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Although the mechanism of action of the antidiabetic drug metformin is still a matter of discussions, it is well accepted that the gut plays an important role. To gain more insights into the mechanisms that occur in the different regions of the intestinal tract in response to metformin treatment, adult male mice were fed a high-fat-high sucrose (HFS) diet for 8 days. Metformin treatment by gavage (300 mg/day/kg body weight) during the HFS diet improved glucose tolerance. Metformin counteracted HFS diet-induced overexpression of a network of genes involved in the transport of glucose and fatty acids in the small intestine, and induced beneficial modification of secondary bile acid profile in the caecum, with reduction in DCA and LCA levels and increased abundance of UDCA and TUDCA, potentially leading to FRX inhibition. In parallel, metformin treatment was associated with specific changes in the microbiota composition in the lumen of the different regions of the gut, counteracting the effects of HFS diet on the abundances of some bacterial groups generally associated with metabolic disturbances (f-Lachnospiraceae, f-Petostreptococcaceae, g-Clostidium). In addition, metformin promotes a strong increase in the abundance of Akkermansia muciniphila in the intestinal lumen all along the gut. Therefore, the present work clearly emphasises the role of all the regions of the intestinal tract in the beneficial action of the antidiabetic drug metformin in a prediabetic mouse model.

Objective: The fat mass and obesity associated (FTO) gene is related to obesity, but the regulation of FTO expression in adipose tissue is not fully understood. We investigated FTO expression in paired subcutaneous and omental adipose tissues (SAT and OAT) from healthy women undergoing gynecological surgeries, and its relation with adiposity and insulin sensitivity. Design and Methods: FTO expression in SAT of type 2 diabetic patients treated or not with Rosiglitazone was also compared. Results: Both the mRNA and protein levels of FTO were higher in OAT from women than in SAT. Only OAT FTO protein levels negatively correlated with BMI and body fat mass, whereas SAT FTO mRNA levels were negatively correlated with subcutaneous fat deposition. In addition, SAT FTO mRNA and protein levels were increased in insulin resistant women (high HOMA) compared to insulin sensitive women (low HOMA), whereas OAT FTO expression was not different between these two subgroups. Interestingly, FTO mRNA levels were increased in SAT of type 2 diabetic patients, and treatment of diabetics with Rosiglitazone improved insulin sensitivity and reduced SAT FTO mRNA levels. Lastly, FTO expression was transiently increased in the early phase of 3T3-L1 cell differentiation, which coincides with the induction of PPARγ2 expression. However, partial reduction of FTO did not impact PPARγ2 expression and adipocyte differentiation. Conclusion: Therefore, FTO gene expression is higher in OAT than in SAT in lean to moderately obese women. OAT FTO expression is associated with adiposity, whereas SAT FTO expression is associated with insulin sensitivity. These associations are independent of an effect of FTO on adipocyte differentiation.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice.We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca(2+) exchange, organelle homeostasis and insulin action.Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca(2+) transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells.Disruption of IP3R-mediated Ca(2+) signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

International audience; Endoplasmic reticulum (ER) stress is proposed as a novel link between elevated fatty acids levels, obesity and insulin resistance in liver and adipose tissue. However, it is unknown whether ER stress also contributes to lipid-induced insulin resistance in skeletal muscle, the major tissue responsible of insulin-stimulated glucose disposal. Here, we investigated the possible role of ER stress in palmitate-induced alterations of insulin action, both in vivo, in gastrocnemius of high-palm diet fed mice, and in vitro, in palmitate-treated C(2)C(12) myotubes. We demonstrated that 8 weeks of high-palm diet increased the expression of ER stress markers in muscle of mice, whereas ex-vivo insulin-stimulated PKB phosphorylation was not altered in this tissue. In addition, exposure of C(2)C(12) myotubes to either tuncamycine or palmitate induced ER stress and altered insulin-stimulated PKB phosphorylation. However, alleviation of ER stress by either TUDCA or 4-PBA treatments, or by overexpressing Grp78, did not restore palmitate-induced reduction of insulin-stimulated PKB phosphorylation in C(2)C(12) myotubes. This work highlights that, even ER stress is associated with palmitate-induced alterations of insulin signaling, ER stress is likely not the major culprit of this effect in myotubes, suggesting that the previously proposed link between ER stress and insulin resistance is less important in skeletal muscle than in adipose tissue and liver.

OBJECTIVE A strong association between genetic variants and obesity was found for the fat mass and obesity-associated gene (FTO). However, few details are known concerning the expression and function of FTO in skeletal muscle of patients with metabolic diseases. RESEARCH DESIGN AND METHODS We investigated basal FTO expression in skeletal muscle from obese nondiabetic subjects and type 1 and type 2 diabetic patients, compared with age-matched control subjects, and its regulation in vivo by insulin, glucose, or rosiglitazone. The function of FTO was further studied in myotubes by overexpression experiments. RESULTS We found a significant increase of FTO mRNA and protein levels in muscle from type 2 diabetic patients, whereas its expression was unchanged in obese or type 1 diabetic patients. Moreover, insulin or glucose infusion during specific clamps did not regulate FTO expression in skeletal muscle from control or type 2 diabetic patients. Interestingly, rosiglitazone treatment improved insulin sensitivity and reduced FTO expression in muscle from type 2 diabetic patients. In myotubes, adenoviral FTO overexpression increased basal protein kinase B phosphorylation, enhanced lipogenesis and oxidative stress, and reduced mitochondrial oxidative function, a cluster of metabolic defects associated with type 2 diabetes. CONCLUSIONS This study demonstrates increased FTO expression in skeletal muscle from type 2 diabetic patients, which can be normalized by thiazolidinedione treatment. Furthermore, in vitro data support a potential implication of FTO in oxidative metabolism, lipogenesis and oxidative stress in muscle, suggesting that it could be involved in the muscle defects that characterize type 2 diabetes.

International audience; Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D). However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFalpha expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H2O2 and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H2O2 production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.

Introduction S’il est bien admis que l’insulino-resistance hepatique (IRH) est associee a des alterations mitochondriales, un stress du reticulum endoplasmique (RE) et une perturbation de l’homeostasie calcique, on ne sait pas a l’heure actuelle si ces defauts representent des facteurs independants. Nous avons teste l’hypothese qu’ils sont inter-relies, via une alteration des echanges calciques au niveaux des points de contact entre les deux oragnites, appeles mitochondria-associated membranes (MAM). Materiels et methodes Sur la base de donnees recentes impliquant une chaperone mitochondriale, la cyclophiline D, dans la regulation de l’homeostasie calcique, du RE et du metabolisme glucidique, nous avons utilise des souris invalidees pour la cypD (cypD-KO) et des approches pharmacologiques et geniques in vitro pour etudier le role de la signalisation calcique mediee par IP3R au niveau des MAM dans l’IRH. Resultats Les souris cypD-KO presentent un stress du RE hepatique responsable d’une steatose, d’une IRH et d’une perturbation de l’homeostasie glucidique, puisque ces alterations sont prevenues par une inhibition du stress du RE (traitement au TUDCA ou surexpression hepatique de la chaperone Grp78). Ce stress du RE est lie a un defaut d’interaction et d’echange calcique entre le RE et la mitochondrie dans les hepatocytes de souris cypD-KO. L’ensemble de ces donnees sont reproduites in vitro dans des cellules HuH7 ou la cypD a ete inhibee soit par des traitements pharmacologiques (cyclosporine et NIM811) soit par un ARNi specifique. Enfin, nous demontrons que les alterations de la signalisation de l’insuline induite par la perte de cypD sont mediees par une activation de la PKCɛepsi; plutot que de la JNK. Conclusion Nos resultats demontrent un nouveau role de la cypD dans les interactions structurales et fonctionnelles entre le RE et la mitochondrie et un role cle de la signalisation calcique mediee par IP3R au niveau des MAM dans l’insulino-resistance hepatique.

Introduction Le gene FTO (fat mass and obesity-associated), fortement associe a l’obesite et au diabete de type 2 dans de nombreuses cohortes, a ete depuis implique dans le controle de la prise alimentaire via STAT3. Dans le foie, l’expression de FTO a ete correlee positivement avec l’expression des genes de la neoglucogenese, et STAT3 est un acteur important au controle de la neoglucogenese hepatique. L’objectif de notre etude etait donc d’evaluer le role de FTO dans le controle de la neoglucogenese hepatique et l’implication de la voie STAT3. Materiels et methodes Nous avons surexprime FTO in vivo dans le foie de souris par infection adenovirale et in vitro dans des cellules HuH7 par transfection transitoire, et etudie les repercussions sur la neoglucogenese et la regulation de la signalisation via STAT3. Resultats Les souris surexprimant FTO (Ad-FTO) specifiquement dans le foie ont une glycemie, une insulinemie et une leptinemie a jeun augmentees. Des tests de tolerance au glucose montrent que ces souris sont intolerantes au glucose. L’expression de genes impliques dans la neoglucogenese (G6P, FOXO1a) est augmentee, alors que l’expression de genes impliques dans la signalisation de la voie leptine/STAT3 (LepR, SOCS3) est diminuee. De plus, la forme phosphorylee de STAT3 est diminuee dans le noyau des souris Ad-FTO. In vitro, la surexpression de FTO dans des HuH7 exprimant le recepteur de la leptine induit des alterations de la reponse a la leptine au niveau de la phosphorylation de STAT3, entrainant une diminution de l’expression de SOCS3 et a une augmentation de la G6P. Conclusion La surexpression hepatique de FTO in vivo et in vitro affecte la signalisation STAT3 regulee par la leptine et induit les genes de la neoglucogenese, suggerant un role important de FTO dans le controle de la neoglucogenese hepatique via la voie STAT3.

Introduction L’obesite est un probleme majeur de sante publique, qui depend de facteurs genetiques et environnementaux. Recemment, des variants genetiques du gene codant pour la proteine FTO (fat mass and obesity associated gene), a la fonction inconnue, ont ete fortement associes a un indice de masse corporelle plus eleve. Le role de ce gene dans le controle du poids est inconnu, mais FTO est fortement present dans le cerveau et dans le tissu adipeux (TA). L’objectif de notre travail etait d’etudier, chez l’homme, la regulation de l’expression de FTO dans le TA et le muscle squelettique, en relation avec l’obesite et le diabete de type 2. Resultats Nous avons demontre que les niveaux d’ARNm de FTO sont augmentes specifiquement dans le muscle et le TA de patients diabetiques, alors qu’ils ne sont pas modifies chez les sujets obeses non diabetiques. L’expression proteique de FTO est egalement augmentee dans le muscle de patients diabetiques. Un traitement a la rosiglitazone (Avendia 8 mg/jour, 12 semaines) restaure en partie l’expression de FTO dans les tissus de patients diabetiques. D’autre part, les niveaux d’ARNm de FTO ne sont pas regules en aigu par l’insuline et le glucose. Enfin, nous avons surexprime FTO dans des HEK 293 et etudie les repercussions sur la sensibilite a l’insuline. Nous avons observe une augmentation de la phosphorylation de la PKB, en situation basale et une absence de regulation par l’insuline, en reponse a la surexpression de FTO, suggerant un controle negatif de la sensibilite a l’insuline par FTO. Conclusion Des experiences supplementaires de signalisation sont en cours pour etayer ces resultats preliminaires qui mettent en evidence un role de FTO dans le controle de la sensibilite a l’insuline dans le muscle squelettique.

Introduction Le stress oxydant associe a l’hyperglycemie est une caracteristique classique du diabete. L’augmentation de la production de radicaux libres (RL) fait intervenir differentes voies intracellulaires, mais les sources precises des RL sont encore mal definies dans les tissus sensibles a l’insuline, comme le muscle squelettique. Le but de notre etude etait donc de caracteriser les sites intracellulaires de production des RL induite par l’hyperglycemie dans le muscle squelettique de souris rendues diabetiques par un traitement a la streptozotocine (STZ). Les effets d’un traitement a l’insuline ou par des antioxydants ont egalement ete etudies. Resultats Les souris traitees a la STZ presentent une forte hyperglycemie associee a un stress oxydant circulant et musculaire. L’augmentation du stress oxydant est liee a une induction de la production de RL au niveau des mitochondries et de la xanthine oxydase (XO). De facon interessante, un traitement a l’insuline diminue l’hyperglycemie et ameliore le stress oxydant circulant et musculaire. Ces effets sont associes a une diminution de la production de RL induite a la fois par les mitochondries et la XO. De plus, la production de RL associee a des concentrations elevees de glucose est inhibee in vitro par la rotenone et l’oxypurinol dans des cellules musculaires, confirmant l’implication du complexe I des mitochondries et de la XO. Enfin, un traitement des souris STZ a l’oxypurinol diminue le stress oxydant systemique et musculaire, validant in vivo l’implication de la XO dans la production de RL associee a l’hyperglycemie. Conclusion L’ensemble de ces travaux demontrent l’implication du complexe I des mitochondries et de la XO dans la production de RL associee a l’hyperglycemie, mettant ainsi en evidence de nouvelles cibles responsables des dommages musculaires associes a l’hyperglycemie.