REGULATION DE l’AMPkinase Fabrizio Andreelli CHU Bichat et INSERM U 695

L’énergie…c’est la vie !!

Lipolyse (à jeun) Lipogenèse (nourri)

TG alimentaires AGL TG AGL + glycérol

Au moins deux phénomènes Résistance à l’insuline Altération de l’insulinosécrétion

Un phénotype stéréotypé… Production hépatique de glucose Stéatose hépatique Insulinorésistance Lipolyse accrue AGL Glycérol Insulinorésistance

C.Tour de taille J Vague 1947

AGL Muscle Stéatose TG AGL Cellule  Lipotoxicité et TAV insulinorésistance TG AGL insulinosécrétion

Relations TAV et NASH Nature 2006

Dépôts lipidiques extra-adipocytaires: muscle Greco AV et al. Diabetes 2002

Dépôts lipidiques extra-adipocytaires: foie

Dépôts lipidiques extra-adipocytaires: beta cell Rat OLEFT Jia MD et al, Diabetes 2000

Cancello R et al. Diabetes 2005

Miyazaki Y et al. AJP 2002

LAYERED Whole-body images such as these allow measurements of total body fat, among other parameters. On the far left, images show all the tissue compartments, each represented by a unique color, for a lean person (top) and an overweight person (bottom). The next images in each series show what's left after removal of subcutaneous adipose tissue (green), muscle tissue (red), and residual tissue (yellow). In the rightmost images, visceral adipose tissue is shown in gray and intramuscular adipose tissue in pink. Images were composed computationally from magnetic resonance imaging scans of cross-sectional slices of a whole body. COURTESY OF THE IMAGE READING CENTER, ST. LUKE'S HOSPITAL OF COLUMBIA UNIVERSITY MEDICAL CENTER

Syndrome métabolique

Obésité - répartition Tour de taille mesuré En position debout et en expiration douce A mi-hauteur entre rebord costal inférieur crête iliaque Risque Risque élevé Homme  94 cm  102 cm Femme  80 cm  88 cm d’après Lean (1995)

Conséquences du syndrome métabolique Insulinorésistance Diabète de type 2 Sd métabolique Risque cardio-vasculaire

Intra-abdominal adiposity and glucose metabolism Insulin 15 1 1,2 1200 1,2 1,2 Area Area 12 1 1,2 1,2 1 1 1,2 800 9 1,2 mmol/L 1 1 pmol/L 1 1,2 1 1,2 6 1 400 1,2 3 1,2 60 120 180 60 120 180 Intra-abdominal adiposity and glucose metabolism These data are from the study described in the previous slide. Plasma glucose and insulin concentrations during an oral glucose tolerance test (the inset graphs show area under the plasma concentration-time curves) were broadly similar in lean subjects and in obese subjects with low intra-abdominal adiposity. The presence of obesity per se therefore did not appear to influence glycaemic status in these subjects. In contrast, plasma glucose concentrations were significantly higher in obese subjects with high intra-abdominal adiposity, compared with the lean controls. While glucose levels generally differed little between obese subjects with high and low intra-abdominal adiposity, a markedly and significantly greater hyperinsulinaemia was required to maintain these glucose levels in the obese/high intra-abdominal adiposity group. These results show that intra-abdominal adiposity is a more important cause of insulin resistance than obesity per se. Pouliot MC, Despres JP, Nadeau A et al. Visceral obesity in men. Associations with glucose tolerance, plasma insulin, and lipoprotein levels. Diabetes 1992;41:826-34. Time (min) Time (min) Non-obese Obese low IAA Obese high IAA IAA: intra-abdominal adiposity Significantly different from 1non-obese, 2obese with low intra-abdominal adiposity levels Pouliot et al 1992 1

AGL Tissu adipeux abdominal

AMPKinase Changements métaboliques Environnement hormonal Effets enzymatiques Effets géniques

Structure de l'AMP-kinase regulatory SU  1, 2, 3 catalytic SU regulatory SU 1, 2   1, 2, 3 1, 2

Structure of AMP-activated protein kinase CaMKK LKB1 AMPK kinase Catalytic subunit  1, 2 N Thr172 C Catalytic domain  binding 1, 2 N C Glycogen binding  binding Regulatory subunit R302Q H383R T400N N488I R531G mutations 2 1, 2, 3 N CBS1 CBS2 CBS3 CBS4 C AMPK AMP/ATP binding R225Q mutation 3

AMP is signal of energy depletion fatty acid oxidation fatty acid synthesis

Regulation of AMP-activated protein kinase • ischemia • hypoxia • glucose deprivation • metabolic poisons  AMP/ATP Adenylate kinase ATP ATP ADP AMP physical exercise     AMP AMP  AMP  Pi ATP LKB1 CaMKK Phosphatases AMPK ADP  H2O     ADP  AMP ATP-consuming AMP = -AMPK ATP AMP   ATP-generation AMP ADP

Regulation of AMP-activated protein kinase CaMKK inactive AMPK LKB1 CBS4 a AMPKK b P  AMP  ATP T172 active AMPK AMP CBS4 : Catalytic subunit : Scaffold/glycogen-binding : Regulatory (AMP-binding)

+ Insuline P IRS-1 PI-3 kinase Glut4 Glucose Glucose AICAR AMPK Ox Stockage

Bergeron R et al. AJP 1999

Bergeron R et al. Diabetes 2001

Le traitement de rongeur par AICAR -augmente la captation intramusculaire de glucose même en présence d’une insulinorésistance *rongeur sain *rongeur obèse

Winder WW. J Appl Physiol 2001

Mu J et al. Mol Cell 2001

Mu J et al. Mol Cell 2001

Mu J et al. 2003

La réduction spécifique de l’activité de l’AMPK dans les muscles -s’associe à une diminution de la captation de glucose contraction dépendante -réduit la synthèse de glycogène -altère sa re-synthèse post-exercice physique -n’altère pas la signalisation de l’insuline musculaire -n’altère pas la sensibilité à l’insuline in vivo ni la glycémie

Les mutations activatrices des sous-unités  de l’AMPK entraînent une accumulation de glycogène intramusculaire (muscle squelettique 3 et cœur 2) et participent à la physiopathologie des syndromes de pré-excitation cardiaque (WPW).

Arad M et al. Circulation 2003

L’activation de l’AMPK musculaire par la contraction ou l’AICAR augmente la synthèse de glycogène musculaire Les mutations activatrices de l’AMPK dans le muscle ou le cœur provoquent l’accumulation de glycogène et favorisent des troubles de conduction

Le traitement de rongeur par AICAR -augmente la captation intramusculaire de glucose -réduit la production hépatique de glucose et la stéatose hépatique *rongeur sain *rongeur obèse

Bergeron R et al. Diabetes 2001

Zhou G et al. J Clin Invest 2001

Acétyl-CoA carboxylase Metformine Glucose Glucose-6P Pyruvate Citrate Krebs FFA FFA + Acyl-CoA AMPK CPT-1 Acétyl-CoA - -Oxydation Acétyl-CoA carboxylase (ACC) Acétyl-CoA Malonyl-CoA

L’activation pharmacologique de l’AMPK -augmente la captation intramusculaire de glucose -réduit la production hépatique de glucose -réduit la lipogenèse hépatique *rongeur sain *rongeur obèse Intérêt thérapeutique dans le diabète de type 2 !!

Phenotypic analysis of AMPK KO mice AMPK1-/- AMPK2-/- KO is embryonic lethal

Cre-loxP Strategy Recombinase from bactériophage P1 = 34bp sequence DNA LoxP LoxP + CRE Activity in vitro Recombinase - without DNA replication - without topo activity - without cofactors +

mice harbouring a complete Total inactivation of 2 AMPK x EIIA promoter ( zygote) CRE mice harbouring a complete inactivation of 2

Characterization of AMPK KO mice Southern blot Southern blot +/- +/+ -/- +/- +/+ -/- 2 wt 1 wt 1 ko 2 ko Western blot Western blot +/+ -/- +/+ -/- +/+ -/- +/+ -/- AMPK1 AMPK1 AMPK2 AMPK2 liver muscle liver muscle Viollet, JCI2003 Jorgensen, JBC2004

a1a2 KO E9.5 dpc E11.5 dpc -> embryonic lethality around 10.5 dpc. Control a1-/- a2-/- E9.5 dpc Alive no morphological differences E11.5 dpc Dead morphological aspect of E10.5 AMPK complexes play an essential role during embryonic development.

AMPK2-/- mice are not obese Body weight % of fat mass Food intake 8 4 40 6 30 3 %of fat mass body weight (g) 4 20 Food intake (g/d)) 2 2 10 1 +/+ -/- +/+ -/- +/+ -/- F Andreelli, J Clin Invest 2001

Glucose tolerance test in AMPK KO mice 350 control * control 220 ** AMPK1-/- AMPK2-/- 300 200 * 250 180 glucose (mg/dl) 200 160 glucose (mg/dl) 150 140 120 100 time (min) time (min) 100 50 20 40 60 80 100 120 20 40 60 80 100 120 0,5 Insulinemia at 20min (ng:ml) Insulinemia at 20min (ng:ml) 0,3 * 0,4 0,3 0,2 0,2 0,1 0,1 +/+ -/- +/+ -/- F Andreelli, J Clin Invest 2001

Metabolic parameters during euglycemic hyperinsulinemic clamp in AMPK2-/- mice -20 20 40 60 80 100 120 Glucose TO HGP Glycolysis Gln Synthesis control AMPK2 -/- ** F Andreelli, J Clin Invest 2001

Muscle glycogen during clamp in AMPK2-/- mice control AMPK2-/- 100 200 300 400 500 600 700 800 Gln synthesis ** 0,25 0,5 Total Gln content * F Andreelli, J Clin Invest 2001

F Andreelli, J Clin Invest 2001 100 120 140 160 180 200 220 20 40 60 80 time (min) * ** glucose (mg/dl) control AMPK2-/- 0,1 0,2 0,3 +/+ -/- Insulinemia at 20min (ng:ml) F Andreelli, J Clin Invest 2001

In vitro glucose transport in AMPK2-/- muscle Deoxyglucose transport (µmol/g/h) 0,5 1 1,5 2 2,5 basal insulin +/+ Soleus EDL -/- F Andreelli, J Clin Invest 2001

In vitro glucose-stimulated insulin secretion 0.05 0.1 0.15 0.2 0.25 insulin release as % content 15 30 45 60 75 90 105 120 Time (min) 7.5 10 20 L-Arg 3 mM glucose AMPK2-/- control Insulin content per islet is similar: 67.2  16.2 ng/islet (AMPK2-/-) versus 63.9  16.5 ng/islet (control) F Andreelli, J Clin Invest 2001

Why are AMPK2-/- mice insulinopenic? AMPK2-/- mice do not present: - hypokaliemia - hyperleptinemia - increased TG content in pancreas  modification of sympathetic and parasympathetic balance activity?

Urinary catecholamine levels in AMPK2-/- mice 10 20 30 40 50 ** urinary epinephrine (ng/d) * urinary dopamine (ng/d) 500 1000 1500 2000 2500 urinary norepinephrine (µg/d) epinephrine norepinephrine dopamine +/+ -/- F Andreelli, J Clin Invest 2001

-adrenergic antagonist treatment of AMPK2-/-mice Glucose tolerance test 50 100 150 200 250 300 20 40 60 Time (min) * ** % of basal glucose saline phentolamine control AMPK2-/- F Andreelli, J Clin Invest 2001

Is impaired glycogen synthesis linked to the lack of AMPK activity in skeletal muscle? use of AMPK-DN mice expressing a dominant inhibitory mutant of AMPK in skeletal muscle. (Mu et al., 2001) AMPK-DN creatine kinase promoter

Glucose tolerance in AMPK-DN mice Glucose tolerance test 500 control AMPK-DN 400 300 serum glucose (mg/dl) 200 100 -30 30 60 90 time (min)

Alpha 2-AMPK KO mice are insulin resistant --> In vivo insulin stimulated glycogen synthesis is reduced Glycogen synthesis rate ng/min.mg Control + Insulin 100 50 - AMPK DN muscle + - AMPK KO Alpha 2 + - * Insulin resistance is not due to lack of muscle AMPK

Altered regulation of metabolism in AMPK2-/- mice glucose insulin high sympathetic tone catecholamine insulin glycogen synthesis F Andreelli, J Clin Invest 2001

Rôle essentiel de la sous-unité alpha 2 dans les actions métaboliques de l’AMPK.

Diabetes 2004

La délétion de la sous-unité alpha 2 de l’AMPK dans tous les tissus altère la sensibilité à l’insuline et prédispose à l’obésité. Intérêt en génétique humaine ?

Quid de l’effet tissu-spécifique de l’AMPK Quid de l’effet tissu-spécifique de l’AMPK ? KO alpha 2 spécifique du foie

Liver a2 KO x 2flox Andreelli F et al, Endocrinology 2006 Liver 2 KO WT liver 2 AMPK1 AMPK2 CRE Albumin promoter 2flox x Liver 2 KO fl/fl; cre+ hypothalamus fl/fl; cre+ adipose tissue fl/fl; cre+ pancreas fl/fl; cre+ muscle fl/fl; cre- liver +/+; cre+ liver fl/fl; cre+ liver fl/fl; cre+ liver wt deleted floxed Andreelli F et al, Endocrinology 2006

Andreelli F et al, Endocrinology 2006

Régulation hormonale de la PHG Insuline - Néoglucogenèse - Leptine Adiponectine

Pas de conséquence sur la signalisation de l’insuline Table adiponectine Pas de conséquence sur la signalisation de l’insuline Perte du contrôle de la PHG par la leptine et l’adiponectine Andreelli F et al, Endocrinology 2006

Leptine Adiponectine Insuline + AMPKalpha 2 Néoglucogenèse

Surexpression de l’AMPkinase

Expression of AMPK2-CA by in vivo adenovirus-mediated gene transfer in mice + Adenovirus AMPK2-CA 48h infection Sacrifice Adenovirus injection 1.109 pfu effect on glycemia analysis of gene expression in fasted/refed conditions Diabetes 2005

CA-AMPK -> injection of adenovirus expressing constitutively active AMPK in control and diabetic mouse models fasted glucose PEPCK mRNA 1.2 300 1.0 250 § 0.8 200 §§ Ad b-gal § Relative mRNA Level Blood Glucose (mg/dl) 0.6 Ad CA-AMPK 150 §§ 0.4 100 §§ §§ 50 0.2 wt ob/ob STZ wt ob/ob STZ Liver AMPK targeting is sufficient to control glycemia in diabetic mice. Diabetes 2005

Acétyl-CoA carboxylase Leptine Adiponectine Metformine TZDs Glucose Glucose-6P Pyruvate Citrate Krebs FFA FFA + Acyl-CoA AMPK CPT-1 Acétyl-CoA - -Oxydation Acétyl-CoA carboxylase (ACC) Acétyl-CoA Malonyl-CoA

Patti ME, Kahn BB. Nature Medicine 2004

Rôle essentiel de la sous-unité alpha 2 de l’AMPK dans les actions métaboliques. Rôle de la SU alpha 1 ?

AMPK1 KO mice are anemic RBC (106/ml) Hb (g/dl) Ht (%) 20 12 60  1 KO 10 *** 16 50 *** *** 8 12 40 6 8 30 4 20 2 4 10 +/+ -/- +/+ -/- +/+ -/- 2 4 6 8 10 12 RBC (106/ml) -/- +/+ 4 8 12 16 20 Hb (g/dl) +/+ -/- 10 20 30 40 50 60 -/- +/+ Ht (%)  2 KO

Splenomegaly in AMPK1 KO mice 200 400 600 800 1000 1200 *** iron (µg/g) +/+ -/- control AMPK1-/-

Expression of AMPK1 isoform in RBC control 1-/- 2-/- AMPK1 AMPK2 Exclusive expression of AMPK1 isoform in RBC = specific role for AMPK1 in RBC integrity.

ATP content in AMPK1 -/- RBC    900 800 700 * 600 500 400 300 200 100 +/+ -/- ATP utilization in RBC  Glucose phosphorylation  Phosphorylation of membrane proteins  Ionic pumps

AMPK AMPK2 brain AMPK1 heart red blood cells adipose tissue muscle food intake insulin sensitivity ion transport cardiac ischemia AMPK adipose tissue muscle Liver lipolysis glucose transport glycogen synthesis hepatic glucose production fatty acid metabolism

University of Toulouse Institut Cochin, Paris University of Toulouse Viollet Benoit Marc Foretz Myriam Bennoun Claire Cheret Axel Kahn Sophie Vaulont Christophe Perrin Rémy Burcelin Howard Hughes Medical Institute Morris J. Birnbaum James Mu Copenhagen Muscle Research Center Sebastian B. Jørgensen Jørgen F.P. Wojtaszewski Erik A. Richter

Les Thiazolidinediones Wilson TM et al, Ann Rev Biochem 2001 Moller DE et al, Int J Obes 2003