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ObjectiveThe pancreatic islets of Langerhans contain distinct cell subtypes including insulin-producing β cells. Although their cell-specific gene expression pattern defines their identity, the underlying molecular network driving this transcriptional specificity is not fully understood. Among the numerous transcriptional regulators, histone deacetylases (HDAC) enzymes are potent chromatin modifiers which directly regulate gene expression through deacetylation of lysine residues within specific histone proteins. The precise molecular mechanisms underlying HDAC effects on cellular plasticity and β-cell identity are currently unknown.MethodsThe pharmacological inhibition of HDAC activity by trichostatin A (TSA) was studied in the mouse Min6 and human EndocBH1 cell lines, as well as primary mouse sorted β cells and human pancreatic islets. The molecular and functional effects of treating these complementary β-cell models with TSA was explored at the epigenomic and transcriptomic level through next-generation sequencing of chromatin immunoprecipitation (ChIP) assays (ChIP-seq) and RNA sequencing (RNA-seq) experiments, respectively.ResultsWe showed that TSA alters insulin secretion associated with β-cell specific transcriptome programming in both mouse and human β-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative β-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers.ConclusionsTaken together, our data indicate that full HDAC activity is required to safeguard the epigenome, to protect against loss of β-cell identity with unsuitable expression of genes associated with alternative cell fates.

Hepatocyte Nuclear Factor-1A (HNF1A) is a master regulator of key glucose-responsive genes in metabolic organs such as the intestine, liver, kidney, and pancreas. Heterozygous mutations in HNF1A cause Maturity-Onset-Diabetes-of-the-Young (HNF1A-MODY) . Mutant carriers are normoglycemic in childhood, but with age, they develop hyperglycemia. Although insulin secretory defects have been extensively studied, it is unclear whether HNF1A deficiency contributes to a progressive decline in alpha cell function. HNF1A-MODY patients display glycosuria and endogenous glucose production linked to reduced renal SGLT2 expression. Since SGLT2 is also expressed in alpha cells and is required for glucagon regulation, we hypothesized that HNF1A coordinates renal and alpha cell SGLT2 activity to maintain glucose homeostasis. Here, we studied Hnf1a regulation of renal Sglt2, Sglt1, and Glut2 in Hnf1a-/- mutant mice, glucagon expression and secretion in siHNF1A transfected human islets and we developed a novel Hnf1a mutant mouse model (Hnf1a+/Δe4-10) . Sglt2 protein was significantly reduced in the kidney of Hnf1a-/- mice, with no changes in Sglt1 or Glut2 protein expression. We observed that HNF1A is heterogeneously expressed in alpha, beta, and delta cells. siHNF1A islets displayed reduced insulin secretion and content, which was rescued by GLP1 treatment. HNF1A deficiency reduced SGLT2 levels, correlating with increased glucagon content and secretion. The Hnf1a+/Δe4-mice were born normoglycemic but became glucose intolerant at 12 weeks of age, mimicking the clinical characteristics of HNF1A-MODY patients. Collectively, these findings indicate that HNF1A deficiency downregulates renal and alpha cell SGLT2 expression, which is associated with an alteration in glucagon secretion in response to glucose stimulation. The new Hnf1a+/Δe4-mouse model may be a useful tool to study the progression of MODY with and without chronic drug treatment. Disclosure A.Acosta-montalvo: None. V.Gmyr: None. M.Cnop: None. J.A.Kerr-conte: None. F.Pattou: None. M.Pontoglio: None. A.Liston: None. C.Bonner: None. C.Saponaro: None. J.Thevenet: None. M.Chiral: None. A.Piron: None. N.Delalleau: None. G.Pasquetti: None. A.Coddeville: None. M.Moreno: None. Funding EFSD/Lilly (2017) awarded to Caroline Bonner, I-SITE ULNE awarded to Caroline Bonner, the European Genomic Institute for Diabetes (ANR-10-LABX-46) , the European Consortium for Islet Transplantation funded by the Juvenile Diabetes Research Foundation. TIGER was developed within the European Union’s Horizon 2020 research and innovation program project T2DSystems, under grant agreement 667191 (to Miriam Cnop) .

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of E2f1 (E2f1

Compromised β-cell function contributes to type 2 diabetes (T2D) development. The glucagon like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential toward T2D treatment, notably by improving β-cell functions. Recent data have shown that the transcription factor E2f1, besides its role as a cell cycle regulator, is involved in glucose homeostasis by modulating β-cell mass, function and identity. Here, we demonstrate a crosstalk between the E2F1, phosphorylation of retinoblastoma protein (pRb) and Glp-1 signaling pathways. We found that β-cell specificE2f1deficient mice (E2f1β−/−) presented with impaired glucose homeostasis and decreased glucose stimulated-insulin secretion mediated by exendin 4 (i.e., GLP1R agonist), which were associated with decreased expression ofGlp1rencoding Glp-1 receptor (GLP1R) inE2f1β−/−pancreatic islets. Decreasing E2F1 transcriptional activity with an E2F inhibitor in islets from nondiabetic humans decreasedGLP1Rlevels and blunted the incretin effect of exendin 4 on insulin secretion. Conversely, overexpressingE2f1in pancreatic β cells increasedGlp1rexpression associated with enhanced insulin secretion mediated by GLP1R agonist. Interestingly, kinome analysis of mouse islets demonstrated that an acute treatment with exendin 4 increased pRb phosphorylation and subsequent E2f1 transcriptional activity. This study suggests a molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways that modulates insulin secretion and glucose homeostasis.

International audience; Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10 days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimu- lates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we fjnd that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

A translational discrepancy exists between animal models used for diabetes research and diabetes in man, thus human relevant models must become a priority. We showed that collectively human islet grafts from 26 donors statistically significantly adapted their function and endocrine and β-cell mass to HFD (high fat diet) in vivo in 233 RAG2KO mice. Human islet compensation occurs at 6 weeks and metabolic dysfunction upon chronic ≥10 weeks HFD. Herein, gene expression studies (GeneChip® Human Gene 2.0 ST Arrays) identified molecular pathways involved. Indeed, human islets compensate to HFD by upregulating hormones, transcription factors, antioxidants, and unfolded protein response genes, and then become dysfunctional at 10 wks -by downregulating these genes. Herein we show Krebs cycle and Mitochondrial electron transport chain was targeted with a transient increase at 6wk and then a drop-in expression at 10 wks; whereas uncouplinggene expression UCP2 and 3 increased. Glycolysis remained upregulated. Explanations for expanded islet and beta mass in HFD (vs. CTL) may be found in cell cycle heatmap. Lastly, a marker of activated stellate cells TAGLN, and matrix genes rose during islet dysfunction (fibrosis?). Conclusion: This human-based model system progressively depicts defects identified in human pre and T2DM that could serve as a “phase 0” in vivo model to test new targets and develop effective therapies in man. Disclosure J.A. Kerr-Conte: None. J. Thevenet: None. G. Pasquetti: None. P. Petit: None. C. Clabaut: None. V. Gmyr: None. C. Bonner: None. S. Gargani: None. F. Pattou: None. Funding JDRF; Programme d’investissements d’avenir; LabEx; European Genomic Institute for Diabetes (ANR-10-LABX-46)

Mutations in HNF1A cause Maturity Onset Diabetes of the Young (HNF1A-MODY). Carriers are normoglycemic in childhood, but with age, they develop hyperglycemia associated with insulin secretory defects and beta cell dysfunction. Although endogenous glucose production has been reported, glucagon in HNF1A-MODY has been less explored. Patients respond well to low dose sulfonylureas such as gliclazide, which increases insulin secretion independent of glucose but may predispose to hypoglycemia. We aimed to investigate whether low dose gliclazide had an effect on glucagon secretion in HNF1A-MODY. Additionally, we explored the potential of GLP-1 to normalize hormone secretion using in vivo and in vitro models of HNF1A deficiency. A 75g oral glucose tolerance test was performed in 7 MODY patients before and 72h after stopping gliclazide. Glucose rise was greater off gliclazide (p=0.003), while c-peptide and glucagon levels were similar between the groups. When adjusting for glucose levels during the OGTT, the change from baseline glucagon levels (Δ glucagon/glucose ratio) showed a significant decrease in those taking gliclazide (p=0.048), while there was no difference in Δ c-peptide/glucose ratio. Hnf1a-/- mice compared to WT mice are hyperglycemic, have decreased plasma insulin levels and hyperglucagonemia that was not suppressed after a glucose challenge (p=0.008). Furthermore, induction of the HNF1A variant Pro291fsInsC in rat INS1 cells reduced insulin and increased glucagon secretion, both of which were improved by GLP-1 treatment. Using the TIGER database we found HNF1A to be heterogeneously expressed in human islets, and siHNF1A reduced insulin secretion at high glucose. Collectively, these findings indicate that HNF1A is also essential for the regulation of glucagon secretion and glucose homeostasis. The mechanism by which GLP-1 normalizes insulin and glucagon secretion needs further investigation but could contribute to a better understanding of treatment response in HNF1A-MODY. Disclosure C. Saponaro: None. A. Acosta-Montalvo: None. L. Anguelova: None. J. Thevenet: None. M. Chiral: None. G. Pasquetti: None. A. Piron: None. M. Cnop: None. V. Gmyr: None. J. Prehn: None. J.A. Kerr-Conte: None. F. Pattou: None. M. Pontoglio: None. K.R. Owen: None. I. Spiliotis: None. C. Bonner: None. Funding European Foundation for the Study of Diabetes/Lilly European Diabetes Research Programme; Oxford Health Sciences Research Committee (1233)

Objective. This retrospective study in 87 clinical islet transplants sought to determine if the quantitative in vivo islet potency assay (QIVIPA) based on human c-peptide measurement in normoglycemic immunodeficient mice transplanted with 1% of clinical islet transplant (n = 165) could predict islet function in man after transplantation (tx). Since each recipient receives a mean of 2.7 grafts in our center, the ultimate goal of this work was to prospectively determine the functional islet mass required in 1–3 transplants in the QIVIPA mice to achieve optimal long-term islet function (beta score ≥ 7) in man. Methods. Islet preparations that met in vitro release criteria (200,000 IEQ, viability ≥ 80%, sterility) were transplanted in severe type 1 diabetic patients (n = 87). 2% of the preparation was used for in vitro quality controls, and 2% for in vivo controls: tx under the kidney capsule of two normoglycemic nude or RAG2 mice where fasting c-peptide and glycemia were measured over 30 days. Human islet function was defined by immediate graft function (hCP increase 7 days after tx), primary graft function (PGF = beta score 1 month after the last tx) and beta score at 1 year. Results. The QIVIPA model is robust and reproducible with two mice transplanted per human islet preparation (P Conclusion. Evidence from this retrospective study on 87 clinical islet transplants in 165 mice suggests that in vivo quantitative islet potency assays like QIVIPA in normoglycemic immunodeficient are a key indicator of the quality of manufactured clinical graft preparations as they predict islet function in man, both early function at 7 days post tx, PGF (beta score 1 month after the last tx), and 1-year beta score even when multiple grafts are transplanted per recipient.

Metformin (MET) is the most prescribed antidiabetic drug but its mechanism of action remains elusive. Recent clinical data point to the gut as MET primary target. In the present study we explored the effect of MET in the gut transport machinery. Using “in vitro” human enterocytes (Caco-2/TC7 cells) we showed that MET transiently reduced the apical density of sodium-glucose transporter 1 (SGLT1) and decreased the absorption of α-Methyl-D-1-glucopyranoside in an AMPK-independent manner, without changes in the post-translational signals. Administered 1hour before a glucose challenge in different animal models, MET reduced the postprandial glucose response (PGR). MET also reduced the urinary appearance of 2-(18F)-fluoro-2-deoxy-D-glucose after the intraluminal administration of the radiotracer. MET effect reducing PGR was maintained in glucose transporter type 2 (GLUT2)-KO mice but abrogated in (SGLT1)-KO animals. In conclusion, we unveiled the reversible effect of MET on SGLT1 trafficking, resulting in a transient decrease of the intestinal glucose absorption.

Introduction La capacite des ilots murins a se regenerer decline avec l'âge. L'âge est un facteur de risque quant au developpement du diabete. Chez l'homme, la replication des ilots pancreatiques decroit avec l'âge sur des coupes pancreatiques post mortem et en culture, autant que la deterioration progressive de la secretion d'insuline en reponse au glucose, et l'augmentation de l'apoptose. Du fait, le vieillissement limiterait l'avenir prometteur de la therapie regenerative des cellules Beta chez l'homme, ainsi que leur survie a long terme apres allo-greffe. Notre laboratoire a developpe precedemment (Diabetologie, 2013), un modele chez la souris immunodeficiente, montrant que les ilots humains issus de donneurs jeunes (16 – 41 ans) etaient capables d'adaptation fonctionnelles (cpeptide humain) et en masse d'ilots a une obesite induite in vivo par regime Hyperlipidique (HFD). L'objectif de cette etude est de determiner si les ilots issus de donneurs âges (57-69 ans) ont la capacite de s'adapter a un regime HFD, et si l'IMC du donneur a une influence. Materiels et Methodes Des souris Rag2-/- ( n =49) ont ete transplantees avec 500 Ilots issus de pancreas de donneurs ages ( n =2) ou ages obeses ( n =5). Les animaux ont ete soumis a un regime Controle ou Hyperlipidique (HFD) et suivis (Poids, glycemie, C-peptide humain) pendant sur 12 semaines. Apres sacrifice, les greffons humains ont ete analyses par morphometrie. Resultats Les ilots humains s'adaptent leur fonction (cpeptide humain) et leur masse d'ilots a l'obesite ( p p 40). Conclusions Les ilots issus de donneurs ages d'IMC normal ou obeses ont la capacite de s'adapter a un environnement obesogene, que ce soit en fonction et en masse endocrine, contrairement a ceux issus de donneurs morbides. Ce travail montre l'importance de la selection des donneurs pour l'allotransplantation d'ilots, ou la therapie regenerative du diabete du type 2.

Introduction Des mutations du gene KCNJ11 sont responsables de differentes formes de diabete precoce. La mutation KCNJ11-E227K est associee a la fois au MODY et au diabete neonatal dans differentes familles, suggerant que le fonds genetique familial contribuerait a moduler le phenotype. Afin de mieux caracteriser les mecanismes induits par cette mutation, nous utilisons les nouvelles techniques de modification ciblee du genome. Materiels et methodes Nous avons derive les cellules-souches pluripotentes induites (iPSC) a partir des cellules mononucleees du sang d’individus porteurs des mutations des genes impliques dans le diabete monogenique. Grâce a la technique des TALE Nucleases et/ou Crispr/Cas9, nous introduisons des cassures double-brin dans l’ADN et la reparation faite par recombinaison homologue afin de realiser la modification nucleotidique specifique de la mutation KCNJ11-E227K. Nous etudierons les iPSC en presence ou non de la mutation E227K pendant leur differenciation en cellules beta-pancreatiques, et l’impact fonctionnel sur leur capacite a secreter l’insuline. Resultats Nous presentons notre strategie de modification ciblee dans une lignee cellulaire qui exprime le gene KCNJ11. Nous avons analyse trois ARNguide de Cas9, et grâce au clonage TOPO-TA, nous avons mis en evidence plusieurs evenements de la reparation de l’ADN par « jonction d’extre-mites non homologues » accompagne de la perte, du gain ou de l’inversion de nucleotides. Ce type de modification n’a pas ete observe aux loci non-specifiques. Pour stimuler la « recombinaison homologue », nous realisons des clonages de donneurs d’ADN avec des fragments d’ADN synthetise. Cette strategie pourra etre facilement adaptee a d’autres regions de genes cibles. Nous utilisons deux etapes de la recombinaison homologue consecutives pour realiser une correction nucleotidique specifique sans alterer l’integrite de l’ADN genomique environnant. Conclusion Les techniques de modification ciblee du genome sont performantes et permettent l’analyse d’une mutation specifique (KCNJ11-E227K) sur un fonds genetique familial lie a la severite du phenotype. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

Studies implicating sodium-glucose cotransporter 2 (SGLT2) inhibitors in glucagon secretion by pancreatic α-cells reported controversial results. We hypothesized that interindividual heterogeneity in SGLT2 expression and regulation may affect glucagon secretion by human α-cells in response to SGLT2 inhibitors. An unbiased RNA-sequencing analysis of 207 donors revealed an unprecedented level of heterogeneity of SLC5A2 expression. To determine heterogeneity of SGLT2 expression at the protein level, the anti-SGLT2 antibody was first rigorously evaluated for specificity, followed by Western blot and immunofluorescence analysis on islets from 10 and 12 donors, respectively. The results revealed a high interdonor variability of SGLT2 protein expression. Quantitative analysis of 665 human islets showed a significant SGLT2 protein colocalization with glucagon but not with insulin or somatostatin. Moreover, glucagon secretion by islets from 31 donors at low glucose (1 mmol/L) was also heterogeneous and correlated with dapagliflozin-induced glucagon secretion at 6 mmol/L glucose. Intriguingly, islets from three donors did not secrete glucagon in response to either 1 mmol/L glucose or dapagliflozin, indicating a functional impairment of the islets of these donors to glucose sensing and SGLT2 inhibition. Collectively, these data suggest that heterogeneous expression of SGLT2 protein and variability in glucagon secretory responses contribute to interindividual differences in response to SGLT2 inhibitors., info:eu-repo/semantics/published