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BackgroundType 2 diabetes (T2D) increases the risk of pancreatic ductal adenocarcinoma (PDAC), which could be due to an epigenetic mechanism.MethodsWe explored the association between T2D and whole pancreas methylation in 141 individuals, of which 28 had T2D, using Illumina MethylationEPIC 850K BeadChip arrays. We performed downstream functional assessment in the rat acinar pancreas cell line AR42J. To further understand the role of our candidate gene in humans, we tested whether null variants were associated with T2D and related traits using the UK biobank.ResultsMethylation analysis identified one significant CpG associated with T2D: hypermethylation in an enhancer inPNLIPRP1, an acinar-specific gene.PNLIPRP1expression was decreased in T2D individuals. Using a rat acinar cell line, we 1/ confirmed decreasedPnliprp1in response to a diabetogenic treatment, and 2/ inPnliprp1knockdown, an up-regulation of cholesterol biosynthesis, cell cycle down-regulation, decreased expression of acinar markers and increased expression of ductal markers pointing towards acinar-to-ductal metaplasia (ADM), a hallmark of PDAC initiation. Using exome data from UK Biobank, we show that rarePNLIPRP1null variants associated with increased glucose, BMI and LDL-cholesterol.Conclusions/interpretationWe present evidence that an epigenetically-regulated gene associates with T2D risk, and might promote ADM and PDAC progression, opening new insights into early prevention of PDAC.

Objective This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue. Methods Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages. Results In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P < 0.05). The expression of several KP enzyme genes (indoleamine 2,3-dioxygenase 1 [IDO1], kynureninase [KYNU], kynurenine 3-monooxygenase [KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P < 0.05), and their expression was induced by proinflammatory cytokines in human primary adipocytes (P < 0.05), except for KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P < 0.05). Conclusions In the context of obesity, the presence of macrophages in adipose tissue may contribute to diverting KP toward KMO activation.

Gene polymorphisms of TCF7L2 are associated with increased risk of type 2 diabetes and transcription factor 7-like 2 (TCF7L2) plays a role in hepatic glucose metabolism. We therefore addressed the impact of TCF7L2 isoforms on hepatocyte nuclear factor 4α (HNF4α) and the regulation of gluconeogenesis genes.Liver TCF7L2 transcripts were analysed by quantitative PCR in 33 non-diabetic and 31 type 2 diabetic obese individuals genotyped for TCF7L2 rs7903146. To analyse transcriptional regulation by TCF7L2, small interfering RNA transfection, luciferase reporter and co-immunoprecipitation assays were performed in human hepatoma HepG2 cells.In livers of diabetic compared with normoglycaemic individuals, five C-terminal TCF7L2 transcripts showed increased expression. The type 2 diabetes risk allele of rs7903146 positively correlated with TCF7L2 expression in livers from normoglycaemic individuals only. In HepG2 cells, transcript and TCF7L2 protein levels were increased upon incubation in high glucose and insulin. Of the exon 13 transcripts, six were increased in a glucose dose-responsive manner. TCF7L2 transcriptionally regulated 29 genes related to glucose metabolism, including glucose-6-phosphatase. In cultured HepG2 cells, TCF7L2 did not regulate HNF4Α and FOXO1 transcription, but did affect HNF4α protein expression. The TCF7L2 isoforms T6 and T8 (without exon 13 and with exon 15/14, respectively) specifically interacted with HNF4α.The different levels of expression of alternative C-terminal TCF7L2 transcripts in HepG2 cells, in livers of normoglycaemic individuals carrying the rs7901346 type 2 diabetes risk allele and in livers of diabetic individuals suggest that these transcripts play a role in the pathophysiology of type 2 diabetes. We also report for the first time a protein interaction in HepG2 cells between HNF4α and the T6 and T8 isoforms of TCF7L2, which suggests a distinct role for these specific alternative transcripts.

Low-grade and chronic inflammation is elicited in white adipose tissue in human obesity. The presence of inflammatory molecules leads to an increased tryptophan catabolism through the induction of indoleamine-2,3-dioxygenase-1 (IDO1). In order to characterize the mechanisms underlying this dysregulation, we have studied 2 mouse models of obesity. Unexpectedly, we did not detect any IDO1 expression in obese or lean mice adipose tissue. In a previous study, we did not find any significant difference in the liver for IDO2 and tryptophan-2,3-dioxygenase (TDO2) gene expression between normal weight and obese patients. IDO2 and TDO2 expression was increased in the liver of high-fat fed mice, but not in ob/ob mice, and was strongly correlated with hydroxysteroid-(11-beta) dehydrogenase-1 (HSD11B1) expression, an enzyme that generates active cortisol within tissues. In conclusion, despite a dysregulation of tryptophan metabolism, obese mice display discrepancies with human obesity metabolism, rendering them inappropriate for further investigations in this animal model.

BACKGROUND:An increase of plasma kynurenine concentrations, potentially bioactive metabolites of tryptophan, was found in subjects with obesity, resulting from low-grade inflammation of the white adipose tissue. Bariatric surgery decreases low-grade inflammation associated with obesity and improves glucose control. OBJECTIVE:Our goal was to determine the concentrations of all kynurenine metabolites after bariatric surgery and whether they were correlated with glucose control improvement. DESIGN:Kynurenine metabolite concentrations, analysed by liquid or gas chromatography coupled with tandem mass spectrometry, circulating inflammatory markers, metabolic traits, and BMI were measured before and one year after bariatric surgery in 44 normoglycemic and 47 diabetic women with obesity. Associations between changes in kynurenine metabolites concentrations and in glucose control and metabolic traits were analysed between baseline and twelve months after surgery. RESULTS:Tryptophan and kynurenine metabolite concentrations were significantly decreased one year after bariatric surgery and were correlated with the decrease of the usCRP in both groups. Among all the kynurenine metabolites evaluated, only quinolinic acid and xanthurenic acid were significantly associated with glucose control improvement. The one year delta of quinolinic acid concentrations was negatively associated with the delta of fasting glucose (p = 0.019) and HbA1c (p = 0.014), whereas the delta of xanthurenic acid was positively associated with the delta of insulin sensitivity index (p = 0.0018). CONCLUSION:Bariatric surgery has induced a global down-regulation of kynurenine metabolites, associated with weight loss. Our results suggest that, since kynurenine monoxygenase diverts the kynurenine pathway toward the synthesis of xanthurenic acid, its inhibition may also contribute to glucose homeostasis.

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.

International audience; We report highly sensitive Bioluminescence Resonance Energy Transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein (YFP), the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced ß-arrestin (ßARR) intramolecular rearrangements and recruitment to G protein-coupled receptors (GPCRs). We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing ßARR assays and offer new options for the development of future BRET assays.

This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue.Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages.In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10(-19) ) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10(-4) ). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P0.05). The expression of several KP enzyme genes (indoleamine 2,3-dioxygenase 1 [IDO1], kynureninase [KYNU], kynurenine 3-monooxygenase [KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P0.05), and their expression was induced by proinflammatory cytokines in human primary adipocytes (P0.05), except for KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P0.05).In the context of obesity, the presence of macrophages in adipose tissue may contribute to diverting KP toward KMO activation.

International audience; Background We previously reported that the low-frequency, loss-of-function variant p.R270H in FFAR4 encoding the lipid sensor GPR120 was associated with obesity. Gpr 120-deficient mice develop obesity and both impaired fasting glucose and glucose intolerance under a high-fat diet. We aimed to assess the contribution of p.R270H to type 2 diabetes (T2D) risk and the variation of glucose-related traits. Methods We genotyped p.R270H in 8996 nondiabetic individuals (among whom 4523 had an oral glucose tolerance test (OGTT)) and in a T2D case-control study including 4725 cases and 4339 controls. The regression models were adjusted for age, sex and body mass index (BMI). Results We found a significant association between p.R270H and increased fasting glucose levels (beta=0.092 +/- 0.05 mmol/L; p=4.13x10(-4)). Furthermore, p.R270H nominally contributed to decreased homeostasis model of pancreatic beta-cell function (HOMA-B; beta=-0.090 +/- 0.06; p=6.01x10(-3)). Despite a high statistical power, we did not find any significant association between p.R270H and T2D risk or the variation of fasting insulin levels, the homeostasis model of insulin resistance or OGTT-derived indices. Conclusions These results suggest that the low-frequency p.R270H variant which inhibits GPR120 activity might influence fasting glucose levels in a normal physiological range. This study does not exclude that other coding mutations in FFAR4 with stronger functional effect than p.R270H may be associated with T2D.

Introduction La chirurgie bariatrique ameliore l'homeostasie glycemique et reduit l'inflammation chronique associee a l'obesite. Consequence de cette inflammation, une augmentation des kynurenines circulantes provenant de la degradation du tryptophane a ete demontree chez les sujets obeses. Certaines kynurenines sont neurotoxiques et pourraient etre diabetogenes. Notre objectif est de determiner si la reduction de poids induite par chirurgie bariatrique permet un retour a l'equilibre de ces metabolites et est correlee a l'amelioration glucidique. Patients et Methodes Les concentrations en kynurenines circulantes de 91 patientes de la cohorte ABOS (47 diabetiques, 44 normoglycemiques) ont ete evaluees a l'inclusion et un an apres la chirurgie bariatrique, par chromatographie liquide ou gazeuse couplee a la spectrometrie de masse. Les associations entre ces concentrations, la CRP (C reactive protein) circulante et les facteurs associes au controle glycemique ont ete evaluees. Resultats Les concentrations du tryptophane, de kynurenine, des acides kynurenique, xanthurenique et quinolinique ont diminue significativement un an apres la chirurgie ( p =2,2×10-10, p =2,7×10-4, p =1,3 × 10-4, p =1,6×10-7 et p =8,4 x 10-5 respectivement) alors que la concentration en serotonine a augmente ( p =2,1×10-5). Ces variations sont significativement correlees a la baisse de la CRP. Les concentrations des differents metabolites ne sont pas significativement differentes entre les deux groupes normoglycemique et diabetique, a l'inclusion ou un an apres chirurgie. Neanmoins apres ajustement par rapport a la perte de poids, les patientes qui ont maintenu une concentration en acide xanthurenique elevee au detriment de l'acide quinolinique, ont significativement moins ameliore leur glycemie a jeun et leur insulino-sensibilite que les patientes pour lesquelles l'acide xanthurenique a plus fortement diminue. Conclusions Cette etude montre que la diminution de l'inflammation associee a la perte de poids apres chirurgie est associee a une diminution globale de l'activation de la voie de kynurenines. Ces donnees suggerent que la baisse d'activite de la kynurenine monoxygenase, enzyme charniere pour l'orientation vers la production d'acide xanthurenique, est associee a une evolution plus favorable de l'homeostasie glycemique un an apres la chirurgie.

Free fatty acids provide an important energy source as nutrients, and act as signalling molecules in various cellular processes. Several G-protein-coupled receptors have been identified as free-fatty-acid receptors important in physiology as well as in several diseases. GPR120 (also known as O3FAR1) functions as a receptor for unsaturated long-chain free fatty acids and has a critical role in various physiological homeostasis mechanisms such as adipogenesis, regulation of appetite and food preference. Here we show that GPR120-deficient mice fed a high-fat diet develop obesity, glucose intolerance and fatty liver with decreased adipocyte differentiation and lipogenesis and enhanced hepatic lipogenesis. Insulin resistance in such mice is associated with reduced insulin signalling and enhanced inflammation in adipose tissue. In human, we show that GPR120 expression in adipose tissue is significantly higher in obese individuals than in lean controls. GPR120 exon sequencing in obese subjects reveals a deleterious non-synonymous mutation (p.R270H) that inhibits GPR120 signalling activity. Furthermore, the p.R270H variant increases the risk of obesity in European populations. Overall, this study demonstrates that the lipid sensor GPR120 has a key role in sensing dietary fat and, therefore, in the control of energy balance in both humans and rodents., 脂肪センサーGPR120が食事性肥満の原因遺伝子であることの発見. 京都大学プレスリリース. 2012-02-20.

Background Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented. Methodology/Principal Findings A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity. Conclusions/Significance The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated.

Introduction La voie de degradation du tryptophane en kynurenines (KP) est deregulee dans l’obesite. L’indoleamine 2,3-dioxygenase 1 (IDO1), l’enzyme cle de cette voie, est activee par l’inflammation et son expression est augmentee dans le tissu adipeux blanc des individus obeses par rapport aux minces. Des 1955, une association entre le diabete et certaines kynurenines avait ete suggeree. Notre objectif est d’analyser les proprietes diabetogenes de ces kynurenines et de caracteriser la voie KP chez des individus obeses normoglycemiques ou diabetiques avant et apres une chirurgie bariatrique. Materiels et methodes 100 individus obeses de la banque ABOS (50 diabetiques et 50 normo-glycemiques) ont ete selectionnes. Les kynurenines ont ete dosees dans les serums, preleves le jour de l’operation et un an plus tard, par chromatographie en phase gazeuse ou liquide couplee a un spectrometre de masse en tandem. La lignee cellulaire beta pancreatique MIN-6 a ete soumise a un test de secretion d’insuline en presence ou non d’acide xanthurenique. Resultats L’activite d’IDO1 evaluee par le rapport kynurenine sur tryptophane diminue significativement (P = 0,0077) apres la chirurgie bariatrique chez les individus normoglycemiques mais pas chez les diabetiques. Cependant, dans les deux groupes, apres chirurgie bariatrique, HbA1c est diminuee significativement (P Discussion Nos resultats montrent une association entre l’amelioration de la valeur d’HbA1c apres chirurgie bariatrique et la diminution de certaines kynurenines inhibitrices de la secretion d’insuline.

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin’s effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.Structured summaryMINT-7714817: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by anti tag co-immunoprecipitation (MI:0007)MINT-7714785: LEPRc (uniprotkb:P48357-2) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714951, MINT-7714744: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714859: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714885, MINT-7714672: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by bioluminescence resonance energy transfer (MI:0012)MINT-7714835: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714914, MINT-7714723, MINT-7714759: LeprB (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714703, MINT-7714936, MINT-7714772: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714872: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by anti tag co-immunoprecipitation (MI:0007)

Introduction Le foie joue un role majeur mais encore mal connu dans le risque de diabete de type 2 (DT2) dont l'heritabilite est en partie expliquee par des facteurs genetiques et epigenetiques. Notre projet a donc pour but d'identifier et de caracteriser des marqueurs epigenetiques du tissu hepatique associes au DT2. Patients et methodes Nous avons analyse les profils de methylation de l'ADN sur plus de 450 000 sites a l'aide d'une puce Infinium Human Methylation 450 BeadChip (Illumina) dans le tissu hepatique de 96 patients DT2 et 96 normoglycemiques francais apparies sur l'âge, l'IMC et le genre. Resultats La methylation globale, en considerant l'ensemble des sites analyses par la puce, n'etait pas differente entre les patients DT2 et les individus controles. Cependant, l'etude site par site a pu mettre en evidence 15 marqueurs epigenetiques potentiels du DT2 (p -7 ). Conclusion Nous avons mis en evidence les premiers marqueurs epigenetiques du DT2 dans le foie. Il est maintenant necessaire d'etablir si ces variations sont partagees entre les differents types cellulaires et de caracteriser les defauts fonctionnels associes.

Introduction Chez l’Homme, 4 isoformes du recepteur a la leptine, l’hormone de satiete, sont obtenues par epissage alternatif du meme gene (OB-Ra, b, c, et d). OB-Rb, la forme longue du recepteur, joue un role majeur dans l’obesite en activant les voies de signalisation responsables de l’homeostasie lipidique. Le role des formes courtes, OB-Ra, c, et d, est encore mal connu. Il est bien etabli que les OB-R homomerisent de facon constitutive grâce a leur partie extracellulaire commune a toutes les isoformes. Cependant, l’existence d’interactions entre la forme longue et les formes courtes d’OB-R reste controversee. Materiels et Methodes Des plasmides exprimant OB-Ra, b et c ont ete transfectes dans les cellules HEK293T afin de suivre leurs interactions par BRET (Bioluminescence Resonance Energy Transfer) en cellules vivantes et par co-immunoprecipitation. La localisation cellulaire des interactions observees a ensuite ete etudiee par BRET sur des preparations de membranes internes et membranes plasmiques de ces cellules. Les experiences ont ete realisees en absence et en presence de leptine afin de determiner son effet sur ces complexes. Resultats Nous montrons que les isoformes courtes interagissent avec OB-Rb de maniere constitutive dans les cellules vivantes. La leptine induit un changement de conformation de ces heteromeres suggerant ainsi leur fonctionnalite. De maniere surprenante les heteromeres d’OB-R ne sont presents qu’a la membrane plasmique, contrairement aux homomeres qui sont localises egalement a l’interieur des cellules. Conclusion Nos resultats suggerent donc que ces heteromeres sont formes par l’association des homomeres OB-Rb avec les homomeres des isoformes courtes a la surface cellulaire ou ils repondent a la leptine. L’existence de ces heteromeres conduit a une reinterpretation des donnees de la litterature, et incite a rechercher leur fonctionnalite, et leur impact potentiel dans l’obesite.

Objectif L’objectif principal de cette etude est d’identifier des marqueurs epigenetiques associes au risque de diabete de type 2 (DT2) par leurs effets sur l’action de l’insuline Patients et methodes Nous avons realise une etude pilote en comparant les profils de methylation de l’ADN sur le genome entier dans le tissu hepatique de 24 normoglycemiques et 24 patients DT2 apparies en utilisant des puces « Infinium HumanMethylation 450 Beadchip » de chez Illumina ® . Resultats De maniere globale, l’ADN du foie des individus DT2 a une tendance a etre demethyle (− 0,4 % ; p = 0,09) par rapport aux echantillons des individus normoglycemiques. Cependant, plus specifiquement, nous avons identifie une hypermethylation significative chez les patients DT2 (+ 2 % ; p = 2 × 10–7) au niveau d’un site sur le chromosome 10q11. Conclusion Nos donnees suggerent qu’il pourrait exister des marqueurs epigenetiques du DT2 dans le foie. Cependant, ces resultats doivent etre confirmes sur un plus grand echantillon et des analyses fonctionnelles restent necessaires afin d’etablir d’eventuelles associations avec l’expression des genes ou des traits metaboliques intermediaires.

Introduction En reponse a l’inflammation chronique induite lors de l’obesite, nous avons montre que le metabolisme du tryptophane est active chez les patients obeses. En particulier, l’indoleamine 2, 3 dioxygenase (IDO1) est surexprimee au niveau du tissu adipeux blanc de ces patients. Certains metabolites issus de cette voie ont des proprietes toxiques (comme l’acide quinolinique) voire diabetogenes. Notre objectif est de caracteriser cette voie dans les adipocytes humains en culture en evaluant l’expression des enzymes impliquees, la production de metabolites potentiellement toxiques et les facteurs susceptibles de les reguler. Materiels et methodes Des preadipocytes humains (Lonza) ont ete differencies en culture et stimules avec des facteurs proinflammatoires (IFN-y) ou par de l’acide quinolinique. L’expression des enzymes de la voie de degradation ainsi que des marqueurs de differenciation et d’inflammation a ete analysee par RT-PCR (Taqman). Resultats L’IFN-y a augmente tres fortement l’expression d’IDO1 dans les adipocytes humains differencies et indifferencies et a permis l’activation de plusieurs enzymes de la voie (IDO2, KYNU, CCBL2, HAAO) susceptibles de generer des metabolites toxiques. De plus, l’addition d’acide quinolinique (1 000nM) potentialise l’expression de la leptine et de CCL2 dans les adipocytes differencies. Conclusion Nos resultats suggerent que l’acide quinolinique produit par l’activation d’IDO1 lors de l’obesite est susceptible d’interagir avec les adipocytes et d’induire une surproduction de composes inflammatoires et de leptine. Une analyse des echantillons de tissu adipeux de patients obeses permettra d’y confirmer la presence de ce produit.

Introduction Les etudes de GWAS sur l’heritabilite du DT2 ont montre le role important du gene TCF7L2. Nous avons montre que plusieurs transcrits alternatifs de TCF7L2 avaient des effets distincts sur la proliferation et la secretion d’insuline des cellules-beta pancreatiques. TCF7L2 est aussi implique dans le metabolisme du glucose des cellules hepatiques (Norton 2011, Savic 2011), d’ou l’interet d’analyser l’expression de TCF7L2 transcrits dans le foie. Patients et methodes Nous avons mesure l’ARNm de TCF7L2 par PCR-quantitative exon-specifique (d’apres Prokunina-Olsson 2009) dans des echantillons de tissu hepatique de patients obeses de la cohorte ABOS (33 normoglycemiques et 31 DT2). Dans les cellules HepG2, nous avons etudie l’expression des genes de la gluconeogenese apres diminution d’expression de TCF7L2 par siRNA. Nous avons egalement analyse les transcrits de TCF7L2 dans ces cellules cultivees avec differentes concentrations de glucose et d’insuline. Resultats Notre analyse montre que les transcrits de TCF7L2 contenant le premier site d’initiation de la transcription (TSS1) sont plus exprimes dans le foie des patients diabetiques que chez les sujets normoglycemiques (augmentation 1,3-fois du rapport TSS1/exon 8, P = 0,002). Grâce a la transfection d’ARNi dans les cellules HepG2, nous avons confirme que la transcription de plusieurs genes de la gluconeogenese, dont G6Pase, est reprimee par TCF7L2. Dans les cellules HepG2 en presence de 1 mM glucose, l’expression de toutes les transcrits de TCF7L2 contenant l’exon 8, dont les TSS1-transcrits, est augmentee d’environ 2 fois avec l’insuline (P = 0,03). D’autre part, le rapport TSS1/exon 8 est toujours augmente par l’insuline dans les cellules HepG2 cultivees avec 1 mM, 5 mM ou 11 mM glucose. Conclusion Nous avons demontre que differents transcrits de TCF7L2 sont induits par l’insuline et par un environnement diabetogene dans les hepatocytes. Ceci suggere que ces transcrits alternatifs pourraient jouer un role important dans les anomalies du metabolisme hepatique du glucose dans le DT2.

Introduction GPR120 est un recepteur couple aux proteines G qui fixe les acides gras libres insatures a longue chaine (particulierement les omega-3). GPR120 jouerait un role important dans l’equilibre de divers mecanismes physiologiques comme l’adipogenese, la regulation de l’appetit ou encore l’appetence alimentaire. Le but de cette etude est d’analyser le role de GPR120 dans le metabolisme energetique de la Souris et de l’Homme. Materiels et methodes Nous avons genere une Souris deficiente pour Gpr120 (KO) et avons etudie son metabolisme (poids-taille, depenses energetiques, masse grasse, glycemie-insulinemie, voies de signalisation impliquees dans l’insulino-resistance, la lipogenese et l’adipogenese), sous differentes conditions (regime standard ou hyperlipidique, tests de tolerance au glucose et a l’insuline). Chez l’Homme, nous avons compare l’expression de GPR120 dans le tissu adipeux omental et sous-cutane, de 14 sujets obeses versus 14 temoins. GPR120 a ete sequence chez 312 individus presentant une obesite severe. Les mutations non-synonymes identifiees ont ete genotypees chez 6 942 individus obeses et 7 654 temoins, et leurs effets sur la fonction de GPR120 ont ete etudies dans des lignees cellulaires. Resultats Sous regime hyperlipidique, la Souris KO developpe une obesite, une intolerance au glucose et une steatose hepatique, avec une diminution de la differenciation et de la lipogenese des adipocytes, et une augmentation de la lipogenese hepatique. L’insulino-resistance de la Souris KO est liee a une diminution du signal insulinique et a une augmentation de l’inflammation du tissu adipeux. Chez l’Homme, nous avons montre que l’expression de GPR120 dans le tissu adipeux etait plus elevee chez les individus obeses (P = 4.0×10 −4 ). Le sequencage de GPR120 a revele une mutation non-synonyme (p. R270H) qui inhibe les voies de signalisation de GPR120 et augmente fortement le risque d’obesite (OR = 1.62; P = 8.0×10 −6 ). Conclusion Notre etude demontre que le senseur lipidique GPR120 joue un role fondamental dans le controle de la balance energetique chez l’Homme et la Souris.