Laurence Ladrière

Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis.

Free fatty acids cause pancreatic β-cell apoptosis and may contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum stress. Reductions in eukaryotic translation initiation factor (eIF) 2α phosphorylation trigger β-cell failure and diabetes. Salubrinal selectively inhibits eIF2α dephosphorylation, protects other cells against endoplasmic reticulum stress-mediated apoptosis, and has been proposed as a β-cell protector. Unexpectedly, salubrinal induced apoptosis in primary β-cells, and it potentiated the deleterious effects of oleate and palmitate. Salubrinal induced a marked eIF2α phosphorylation and potentiated the inhibitory effects of free fatty acids on protein synthesis and insulin release. The synergistic activation of the PERK-eIF2α branch of the endoplasmic reticulum stress response, but not of the IRE1 and activating transcription factor-6 pathways, led to a marked induction of activating transcription factor-4 and the pro-apoptotic transcription factor CHOP. Our findings demonstrate that excessive eIF2α phosphorylation is poorly tolerated by β-cells and exacerbates free fatty acid-induced apoptosis. This modifies the present paradigm regarding the beneficial role of eIF2α phosphorylation in β-cells and must be taken into consideration when designing therapies to protect β-cells in type 2 diabetes.

Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes.

Aims/hypothesisBeta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.MethodsChemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28xa0mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.ResultsMicroarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate.Conclusions/interpretationSaturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.

RNA-sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate

Pancreatic β-cell dysfunction and death are central in the pathogenesis of type 2 diabetes (T2D). Saturated fatty acids cause β-cell failure and contribute to diabetes development in genetically predisposed individuals. Here we used RNA sequencing to map transcripts expressed in five palmitate-treated human islet preparations, observing 1,325 modified genes. Palmitate induced fatty acid metabolism and endoplasmic reticulum (ER) stress. Functional studies identified novel mediators of adaptive ER stress signaling. Palmitate modified genes regulating ubiquitin and proteasome function, autophagy, and apoptosis. Inhibition of autophagic flux and lysosome function contributed to lipotoxicity. Palmitate inhibited transcription factors controlling β-cell phenotype, including PAX4 and GATA6. Fifty-nine T2D candidate genes were expressed in human islets, and 11 were modified by palmitate. Palmitate modified expression of 17 splicing factors and shifted alternative splicing of 3,525 transcripts. Ingenuity Pathway Analysis of modified transcripts and genes confirmed that top changed functions related to cell death. Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of transcription factor binding sites in palmitate-modified transcripts revealed a role for PAX4, GATA, and the ER stress response regulators XBP1 and ATF6. This human islet transcriptome study identified novel mechanisms of palmitate-induced β-cell dysfunction and death. The data point to cross talk between metabolic stress and candidate genes at the β-cell level.

Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction.

Pancreatic β‐cell dysfunction is central to the pathogenesis of type 2 diabetes, and the loss of functional β‐cell mass in type 2 diabetes is at least in part secondary to increased β‐cell apoptosis. Accumulating evidence suggests that endoplasmic reticulum (ER) stress is present in β‐cells in type 2 diabetes. Free fatty acids (FFAs) cause ER stress and are putative mediators of β‐cell dysfunction and death. In this review, we discuss the molecular mechanisms underlying ER stress induced by saturated and unsaturated FFAs. Oleate and palmitate trigger ER stress through ER Ca2+ depletion and build‐up of unfolded proteins in the secretory pathway. Saturated and unsaturated FFAs elicit a differential signal transduction in the three branches of the ER stress response, resulting in different survival/apoptosis outcomes. The protection of β‐cells against FFAs through the interference with ER stress signalling has opened novel therapeutic perspectives for type 2 diabetes. Chemical chaperones, salubrinal and glucagon‐like peptide‐1 (GLP‐1) analogues have been used to protect β‐cells from lipotoxic ER stress. Importantly, the pro‐ and antiapoptotic effects of these compounds are cell and context dependent.

Stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester.

The role currently ascribed to the accumulation of l-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to l-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on d-glucose metabolism, more severe inhibition of the catabolism of endogenous l-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-meadiated entry of l-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.The role currently ascribed to the accumulation of L-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to L-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on D-glucose metabolism, more severe inhibition of the catabolism of endogenous L-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-mediated entry of L-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.

An update on lipotoxic endoplasmic reticulum stress in pancreatic β-cells

The UPR (unfolded protein response) or ER (endoplasmic reticulum) stress response was first described 20 years ago. The field of ER stress has expanded tremendously since, moving from basic biology in yeast to human neurodegenerative, inflammatory, cardiovascular and neoplastic diseases. The ER stress response has also been implicated in diabetes development, affecting both insulin production by pancreatic beta-cells and insulin sensitivity in peripheral tissues. In the present mini-review, we focus on recent progress in the field of ER stress in pancreatic beta-cells. Recent advances in the understanding of lipotoxic ER stress and beta-cell recovery from ER stress are discussed.

Microarray analysis of isolated human islet transcriptome in type 2 diabetes and the role of the ubiquitin–proteasome system in pancreatic beta cell dysfunction

To shed light on islet cell molecular phenotype in human type 2 diabetes (T2D), we studied the transcriptome of non-diabetic (ND) and T2D islets to then focus on the ubiquitin-proteasome system (UPS), the major protein degradation pathway. We assessed gene expression, amount of ubiquitinated proteins, proteasome activity, and the effects of proteasome inhibition and prolonged exposure to palmitate. Microarray analysis identified more than one thousand genes differently expressed in T2D islets, involved in many structures and functions, with consistent alterations of the UPS. Quantitative RT-PCR demonstrated downregulation of selected UPS genes in T2D islets and beta cell fractions, with greater ubiquitin accumulation and reduced proteasome activity. Chemically induced reduction of proteasome activity was associated with lower glucose-stimulated insulin secretion, which was partly reproduced by palmitate exposure. These results show the presence of many changes in islet transcriptome in T2D islets and underline the importance of the association between UPS alterations and beta cell dysfunction in human T2D.

Central role and mechanisms of β-cell dysfunction and death in friedreich ataxia-associated diabetes.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused in almost all cases by homozygosity for a GAA trinucleotide repeat expansion in the frataxin gene. Frataxin is a mitochondrial protein involved in iron homeostasis. FRDA patients have a high prevalence of diabetes, the pathogenesis of which is not known. We aimed to evaluate the relative contribution of insulin resistance and β‐cell failure and the pathogenic mechanisms involved in FRDA diabetes.

MicroRNAs miR-23a-3p, miR-23b-3p, and miR-149-5p Regulate the Expression of Proapoptotic BH3-Only Proteins DP5 and PUMA in Human Pancreatic β-Cells

Type 1 diabetes (T1D) is an autoimmune disease leading to β-cell destruction. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and organ formation. They participate in the pathogenesis of several autoimmune diseases, but the nature of miRNAs contributing to β-cell death in T1D and their target genes remain to be clarified. We performed an miRNA expression profile on human islet preparations exposed to the cytokines IL-1β plus IFN-γ. Confirmation of miRNA and target gene modification in human β-cells was performed by real-time quantitative PCR. Single-stranded miRNAs inhibitors were used to block selected endogenous miRNAs. Cell death was measured by Hoechst/propidium iodide staining and activation of caspase-3. Fifty-seven miRNAs were detected as modulated by cytokines. Three of them, namely miR-23a-3p, miR-23b-3p, and miR-149-5p, were downregulated by cytokines and selected for further studies. These miRNAs were found to regulate the expression of the proapoptotic Bcl-2 proteins DP5 and PUMA and consequent human β-cell apoptosis. These results identify a novel cross talk between a key family of miRNAs and proapoptotic Bcl-2 proteins in human pancreatic β-cells, broadening our understanding of cytokine-induced β-cell apoptosis in early T1D.

Phenylpropenoic acid glucoside augments pancreatic beta cell mass in high‐fat diet‐fed mice and protects beta cells from ER stress‐induced apoptosis

SCOPEnA major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established.nnnMETHODS AND RESULTSnMice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals.nnnCONCLUSIONSnWe identified an antidiabetic phytochemical that protects pancreatic beta cells from ER stress and apoptosis induced by high-fat diet/lipotoxicity. At the tissue level, this led to a tripling of beta cell mass. At the molecular level, the protective effect of the phytochemical was mediated by increasing BCL2 expression in beta cells.