condition G25. FAS seems to be degraded by the proteasome in the liver Team of Pr Tony Lefebvre Pr Annick Pierce Dr Ikram El Yazidi Dr Anne Sophie Vercoutter Edouart Dr Agata Steenackers Dr Nao Yamakawa Moyira Aquino-gil Maité Leturcq Anne-Marie Mir Marlène Mortuaire Jeanne Vermuse 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)
