Pancreatic ß-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF).

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic ß-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote ß-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human ß-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in ß-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of ß-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in ß-cells should be explored as a cytoprotective strategy in type 1 diabetes.

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

OBJECTIVE: 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 ß-cell failure and the pathogenic mechanisms involved in FRDA diabetes. METHODS: Forty-one FRDA patients, 26 heterozygous carriers of a GAA expansion, and 53 controls underwent oral and intravenous glucose tolerance tests. ß-Cell proportion was quantified in postmortem pancreas sections from 9 unrelated FRDA patients. Using an in vitro disease model, we studied how frataxin deficiency affects ß-cell function and survival. RESULTS: FRDA patients had increased abdominal fat and were insulin resistant. This was not compensated for by increased insulin secretion, resulting in a markedly reduced disposition index, indicative of pancreatic ß-cell failure. Loss of glucose tolerance was driven by ß-cell dysfunction, which correlated with abdominal fatness. In postmortem pancreas sections, pancreatic islets of FRDA patients had a lower ß-cell content. RNA interference-mediated frataxin knockdown impaired glucose-stimulated insulin secretion and induced apoptosis in rat ß cells and human islets. Frataxin deficiency sensitized ß cells to oleate-induced and endoplasmic reticulum stress-induced apoptosis, which could be prevented by the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. INTERPRETATION: Pancreatic ß-cell dysfunction is central to diabetes development in FRDA as a result of mitochondrial dysfunction and higher sensitivity to metabolic and endoplasmic reticulum stress-induced ß-cell death.

In depth functional characterization of human induced pluripotent stem cell-derived beta cells in vitro and in vivo.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

DNAJC3 deficiency induces ß-cell mitochondrial apoptosis and causes syndromic young-onset diabetes.

OBJECTIVE: DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic ß-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of ß-cell failure in this syndrome. METHODS: Whole exome sequencing was performed and identified variants were confirmed by Sanger sequencing. DNAJC3 was silenced by RNAi in INS-1E cells, primary rat ß-cells, human islets, and induced pluripotent stem cell-derived ß-cells. ß-cell function and apoptosis were assessed, and potential mediators of apoptosis examined. RESULTS: The two patients presented with juvenile-onset diabetes, short stature, hypothyroidism, neurodegeneration, facial dysmorphism, hypoacusis, microcephaly and skeletal bone deformities. They were heterozygous compound and homozygous for novel loss-of-function mutations in DNAJC3. DNAJC3 silencing did not impair insulin content or secretion. Instead, the knockdown induced rat and human ß-cell apoptosis and further sensitized cells to endoplasmic reticulum stress, triggering mitochondrial apoptosis via the pro-apoptototic Bcl-2 proteins BIM and PUMA. CONCLUSIONS: This report confirms previously described features and expands the clinical spectrum of syndromic DNAJC3 diabetes, one of the five monogenic forms of diabetes pertaining to the PERK pathway of the endoplasmic reticulum stress response. DNAJC3 deficiency may lead to ß-cell loss through BIM- and PUMA-dependent activation of the mitochondrial pathway of apoptosis.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Guanabenz Sensitizes Pancreatic ß Cells to Lipotoxic Endoplasmic Reticulum Stress and Apoptosis.

Deficient as well as excessive/prolonged endoplasmic reticulum (ER) stress signaling can lead to pancreatic ß cell failure and the development of diabetes. Saturated free fatty acids (FFAs) such as palmitate induce lipotoxic ER stress in pancreatic ß cells. One of the main ER stress response pathways is under the control of the protein kinase R-like endoplasmic reticulum kinase (PERK), leading to phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α). The antihypertensive drug guanabenz has been shown to inhibit eIF2α dephosphorylation and protect cells from ER stress. Here we examined whether guanabenz protects pancreatic ß cells from lipotoxicity. Guanabenz induced ß cell dysfunction in vitro and in vivo in rodents and led to impaired glucose tolerance. The drug significantly potentiated FFA-induced cell death in clonal rat ß cells and in rat and human islets. Guanabenz enhanced FFA-induced eIF2α phosphorylation and expression of the downstream proapoptotic gene C/EBP homologous protein (CHOP), which mediated the sensitization to lipotoxicity. Thus, guanabenz does not protect ß cells from ER stress; instead, it potentiates lipotoxic ER stress through PERK/eIF2α/CHOP signaling. These data demonstrate the crucial importance of the tight regulation of eIF2α phosphorylation for the normal function and survival of pancreatic ß cells.

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 ß-Cells.

Type 1 diabetes (T1D) is an autoimmune disease leading to ß-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 ß-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-1ß plus IFN-γ. Confirmation of miRNA and target gene modification in human ß-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 ß-cell apoptosis. These results identify a novel cross talk between a key family of miRNAs and proapoptotic Bcl-2 proteins in human pancreatic ß-cells, broadening our understanding of cytokine-induced ß-cell apoptosis in early T1D.

High-throughput screening and bioinformatic analysis to ascertain compounds that prevent saturated fatty acid-induced ß-cell apoptosis.

Pancreatic ß-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause ß-cell death and develop novel approaches to prevent it, a high-throughput screen on the ß-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes.

Disruption of the gamma-interferon signaling pathway at the level of signal transducer and activator of transcription-1 prevents immune destruction of beta-cells.

beta-cells under immune attack are destroyed by the aberrant activation of key intracellular signaling cascades. The aim of the present study was to evaluate the contribution of the signal transducer and activator of transcription (STAT)-1 pathway for beta-cell apoptosis by studying the sensitivity of beta-cells from STAT-1 knockout (-/-) mice to immune-mediated cell death in vitro and in vivo. Whole islets from STAT-1-/- mice were completely resistant to interferon (IFN)-gamma (studied in combination with interleukin [IL]-1beta)-mediated cell death (92 +/- 4% viable cells in STAT-1-/- mice vs. 56 +/- 3% viable cells in wild-type controls, P < or = 0.001) and had preserved insulin release after exposure to IL-1beta and IFN-gamma. Moreover, analysis of cell death in cytokine-exposed purified beta-cells confirmed that protection was due to absence of STAT-1 in the beta-cells themselves. Deficiency of STAT-1 in islets completely prevented cytokine-induced upregulation of IL-15, interferon inducible protein 10, and inducible nitric oxide synthase transcription but did not interfere with monocyte chemoattractant protein 1 and macrophage inflammatory protein 3alpha expression. In vivo, STAT-1-/- mice were partially resistant to development of diabetes after multiple low-dose streptozotocin injections as reflected by mean blood glucose at 12 days after first injection (159 +/- 28 vs. 283 +/- 81 mg/dl in wild-type controls, P < or = 0.05) and diabetes incidence at the end of the follow-up period (39 vs. 73% in wild-type controls, P < or = 0.05). In conclusion, the present results indicate that STAT-1 is a crucial transcription factor in the process of IFN-gamma-mediated beta-cell death and the subsequent development of immune-mediated diabetes.

Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress.

Apoptosis is probably the main form of beta-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to beta-cell destruction through nuclear factor-kappaB (NF-kappaB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are beta-cell toxic also via NF-kappaB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced beta-cell apoptosis and determine whether TNFalpha, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-kappaB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified beta-cells by oleate, palmitate, and/or cytokines (IL-1beta, interferon-gamma, TNFalpha). Palmitate and IL-1beta induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFalpha did not potentiate FFA toxicity in primary beta-cells. The NF-kappaB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1beta but not by FFAs. Cytokines activated NF-kappaB in INS-1E and beta-cells, but FFAs did not. Moreover, FFAs did not enhance NF-kappaB activation by TNFalpha. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA- and cytokine-induced beta-cell death but the mechanisms involved are different. Whereas cytokines induce NF-kappaB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-kappaB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of beta-cell death in T1DM and T2DM.

Metabolism of 13C-enriched D-fructose in hepatocytes from Goto-Kakizaki rats.

This study aims at assessing the conversion of exogenous D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]-fructose (10 mM) to 13C-enriched and either hydrogenated or deuterated D-glucose, L-lactate and L-alanine released by rat liver cells prepared from Goto-Kakizaki rats and incubated for 120 min in the presence of unlabelled D-glucose (also 10 mM) and D2O. The results of this study are relevant to the relative contribution of fructokinase and hexokinase isoenzyme to the phosphorylation of D-fructose, the capacity of D-glucose to confer to glucokinase positive cooperativity towards D-fructose, the circulation of D-fructose 6-phosphate in the pentose phosphate pathway, the regulation of the cytosolic NADD/NADH ratio, the respective fate of D-fructose-derived D-glyceraldehyde and dihydroxyacetone phosphate, the deuteration of fructose-derived glycolytic intermediates at the phosphoglucoisomerase, phosphomannoisomerase, enolase, pyruvate kinase and glutamate-alanine transaminase levels, and the unequal generation of L-[1-13C]lactate by cells exposed to D-[1-13C]fructose or D-[6-13C]fructose versus D-[2-13C]-fructose.

Pharmacodynamics, insulinotropic action and hypoglycemic effect of nateglinide and glibenclamide in normal and diabetic rats.

Control and streptozotocin-induced diabetic rats, as well as GK rats, received a single oral administration of either nateglinide (50 microg/g body wt.) or glibenclamide (1.0 microg/g body wt.). The plasma D-glucose and insulin concentrations, as well as the content of plasma, liver and pancreas in either nateglinide or glibenclamide were measured 60 min or 24 hours after the administration of these antidiabetic agents. At the 60th min, the plasma, hepatic and pancreatic content of nateglinide largely exceeded that of glibenclamide. At the 24th hour, however, the plasma concentration, as well as liver and pancreas content, of nateglinide became negligible, whilst that of glibenclamide exceeded the values recorded at the 60th min. A comparable pattern characterized the insulinotropic action and hypoglycemic effect of these two antidiabetic agents. This study thus emphasizes the vastly different pharmacodynamics of nateglinide and glibenclamide in both control and diabetic rats.

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

SCOPE: A 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. METHODS AND RESULTS: Mice 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. CONCLUSIONS: We 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.

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

Pancreatic ß-cell dysfunction and death are central in the pathogenesis of type 2 diabetes (T2D). Saturated fatty acids cause ß-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 ß-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 ß-cell dysfunction and death. The data point to cross talk between metabolic stress and candidate genes at the ß-cell level.

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.

Mild endoplasmic reticulum stress augments the proinflammatory effect of IL-1ß in pancreatic rat ß-cells via the IRE1α/XBP1s pathway.

The prevalence of obesity and type 1 diabetes in children is increasing worldwide. Insulin resistance and augmented circulating free fatty acids associated with obesity may cause pancreatic ß-cell endoplasmic reticulum (ER) stress. We tested the hypothesis that mild ER stress predisposes ß-cells to an exacerbated inflammatory response when exposed to IL-1ß or TNF-α, cytokines that contribute to the pathogenesis of type 1 diabetes. INS-1E cells or primary rat ß-cells were exposed to a low dose of the ER stressor cyclopiazonic acid (CPA) or free fatty acids, followed by low-dose IL-1ß or TNF-α. ER stress signaling was inhibited by small interfering RNA. Cells were evaluated for proinflammatory gene expression by RT-PCR and ELISA, gene reporter activity, p65 activation by immunofluorescence, and apoptosis. CPA pretreatment enhanced IL-1ß- induced, but not TNF-α-induced, expression of chemokine (C-C motif) ligand 2, chemokine (C-X-C motif) ligand 1, inducible nitric oxide synthase, and Fas via augmented nuclear factor κB (NF-κB) activation. X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1, but not CCAAT/enhancer binding protein homologous protein, knockdown prevented the CPA-induced exacerbation of NF-κB-dependent genes and decreased IL-1ß-induced NF-κB promoter activity. XBP1 modulated NF-κB activity via forkhead box O1 inhibition. In conclusion, rat ß-cells facing mild ER stress are sensitized to IL-1ß, generating a more intense and protracted inflammatory response through inositol-requiring enzyme 1/XBP1 activation. These observations link ß-cell ER stress to the triggering of exacerbated local inflammation.

Perturbation of glycerol metabolism in hepatocytes from n3-PUFA-depleted rats.

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹4C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹4C]glycerol uptake could be explained by an observed increase in glycerol kinase activity.

Enhanced signaling downstream of ribonucleic Acid-activated protein kinase-like endoplasmic reticulum kinase potentiates lipotoxic endoplasmic reticulum stress in human islets.

BACKGROUND: Free fatty acids cause pancreatic beta-cell apoptosis and may contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. Eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation is an adaptive response to ER stress, and reductions in eIF2alpha phosphorylation trigger beta-cell failure. Salubrinal inhibits eIF2alpha dephosphorylation and has been proposed as a novel therapy for diabetes. OBJECTIVE: The objective of the study was to examine whether salubrinal modulates human islet susceptibility to lipotoxicity. STUDY DESIGN: Human islets were treated with oleate or palmitate, alone or in combination with salubrinal, and examined for apoptosis, ultrastructure, and gene expression. RESULTS: Salubrinal enhanced signaling downstream of eIF2alpha and markedly induced the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein, but it did not induce the inositol requiring-1alpha or activating transcription factor 6 ER stress pathways. Salubrinal potentiated the deleterious effects of oleate and palmitate in human islets. This proapoptotic effect involved ER dilation and mitochondrial rounding and fragmentation. CONCLUSIONS: Excessive eIF2alpha phosphorylation is poorly tolerated by human islets and exacerbates fatty acid-induced apoptosis through ER and mitochondrial mechanisms. This should be taken into consideration when designing approaches to pharmacologically modulate the beta-cell ER stress response in type 2 diabetes.

Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells.

OBJECTIVE: Cytokines contribute to pancreatic beta-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in beta-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes. CONCLUSIONS: The present study doubles the number of known genes expressed in primary beta-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in beta-cells. It also shows that cytokines modify alternative splicing in beta-cells, opening a new avenue of research for the field.