Regulation of hepatic Fatty Acid Synthase properties by

Regulation of hepatic Fatty Acid Synthase properties by O-Glc. NAcylation 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 FR 3688, Lille 1 University, 59655 Villeneuve d’Ascq, France

Control of blood glucose by the liver A l’état nourri Fasted 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 glycogenolysis gluconeogenesis β-oxydation Fatty Acid + glucagon adipocytes glycogenesis 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 : Acetyl. Co. A Carboxylase GLUT Glucose CYTOSOL Citrate Acetyl-Co. A Glucose-6 -P Pyruvate ACC Malonyl-Co. A FAS Acyl-Co. A MITOCHONDRIA Acetyl-Co. A 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 : acetyl. Co. A + 7 malonyl. Co. A + 14 NADPH, H + palmitoyl. Co. A (C 16: 0) + 7 CO 2 + 14 NADP++ 7 Co. A

Regulation of FAS Ø At the transcriptional level by the transcription factors, Ch. REBP and SREBP-1 c insulin glucose + Ch. REBP SREBP-1 c + insulin Glycolysis G 6 P PEP L-PK + Pyruvate + citrate + ACC, FAS, SCD -1 FA Lipogenesis Hepatocytes Ø At the post translational level by phosphorylation

Functions of O-Glc. NAcylation … Hundreds proteins bearing O-Glc. NAc 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-Glc. NAcylation) O-Glc. NAcylation UDP-Glc. NAc ATP ADP OH OGT (O-Glc. NAc transferase) S/T Kinase S/T OGA (O-Glc. NAcase) H 2 O S/T Phosphatase Pi Glc. NAc H 2 O The Hexosamine Biosynthesis Pathway (HBP) Glc G 6 P F 6 P GFAT Gln Glu Glc. NH 26 P Ac. T Acetyl Co. A Glc. NAc 6 P HSCo. A UDP-Glc. NAc PPase Glc. NAc 1 P UTP PPi UDPGlc. NAc

Hypothesis • FAS expression and O-Glc. NAcylation level depend on glucose concentrations Relation between O-Glc. NAcylation, expression and activity of FAS

Ø Is FAS O-Glc. NAcylated ? q Relations between FAS O-Glc. NAcylation and nutritional conditions Ø Do O-Glc. NAcylation levels interfere with: q FAS expression q FAS activity q FAS stabilization

O-Glc. NAcylation levels and FAS expression in physiopathological models Model 1 : Use of mice C 57 BL 6 wild type or ob/ob Day 3 h 15 h 3 h Fasted C 57 BL 6 Fasted HCHO Fasted Ob/ob Fasted HCHO 15 h Night Group 1 : C 57 Bl 6 Fasted Group 2 : C 57 Bl 6 Refed Group 3 : ob/ob Fasted Group 4 : ob/ob Refed

O-Glc. NAcylated protein levels and FAS expression C 57 BL 6 Refed ob/ob C 57 BL 6 Fasted 17013010070 - WB: O-Glc. NAc 55 WB: FAS 17055 - Quantification *** 3 C 57 *** Ob/ob C 57 Ob/ob 2. 5 0. 5 C 57 BL 6 *** 0. 45 ob/ob 0. 4 3 FAS/GAPDH O-Glc. NAc/GAPDH 3. 5 * FAS ARNm * 2. 5 2 1. 5 1 2 0. 35 Relative m. RNA level FAS / cyclophilin 4 WB: GAPDH 35 - 1. 5 1 0. 5 0. 3 0. 25 0. 2 0. 15 * 0. 1 0. 05 0 0 Fasted Refed

O-Glc. NAcylation levels and FAS expression in physiopathological models Model 2 : Use of mice C 57 BL 6 fed a chow diet or fed a High Carbohydrate Diet. 12 weeks Fasted Chow Diet (CD) 65% carbohydrate, 24% protein, 11% fat Fasted High Carbohydrate Diet (HCD) 75% carbohydrate, 22% protein, 3% fat HCHO Fasted HCHO Group 1 : CD Fasted Group 2 : CD Refed Group 3 : HCD Fasted Group 4 : HCD Refed

O-Glc. NAcylated protein levels and FAS expression Refed CD HCD CD Fasted 1701301007055 - WB: O-Glc. NAc WB: FAS 17055 - FAS ARNm CD CD HCD 0. 07 CD *** HCD 0. 06 Relative m. RNA level FAS / cyclophilin Quantification WB: GAPDH 35 - 0. 05 0. 04 0. 03 NS 0. 02 0. 01 Fasted Refed 0 Fasted Refed

Interaction FAS/OGT Fasted ob C 57 ob Refed Fasted C 57 CD HCD CD WB: FAS 170 - WB: FAS Input 130100 - 170130 - WB: OGT 100 Ig. G 170130100 - Refed WB: FAS WB: OGT Co. IP OGT 170130100 - FAS and OGT are partners of interaction Input WB: OGT WB: FAS WB: OGT Co. IP OGT

FAS O-Glc. NAcylation q Use of lectins : Wheat Germ Agglutinin + - - + Glc. NAc 0. 5 M HCD + - + Glc. NAc 0. 5 M Input 170 - WB: FAS WGA beads 170 - Refed CD C 57 BL 6 ob/ob C 57 BL 6 - Fasted CD Refed Fasted 170170 - WB: FAS WGA beads q click chemistry UDP-Gal. NAz UDP N 3 biotin UDP Y 289 L Gal. T 1 N 3 Avidinbead

FAS O-Glc. NAcylation q Use of lectins : Wheat Germ Agglutinin + - HCD Refed CD C 57 BL 6 ob/ob C 57 BL 6 - Fasted - + Glc. NAc 0. 5 M + - + CD Refed Fasted - WB: FAS WGA beads 170 - Glc. NAc 0. 5 M Input 170 - + 170 - WB: FAS WGA beads 170 - q click chemistry 20 - + Gal. NAz / biotin alkyn Input WB: Cristallin 20 - + Gal. NAz / biotin alkyne Fasted Refed CD HCD CD Input Avidin beads WB: Avidin-HRP WB: FAS Avidin beads

O-Glc. NAcylation levels and FAS expression in physiopathological models Model 3 : Use of mice C 57 BL 6 presenting an inhibition of OGA Day 0 Daily injection of PBS/Thiamet. G 20 mg/ kg/ day Day 14 Fasted HCHO Group 1 : Fasted Group 2 : Refed

O-Glc. NAcylated protein levels and FAS expression Fasted PBS Thiamet. G Refed PBS WB : FAS 130 100 WB : O-Glc. NAc 70 50 40 40 WB : GAPDH 35 Quantification FAS ARNm 30 PBS *** Thiamet. G Relative m. RNA level FAS / cyclophilin 25 20 15 10 NS 5 0 Fasted Refed

Ø Ex vivo q Cancer human hepatocytes cell line : Hep. G 2 q Mouse primary hepatocytes cultures Glc Fru Gln Glc. NH 2 Gln Glu Glc G 6 P F 6 P GFAT Glc. NH 26 P Glc. NAc 6 P Ser/Thr Thiamet. G NBut. GT Mouse primary hepatocytes culture Expression of FAS Glc. NAc 1 P UDP-Glc. NAc OGT OGA Ser/Thr O-Glc. NAc

Role of O-Glc. NAcylation on FAS stabilisation Hep. G 2 Kinetic with cycloheximide CHX + NBut. GT CHX O-Glc. NAc Time (h) 0 S/T CHX translation NBut. GT OGA S/T 1 2 6 14 0 24 1 2 6 14 270 WB : FAS 130 100 70 WB : O-Glc. NAc 55 PROTEIN WB : GAPDH 35 Liver mice Treatment with β-N-acetylglucosaminidase O-Glc. NAc S/T k. Da 270 - Fasted Refed - - + + Glucosaminidase WB : FAS 130 - Glucosaminidase 100 - WB : O-Glc. NAc 70 - S/T 24 5535 - WB : GAPDH

Role of O-Glc. NAcylation on FAS activity FAS O-Glc. NAcylation Refed - + Glc. NAc 0. 5 M - + CD C 57 BL 6 + Refed HCD - + Fasted CD - ob/ob C 57 BL 6 Fasted - + Input 170 - Glc. NAc 0. 5 M Input 170 WGA beads 170 - WB: FAS *** 180 *** 10 8 6 4 2 0 Fasted Refed *** Fasted µmoles of NAPDH oxydized. min-1. mg-1 of liver 12 PBS Thiamet. G CD HCD C 57 BL 6 ob/ob µmoles of NAPDH oxydized. min-1. mg-1 of liver FAS activity Refed 160 * 140 120 100 80 60 40 20 0 Fasted Refed

Conclusions FAS regulation : q by transcription factors Glucose Glycolysis PK pyruvate Ch. REBP Lipogenesis ACC, FAS Fatty acid SREBP-1 c q by O-Glc. NAcylation Ø FAS interacts with OGT and it’s O-Glc. NAcylated ØRoles of the O-Glc. NAcylation : q Increase of global O-Glc. NAcylation levels is correlated with an increase of FAS expression through a reduction of its degradation. q FAS activity is in correlation with its O-Glc. NAcylation

Perspectives Relation O-Glc. NAcylation/ubiquitinylation of FAS O-Glc. NAcylation Stabilisation S/T Phosphorylation Ubiquitinylation Degradation S/T G 5 ø 130100705540170130100 - 1704035 - Glc. NH 2 + - + G 25 i - MG 132 WB : O-Glc. NAc • Perform si. RNA to silent OGT WB : ub • Evaluate FAS ubiquitinylation in function of O-Glc. NAcylation levels WB : FAS WB : GAPDH The MG 132 can restore FAS expression at G 5 similar to the condition G 25. 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)