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Using13C6glucose labeling coupled to GC-MS and 2D1H-13C HSQC NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning steady state insulin release and β cell function. In both mouse and human islets, the contribution of glucose to the TCA cycle is similar. Pyruvate-fueling of the TCA cycle is found to be mediated primarily by the activity of pyruvate dehydrogenase (PDH), with only a limited contribution from pyruvate carboxylase (PC). While conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in both species, lactate accumulation via this route is six-fold higher in human islets. Transcriptomic analysis reveals that human β cells specifically express lactate dehydrogenase B (LDHB) at high levels, in keeping with the phenotype of patients harboring gain-of-function mutations in MCT1/ SLC16A1 (HHF7). Thus, glycolytically-derived acetyl CoA preferentially feeds the TCA cycle in both mouse and human β cells. However, human β cells possess the machinery needed to generate extra-mitochondrial lactate, which might reflect a key mechanism to balance the reducing activity of NADH-producing pathways.

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) .

BACKGROUND Antithrombin (AT) III physiological levels are decreased during septic shock and supplementation therapy could therefore be beneficial. OBJECTIVE We hypothesized that the use of recombinant human AT could reduce disseminated intravascular coagulation (DIC) occurrence. METHODS We conducted a randomized open label controlled experimental study. Ten female "Large White" pigs were challenged with i.v. infusion of Escherichia coli endotoxin. Two groups of 5 pigs were randomly assigned to receive either recombinant human AT 100 U/kg over 30 min (ATryn group) or 0.9% saline (control group). AT III levels, coagulation, hemostasis, inflammation parameters, hemodynamics, and microcirculatory parameters were measured over a 5-h period. Immediately after euthanasia, kidneys were withdrawn for histology evaluation. Statistical analysis was performed with nonparametric tests and Dunn's test for multiple comparisons. RESULTS AT III activity was significantly higher in the ATryn group than in the control group from 60% (213% [203-223] vs. 104% [98-115], P = 0.008, respectively) to 300 min (115% [95-124] vs. 79% [67-93], P = 0.03). Recombinant human AT supplementation had no impact on hemodynamics, microcirculatory parameters, and sequential changes of coagulation parameters (platelet count, fibrinogen level, thrombin-AT complexes, and von Willebrand factor). Interleukin 6 and tumor necrosis factor α values were statistically the same for both groups throughout the study. Percentage of thrombosed glomeruli and percentage of thrombosed capillary in glomerulus were not significantly different between both groups. CONCLUSIONS In our model of endotoxic shock, a single low dose of recombinant human AT did not prevent DIC occurrence, severity, inflammatory profile, or hemodynamic alterations.

International audience; Liraglutide, an anti-diabetic drug and agonist of the glucagon-like peptide one receptor (GLP1R), has recentlybeen approved to treat obesity in individuals with or without type 2 diabetes. Despite its extensive metabolicbenefits, the mechanism and site of action of liraglutide remain unclear. Here, we demonstrate that liraglutideis shuttled to target cells in the mouse hypothalamus by specialized ependymoglial cells called tanycytes,bypassing the blood-brain barrier. Selectively silencing GLP1R in tanycytes or inhibiting tanycytic transcytosisby botulinum neurotoxin expression not only hampers liraglutide transport into the brain and its activationof target hypothalamic neurons, but also blocks its anti-obesity effects on food intake, body weight and fatmass, and fatty acid oxidation. Collectively, these striking data indicate that the liraglutide-induced activationof hypothalamic neurons and its downstream metabolic effects are mediated by its tanycytic transport intothe mediobasal hypothalamus, strengthening the notion of tanycytes as key regulators of metabolic homeostasis.

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.

With the increasing prevalence of type 2 diabetes (T2D), therapies aimed at delineating diabetes pathophysiology and understanding their mechanisms of action are of critical importance. As such, growing interest in the clinical pharmacology of sodium-glucose cotransporter 2 (SGLT2) and its inhibition by gliflozins in the treatment of T2D is becoming increasingly evident. SGLT2 inhibition results in urinary glucose excretion, thereby reducing blood glucose levels. The importance of this homoeostasis mechanism is evident from several clinical trials demonstrating that patients taking this class of compounds have reductions in glycaemia, body weight and blood pressure compared with other antidiabetic agents. Yet, while such outcomes are very encouraging, some studies have reported elevated plasma glucagon levels and endogenous glucose production (EGP), two traits that are already prevalent in T2D. However, these findings were later explained by the specific expression of SGLT2 by pancreatic alpha cells, where glucagon secretion is directly regulated. Although conflicting data are now emerging on SGLT2 regulation of glucagon secretion, as SGLT2 is not expressed in the intestines, circulating glucagon concentrations are most likely of pancreatic origin. Thus, the present review considers the mechanism of action of SGLT2 inhibitors in the regulation of glucagon secretion, and the discrepancies in data from mice compared with people. The pragmatic use of human islets to accurately decipher SGLT2 inhibition in the regulation of glucagon secretion is also discussed.

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.

Total pancreatectomy with intraportal islet autotransplantation (TPIAT) rather than partial pancreatectomy could represent a major shift in the management of patients with resectable pancreatic ductal adenocarcinoma (PDAC) when risks of postoperative pancreatic fistula are well identified. This approach provides a theoretical risk of tumor cell dissemination when islet cells are transplanted into the portal vein. Our objective was to explore the safety of TPIAT in PDAC in a mouse preclinical model of subcutaneous xenotransplantation of human cells isolated from pancreatic specimen during partial pancreatectomy performed for PDAC.Patients requiring pancreatectomy for PDAC were prospectively included. Immunocompromised mice were transplanted with pancreatic cells isolated from the nonmalignant part of the surgical specimen (experimental group). Results were compared with pancreatic tumor implants (control group). Pancreatic grafts were explanted at 6 weeks for histological analyses.Nine patients were included, and 31 mice were transplanted. In the experimental group, explants were microscopically devoid of tumor cell, and no metastasis was observed. In the control group, all explants were composed of tumor.We report in a preclinical model the absence of local and distant spreading of malignant cells after pancreatic islets xenograft isolated from PDAC patients. These data supports the oncological safety of TPIAT as valuable alternative to partial pancreatectomy for PDAC patients with a high risk of postoperative pancreatic fistula.

Objective Aberrant hepatic glucose production contributes to the development of hyperglycemia and is a hallmark of type 2 diabetes. In a recent study, we showed that the transcription factor E2F1, a component of the cell cycle machinery, contributes to hepatic steatosis through the transcriptional regulation of key lipogenic enzymes. Here, we investigate if E2F1 contributes to hyperglycemia by regulating hepatic gluconeogenesis. Methods We use different genetic models to investigate if E2F1 regulates gluconeogenesis in primary hepatocytes and in vivo. We study the impact of depleting E2F1 or inhibiting E2F1 activity in diabetic mouse models to evaluate if this transcription factor contributes to hyperglycemia during insulin resistance. We analyze E2F1 mRNA levels in the livers of human diabetic patients to assess the relevance of E2F1 in human pathophysiology. Results Lack of E2F1 impaired gluconeogenesis in primary hepatocytes. Conversely, E2F1 overexpression increased glucose production in hepatocytes and in mice. Several genetic models showed that the canonical CDK4-RB1-E2F1 pathway is directly involved in this regulation. E2F1 mRNA levels were increased in the livers from human diabetic patients and correlated with the expression of the gluconeogenic enzyme Pck1. Genetic invalidation or pharmacological inhibition of E2F1 improved glucose homeostasis in diabetic mouse models. Conclusions Our study unveils that the transcription factor E2F1 contributes to mammalian glucose homeostasis by directly controlling hepatic gluconeogenesis. Together with our previous finding that E2F1 promotes hepatic steatosis, the data presented here show that E2F1 contributes to both hyperlipidemia and hyperglycemia in diabetes, suggesting that specifically targeting E2F1 in the liver could be an interesting strategy for therapies against type 2 diabetes., Highlights • Genetic manipulation of the CDK4-RB1-E2F1 pathway in hepatocytes results in changes in gluconeogenic gene expression and glucose production. • Overexpression of E2F1 promotes gluconeogenesis in primary hepatocytes and in liver. • E2F1 mRNA levels are increased in the livers of diabetic patients and correlate with PCK1 gene expression. • Genetic depletion and pharmacological inhibition of E2F1 reduces hyperglycemia in diabetic mice.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia resulting from a deficiency in insulin signaling, because of insulin resistance and/or defects in insulin secretion; it is also associated with increases in glucagon and endogenous glucose production (EGP). Gliflozins, including dapagliflozin, are a new class of approved oral antidiabetic agents that specifically inhibit sodium-glucose co-transporter 2 (SGLT2) function in the kidney, thus preventing renal glucose reabsorption and increasing glycosuria in diabetic individuals while reducing hyperglycemia. However, gliflozin treatment in subjects with T2D increases both plasma glucagon and EGP by unknown mechanisms. In spite of the rise in EGP, T2D patients treated with gliflozin have lower blood glucose levels than those receiving placebo, possibly because of increased glycosuria; however, the resulting increase in plasma glucagon levels represents a possible concerning side effect, especially in a patient population already affected by hyperglucagonemia. Here we demonstrate that SGLT2 is expressed in glucagon-secreting alpha cells of the pancreatic islets. We further found that expression of SLC5A2 (which encodes SGLT2) was lower and glucagon (GCG) gene expression was higher in islets from T2D individuals and in normal islets exposed to chronic hyperglycemia than in islets from non-diabetics. Moreover, hepatocyte nuclear factor 4-α (HNF4A) is specifically expressed in human alpha cells, in which it controls SLC5A2 expression, and its expression is downregulated by hyperglycemia. In addition, inhibition of either SLC5A2 via siRNA-induced gene silencing or SGLT2 via dapagliflozin treatment in human islets triggered glucagon secretion through KATP channel activation. Finally, we found that dapagliflozin treatment further promotes glucagon secretion and hepatic gluconeogenesis in healthy mice, thereby limiting the decrease of plasma glucose induced by fasting. Collectively, these results identify a heretofore unknown role of SGLT2 and designate dapagliflozin an alpha cell secretagogue.

Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method ( p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number ( r2 = 0.91) and total islet number ( r2 = 0.88) and thus increased to r2 = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method ( p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method ( p < 0.001). The ADIA method also detected small islets between 10 and 50 μm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

In allogenic islet transplantation (IT), high purity of islet preparations and low contamination by non-islet cells are generally favored. The aim of the present study was to analyze the relation between the purity of transplanted preparations, and graft function during 5 years post-IT. Twenty-four type 1 diabetic patients, followed-up 5 years after IT, were enrolled. Metabolic parameters and daily insulin requirements were compared between patients who received islet preparations with a mean purity < 50% (LOW purity) or ≥50% (HIGH purity). We also analyzed blood levels of carbohydrate 19-9 (CA 19-9) - a biomarker of pancreatic ductal cells - and glucagon, before and after IT. At 5 years, mean HbA1c levels (p=0.01) and daily insulin requirements (p=0.03) were lower in the LOW purity group. Insulin independence was more frequent in the LOW purity group (p

Human islet transplantation is a viable treatment option for type 1 diabetes mellitus (T1DM). However, pancreatic islet inflammation after transplantation induced by innate immune responses is likely to hinder graft function. This is mediated by incompatibility between islets and the blood interface, known as instant blood-mediated inflammatory reaction (IBMIR). Herein we hypothesized that portal venous administration of islet cells with human recombinant antithrombin (ATryn®), a serine protease inhibitor (serpin), which plays a central role in the physiological regulation of coagulation and exerts indirect anti-inflammatory activities, may offset coagulation abnormalities such as disseminated intravascular coagulation (DIC) and IBMIR. The current prospective, randomized experiment was conducted using an established preclinical pig model. Three groups were constituted for digested pancreatic tissue transplantation (0.15 ml/kg): control, NaCl 0.9% ( n = 7); gold standard, heparin (25 UI/kg) ( n = 7); and human recombinant ATryn® (500 UI/kg) ( n = 7). Blood samples were collected over time (T0 to 24 h), and biochemical, coagulation, and inflammatory parameters were evaluated. In both the control and heparin groups, one animal died after a portal thrombosis, while no deaths occurred in the ATryn®-treated group. As expected, islet transplantation was associated with an increase in plasma IL-6 or TNF-α levels in all three groups. However, DIC was only observed in the control group, an effect that was suppressed after ATryn® administration. ATryn® administration increased antithrombin activity by 800%, which remained at 200% for the remaining period of the study, without any hemorrhagic complications. These studies suggest that coadministration of ATryn® and pancreatic islets via intraportal transplantation may be a valuable therapeutic approach for DIC without risk for islets and subjects.

Plaisance, Valérie et al., Elevated plasma concentration of the pro-Atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-Treatment with the N-Acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment., This work was supported by the “European Genomic Institute for Diabetes” (E.G.I.D., ANR-10-LABX-46), European Commission, the Regional Council Nord Pas de Calais and the European Regional Development Fund.

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease.

HNF1A-maturity onset diabetes of the young (HNF1A-MODY) is caused by mutations in Hnf1a gene encoding the transcription factor hepatocyte nuclear factor 1alpha (HNF1A). An increased rate of apoptosis has been associated with the decrease in beta-cell mass that is a hallmark of HNF1A-MODY and other forms of diabetes. In a cellular model of HNF1A-MODY, we have recently shown that signalling through mammalian target of rapamycin (mTOR) is decreased by the overexpression of a dominant-negative mutant of HNF1A (DN-HNF1A). mTOR is a protein kinase which has important roles in cell metabolism and growth, but also in cell survival, where it has been shown to be both protective and detrimental. Here, we show that pharmacological inhibition of mTOR activity with rapamycin protected INS-1 cells against DN-HNF1A-induced apoptosis. Rapamycin also prevented DN-HNF1A-induced activation of AMP-activated protein kinase (AMPK), an intracellular energy sensor which we have previously shown to mediate DN-HNF1A-induced apoptosis. Conversely, activation of mTOR with leucine potentiated DN-HNF1A-induced apoptosis. Gene silencing of raptor (regulatory associated protein of mTOR), a subunit of mTOR complex 1 (mTORC1), also conferred protection on INS-1 cells against DN-HNF1A-induced apoptosis, confirming that mTORC1 mediates the protective effect. The potential relevance of this effect with regards to the clinical use of rapamycin as an immunosuppressant in diabetics post-transplantation is discussed.

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A–maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation.

Proteasomal stress is believed to contribute to the pathology of ischemic brain injury and several neurodegenerative disorders, but can activate both cytoprotective and cell death-inducing pathways. Here we have utilized the complex environment of organotypic hippocampal slice cultures (OHSCs) to investigate the stress responses activated in different neuronal populations following proteasome inhibition. Incubation of OHSCs with the specific proteasome inhibitors, epoxomicin or bortezomib led to a selective injury of the CA1 pyramidal neurons although similarly increased levels of poly-ubiquitinylated proteins were detected throughout all regions of the hippocampus. Micro-dissection, quantitative PCR and immunohistochemical analyses of epoxomicin-treated OHSCs identified a selective activation of cytoprotective genes in non-vulnerable regions, and a selective activation of p53 target genes within the CA1. Genetic deletion of the pro-apoptotic p53 target gene, p53-upregulated modulator of apoptosis (puma), significantly reduced injury within the CA1 following proteasomal inhibition. Activation of cytoprotective genes by treatment with inducers of heat shock protein 70 inhibited the selective activation of p53 signaling within the CA1 and protected CA1 neurons from epoxomicin-induced cell death. In summary, we demonstrate that the reciprocal activation of p53/p53-upregulated modulator of apoptosis and heat shock protein 70 signalling determines the selective vulnerability of neurons to proteasome inhibition.

Disturbances in cellular ion gradients by excitotoxicity promote apoptosis through activation of the Bcl-2 family member Bim., Excitotoxicity after glutamate receptor overactivation induces disturbances in cellular ion gradients, resulting in necrosis or apoptosis. Excitotoxic necrosis is triggered by rapid, irreversible ATP depletion, whereas the ability to recover cellular bioenergetics is suggested to be necessary for the activation of excitotoxic apoptosis. In this study, we demonstrate that even a transient decrease in cellular bioenergetics and an associated activation of adenosine monophosphate–activated protein kinase (AMPK) is necessary for the activation of excitotoxic apoptosis. We show that the Bcl-2 homology domain 3 (BH3)–only protein Bim, a proapoptotic Bcl-2 family member, is activated in multiple excitotoxicity paradigms, mediates excitotoxic apoptosis, and inhibits delayed Ca2+ deregulation, mitochondrial depolarization, and apoptosis-inducing factor translocation. We demonstrate that bim activation required the activation of AMPK and that prolonged AMPK activation is sufficient to induce bim gene expression and to trigger a bim-dependent cell death. Collectively, our data demonstrate that AMPK activation and the BH3-only protein Bim couple transient energy depletion to stress-induced neuronal apoptosis.

Mutations in the HNF1A (previously known as TCF1) gene encoding hepatocyte nuclear factor-1alpha (HNF1A) lead to the development of maturity-onset diabetes of the young, type 3 (HNF1A-MODY), characterised by impaired insulin secretion and a reduction in beta cell mass. HNF1A plays an important role in pancreatic beta cell differentiation and survival. The mammalian target of rapamycin (mTOR) is a central growth factor- and nutrient-activated protein kinase controlling cell metabolism, growth and survival. We investigated the role of mTOR inactivation in the decline in beta cell mass in a cellular model of HNF1A-MODY.Previously we showed that suppression of HNF1A function via expression of a dominant-negative mutant (DN-HNF1A) decreases insulin gene transcription in insulinoma (INS-1) cells. We investigated the signalling of two distinct mTOR protein complexes, mTORC1 and mTORC2, in response to DN-HNF1A induction.We observed delayed inactivation of mTORC2 48 h after DN-HNF1A induction, evidenced by a reduction in serine 473 phosphorylation of thymoma viral proto-oncogene 1 (AKT1). We also observed an early inactivation of mTORC1 24 h after DN-HNF1A induction, which was detected by decreases in threonine 389 phosphorylation of p70 ribosomal protein S6 kinase (S6K1) and serine 65 phosphorylation of translational inhibitor eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Flow cytometry and gene expression analysis demonstrated a pre-apoptotic decrease in INS-1 cell size in response to DN-HNF1A induction, and an increase in the level of the mTORC1-regulated cell-cycle inhibitor, cyclin-dependent kinase inhibitor 1B p27.Our data suggest that mTOR kinase and signalling through mTORC1 are highly sensitive to suppression of HNF1A function, and may contribute to disturbance of cell-size regulation and cell-cycle progression in HNF1A-MODY.

E2F transcription factors are known regulators of the cell cycle, proliferation, apoptosis, and differentiation. Here, we reveal that E2F1 plays an essential role in liver physiopathology through the regulation of glycolysis and lipogenesis. We demonstrate that E2F1 deficiency leads to a decrease in glycolysis and de novo synthesis of fatty acids in hepatocytes. We further demonstrate that E2F1 directly binds to the promoters of key lipogenic genes, including Fasn, but does not bind directly to genes encoding glycolysis pathway components, suggesting an indirect effect. In murine models, E2F1 expression and activity increased in response to feeding and upon insulin stimulation through canonical activation of the CDK4/pRB pathway. Moreover, E2F1 expression was increased in liver biopsies from obese, glucose-intolerant humans compared with biopsies from lean subjects. Finally, E2f1 deletion completely abrogated hepatic steatosis in different murine models of nonalcoholic fatty liver disease (NAFLD). In conclusion, our data demonstrate that E2F1 regulates lipid synthesis and glycolysis and thus contributes to the development of liver pathology.

The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic β cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and β cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent β cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic β cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive β cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment.

ObjectiveOvarian cancer prognosis remains dire after primary therapy. Recurrence rates are disappointingly high as 60% of women with advanced epithelial ovarian cancer considered in remission will develop recurrent disease within 5 years. Special attention to undetected peritoneal metastasis and residual tumorous cells during surgery is necessary as they are the main predictive factors of recurrences. Folate receptor α (FRα) shows promising prospects in targeting ovarian cancerous cells. Our aim was to determine if the Fischer model described by Rose et al could be used to evaluate folate-targeted therapies in preclinical studies.MethodsNuTu-19 epithelial ovarian cancer cell line was used to induce peritoneal carcinomatosis in female Fischer 344 rats. FRα expression by NuTu-19 cells was assessed in vitro by immunofluorescence using “Cytospin®” protocol. In vitro folate-targeted compound uptake by NuTu-19 cells was evaluated by incubation of FRα-positive ovarian cancer cell lines (NuTu-19/SKOV-3/OVCAR-3/IGROV-1) with or without (control) a folate-targeted photosensitizer. Intracellular incorporation was assessed by confocal microscopy. Determination of in vivo FRα tissue expression by several organs of the peritoneal cavity was studied by immunohistochemistry.ResultsNuTu-19 cells express FRα which allows intracellular incorporation of folate-targeted compound by endocytosis. FRα is expressed in tumor tissue, ovary, and liver. Peritoneum, colon, small intestine, and kidney do not express the receptor.ConclusionsFemale Fischer 344 rat is an inexpensive reproducible and efficient preclinical model to study ovarian peritoneal carcinomatosis folate-targeted therapies.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic β-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

Aims/hypothesis: Inactivating mutations in Tcf1, which encodes the transcription factor hepatocyte nuclear factor (HNF)-1alpha, cause maturity-onset diabetes of the young-3. We have previously shown that a dominant- negative mutant (DN-HNF-1alpha) renders INS-1 insuli- noma cells sensitive to the mitochondrial apoptosis pathway, but the underlying alterations in signal transduc- tion remain unknown. Materials and methods: Using a reverse tetracycline-dependent transactivator system, DN- HNF-1alpha-induced apoptosis was assessed by immuno- blotting and caspase assays. Alterations in AKT1 kinase/ protein kinase B (AKT1) survival signalling during DN- HNF-1alpha-induced apoptosis were investigated by phos- pho-specific immunodetection and transient transfection experiments. Results: Induction of DN-HNF-1alpha caused significant changes in the activation-specific phosphoryla- tion status of AKT1 that were preceded by a down- regulation of Ins1 gene transcription. Phosphorylation of AKT1 at Ser473 was dramatically reduced after 36 to 48 h of DN-HNF-1alpha induction and coincided with maximal apoptosis activation. Overexpression of a constitutively active mutant of Akt1 rescued INS-1 cells from DN-HNF- 1alpha-induced apoptosis, while ectopic expression of a dominant-negative mutant mimicked the effect of DN- HNF-1alpha on apoptosis activation. Pharmacological sup- pression of growth factor survival signalling through administration of the phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin accelerated the induction of apoptosis by DN-HNF-1alpha. Conclusions/interpretation: These data suggest that a decrease in PI-3K/AKT1 survival signalling mediates DN-HNF-1alpha-induced apoptosis in insulin- secreting cells. Keywords AKT1 . Apoptosis . HNF-1alpha . INS-1 . MODY3 . TCF1

Pancreatic islet dysfunction and death, which are key characteristics of type 1 diabetes mellitus (T1DM), are now befalling also in the pathogenesis of type 2 diabetes mellitus (T2DM). Prospective studies of high-risk patients or newly diagnosed with T2DM imply that disease progression correlates with a relatively modest deterioration of insulin resistance, followed by a steep decline in residual beta cell function [1, 2], while others argue that a decrease in beta cell mass is the key contributor to the progression of T2DM [3]. It is indeed plausible to consider, that both scenarios, loss of function and loss of mass, are at work in T2DM [4]. With respect to the latter, a decrease in beta cell mass may be caused by defects in beta cell differentiation and proliferation, a decrease in beta cell size, or an increased rate of beta cell apoptosis [3, 5]. It is important to emphasize, however, that a decrease in beta cell mass does not necessarily suggest that new beta cells are not generated during disease progression. In fact, Dor and colleagues argue that beta cell mass is not static but in a constant process of cell death and renewal [6]. Conversely, Talchai et al., argue that beta cells do not undergo apoptosis, but instead cheat cell death by dedifferentiating into alpha-like cells [7]. Although it is apparent that beta cell mass regeneration does occur, it is not clear whether this happens through self-renewal of mature beta cells or through neogenesis of progenitor cells residing in the pancreas or recruited from other near-by tissues such as the liver [8]. PSP/reg (regenerating protein) belongs to the Regenerating family of proteins [9]. However, regenerating gene 1 (Reg 1) and pancreatic stone protein (PSP) have been discovered independently in the fields of diabetes and pancreatitis [10]. Sequence analysis later revealed that Reg1 and PSP are indeed identical [11], and for clarification purposes Graf and colleagues suggested that the combined terms of PSP/reg to be used in the future [10]. PSP/reg has been shown to be physiologically secreted from rat pancreatic acinar cells [12], but it’s also expressed in human non-beta islet cells [13] and in the liver [14]. Its regenerative capabilities were first identified in an unbiased screening study of genes involved in beta cell regeneration [9]. Additionally, PSP/reg has been shown to increase beta cell mass and to stimulate beta cell proliferation under physiological conditions and in rodent models of diabetes [9, 10]. A more recent study has shown that apoptotic beta cells secrete factor(s) that can stimulate PSP/reg gene induction in neighboring islet cells, which in turn are able to facilitate islet/beta cell regeneration [15]. Of note, these observations were observed in rodents, which unlike humans, have a small percentage of alpha and delta cells compared to beta (80–90 % beta) [16]. Collectively, these findings led to the conclusions that PSP/reg may be a protein involved in neogenesis and regeneration [10, 11] and may act as a growth mediator stimulating the proliferation of beta cells [9, 10]. Evidently, activation of PSP/ reg gene is thought to be an important event in islet cell regeneration and diabetogenesis. However, the mechanisms involved remain largely unknown, and more importantly, whether the ‘‘regenerating’’ neighboring islets cells are of alpha, beta, or delta origin remains an enigma. In this issue of Endocrine, Yang and colleagues describe the prognostic value of PSP/reg serum concentration in patients at different clinical stages of T2DM as well as C. Bonner (&) European Genomic Institute for Diabetes (EGID), FR 3508, 59000 Lille Cedex, France e-mail: caroline.bonner@univ-lille2.fr

BACKGROUND Pancreatic islet transplantation offers a promising biotherapy for the treatment of type 1 diabetes, but this procedure has met significant challenges over the years. One such challenge is to address why primary graft function still remains inconsistent after islet transplantation. Several variables have been shown to affect graft function, but the impact of procedure-related complications on primary and long-term graft functions has not yet been explored. METHODS Twenty-six patients with established type 1 diabetes were included in this study. Each patient had two to three intraportal islet infusions to obtain 10,000 islet equivalent (IEQ)/kg in body weight, equaling a total of 68 islet infusions. Islet transplantation consisted of three sequential fresh islet infusions within 3 months. Islet infusions were performed surgically or under ultrasound guidance, depending on patient morphology, availability of the radiology suite, and patient medical history. Prospective assessment of adverse events was recorded and graded using "Common Terminology Criteria for adverse events in Trials of Adult Pancreatic Islet Transplantation." RESULTS There were no deaths or patients dropouts. Early complications occurred in nine of 68 procedures. β score 1 month after the last graft and optimal graft function (β score ≥7) rate were significantly lower in cases of procedure-related complications (P = 0.02, P = 0.03). Procedure-related complications negatively impacted graft function (P = 0.009) and was an independent predictive factor of long-term graft survival (P = 0.033) in multivariate analysis. CONCLUSION Complications occurring during radiologic or surgical intraportal islet transplantation significantly impair primary graft function and graft survival regardless of their severity.

Background There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A. Methods Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use. Results Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects. Conclusion Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects.