Fabrice Moore

Initiation and execution of lipotoxic ER stress in pancreatic beta-cells.

Free fatty acids (FFA) cause apoptosis of pancreatic β-cells and might contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary β-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca2+ stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor β-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary β-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced β-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca2+ depletion. The IRE1 and resulting JNK activation contribute to β-cell apoptosis. PERK activation by palmitate also contributes to β-cell apoptosis via CHOP.

Peripheral and Islet Interleukin-17 Pathway Activation Characterizes Human Autoimmune Diabetes and Promotes Cytokine-Mediated β-Cell Death

OBJECTIVE CD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells. RESEARCH DESIGN AND METHODS Peripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined. RESULTS A total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ–induced and tumor necrosis factor (TNF)-α/IFN-γ–induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB. CONCLUSIONS Circulating IL-17+ β-cell–specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.

Induction of nuclear factor-κB and its downstream genes by TNF-α and IL-1β has a pro-apoptotic role in pancreatic beta cells

Aims/hypothesisIL-1β and TNF-α contribute to pancreatic beta cell death in type 1 diabetes. Both cytokines activate the transcription factor nuclear factor-κB (NF-κB), but recent observations suggest that NF-κB blockade prevents IL-1β + IFN-γ- but not TNF-α + IFN-γ-induced beta cell apoptosis. The aim of the present study was to compare the effects of IL-1β and TNF-α on cell death and the pattern of NF-κB activation and global gene expression in beta cells.MethodsCell viability was measured after exposure to IL-1β or to TNF-α alone or in combination with IFN-γ, and blockade of NF-κB activation or protein synthesis. INS-1E cells exposed to IL-1β or TNF-α in time course experiments were used for IκB kinase (IKK) activation assay, detection of p65 NF-κB by immunocytochemistry, real-time RT-PCR and microarray analysis.ResultsBlocking NF-κB activation protected beta cells against IL-1β + IFNγ- or TNFα + IFNγ-induced apoptosis. Blocking de novo protein synthesis did not increase TNF-α- or IL-1β-induced beta cell death, in line with the observations that cytokines induced the expression of the anti-apoptotic genes A20, Iap-2 and Xiap to a similar extent. Microarray analysis of INS-1E cells treated with IL-1β or TNF-α showed similar patterns of gene expression. IL-1β, however, induced a higher rate of expression of NF-κB target genes putatively involved in beta cell dysfunction and death and a stronger activation of the IKK complex, leading to an earlier translocation of NF-κB to the nucleus.Conclusions/interpretationNF-κB activation in beta cells has a pro-apoptotic role following exposure not only to IL-1β but also to TNF-α. The more marked beta cell death induced by IL-1β is explained at least in part by higher intensity NF-κB activation, leading to increased transcription of key target genes.

PTPN2, a Candidate Gene for Type 1 Diabetes, Modulates Interferon-γ–Induced Pancreatic β-Cell Apoptosis

OBJECTIVE The pathogenesis of type 1 diabetes has a strong genetic component. Genome-wide association scans recently identified novel susceptibility genes including the phosphatases PTPN22 and PTPN2. We hypothesized that PTPN2 plays a direct role in β-cell demise and assessed PTPN2 expression in human islets and rat primary and clonal β-cells, besides evaluating its role in cytokine-induced signaling and β-cell apoptosis. RESEARCH DESIGN AND METHODS PTPN2 mRNA and protein expression was evaluated by real-time PCR and Western blot. Small interfering (si)RNAs were used to inhibit the expression of PTPN2 and downstream STAT1 in β-cells, allowing the assessment of cell death after cytokine treatment. RESULTS PTPN2 mRNA and protein are expressed in human islets and rat β-cells and upregulated by cytokines. Transfection with PTPN2 siRNAs inhibited basal- and cytokine-induced PTPN2 expression in rat β-cells and dispersed human islets cells. Decreased PTPN2 expression exacerbated interleukin (IL)-1β + interferon (IFN)-γ–induced β-cell apoptosis and turned IFN-γ alone into a proapoptotic signal. Inhibition of PTPN2 amplified IFN-γ–induced STAT1 phosphorylation, whereas double knockdown of both PTPN2 and STAT1 protected β-cells against cytokine-induced apoptosis, suggesting that STAT1 hyperactivation is responsible for the aggravation of cytokine-induced β-cell death in PTPN2-deficient cells. CONCLUSIONS We identified a functional role for the type 1 diabetes candidate gene PTPN2 in modulating IFN-γ signal transduction at the β-cell level. PTPN2 regulates cytokine-induced apoptosis and may thereby contribute to the pathogenesis of type 1 diabetes.

Nicotine protects kidney from renal ischemia/reperfusion injury through the cholinergic anti-inflammatory pathway.

Kidney ischemia/reperfusion injury (I/R) is characterized by renal dysfunction and tubular damages resulting from an early activation of innate immunity. Recently, nicotine administration has been shown to be a powerful inhibitor of a variety of innate immune responses, including LPS-induced toxaemia. This cholinergic anti-inflammatory pathway acts via the α7 nicotinic acetylcholine receptor (α7nAChR). Herein, we tested the potential protective effect of nicotine administration in a mouse model of renal I/R injury induced by bilateral clamping of kidney arteries. Renal function, tubular damages and inflammatory response were compared between control animals and mice receiving nicotine at the time of ischemia. Nicotine pretreatment protected mice from renal dysfunction in a dose-dependent manner and through the α7nAChR, as attested by the absence of protection in α7nAChR-deficient mice. Additionally, nicotine significantly reduced tubular damages, prevented neutrophil infiltration and decreased productions of the CXC-chemokine KC, TNF-α and the proinflammatory high-mobility group box 1 protein. Reduced tubular damage in nicotine pre-treated mice was associated with a decrease in tubular cell apoptosis and proliferative response as attested by the reduction of caspase-3 and Ki67 positive cells, respectively. All together, these data highlight that nicotine exerts a protective anti-inflammatory effect during kidney I/R through the cholinergic α7nAChR pathway. In addition, this could provide an opportunity to overcome the effect of surgical cholinergic denervation during kidney transplantation.

STAT1 is a master regulator of pancreatic beta-cell apoptosis and islet inflammation.

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Cytokines Interleukin-1β and Tumor Necrosis Factor-α Regulate Different Transcriptional and Alternative Splicing Networks in Primary β-Cells

OBJECTIVE Cytokines contribute to pancreatic β-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)-1β + interferon (IFN)-γ and tumor necrosis factor (TNF)-α + IFN-γ in primary rat β-cells. RESEARCH DESIGN AND METHODS Fluorescence-activated cell sorter–purified rat β-cells were exposed to IL-1β + IFN-γ or TNF-α + IFN-γ 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 β-cells, with temporal differences in the number of genes modulated by IL-1β + IFNγ or TNF-α + IFN-γ. 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 β-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-α, 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 β-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in β-cells. It also shows that cytokines modify alternative splicing in β-cells, opening a new avenue of research for the field.

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis.

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.

Cytokines Tumor Necrosis Factor-α and Interferon-γ Induce Pancreatic β-Cell Apoptosis through STAT1-mediated Bim Protein Activation

Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing β-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of β-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent β-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced β-cell apoptosis. DP5 knock-out mice had increased β-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human β-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of β-cell apoptosis during insulitis.

PTPN2, a Candidate Gene for Type 1 Diabetes, Modulates Pancreatic β-Cell Apoptosis via Regulation of the BH3-Only Protein Bim

OBJECTIVE Genome-wide association studies allowed the identification of several associations between specific loci and type 1 diabetes (T1D). However, the mechanisms by which most candidate genes predispose to T1D remain unclear. We presently evaluated the mechanisms by which PTPN2, a candidate gene for T1D, modulates β-cell apoptosis after exposure to type I and II interferons (IFNs), cytokines that contribute to β-cell loss in early T1D. RESEARCH DESIGN AND METHODS Small interfering RNAs were used to inhibit PTPN2, STAT1, Bim, and Jun NH2-terminal kinase 1 (JNK1) expression. Cell death was assessed by Hoechst and propidium iodide staining. BAX translocation, Bim phosphorylation, cytochrome c release, and caspases 9 and 3 activation were measured by Western blot or immunofluorescence. RESULTS PTPN2 knockdown exacerbated type I IFN–induced apoptosis in INS-1E, primary rat, and human β-cells. PTPN2 silencing and exposure to type I and II IFNs induced BAX translocation to the mitochondria, cytochrome c release, and caspase 3 activation. There was also an increase in Bim phosphorylation that was at least in part regulated by JNK1. Of note, both Bim and JNK1 knockdown protected β-cells against IFN-induced apoptosis in PTPN2-silenced cells. CONCLUSIONS The present findings suggest that local IFN production may interact with a genetic factor (PTPN2) to induce aberrant proapoptotic activity of the BH3-only protein Bim, resulting in increased β-cell apoptosis via JNK activation and the intrinsic apoptotic pathway. This is the first indication of a direct interaction between a candidate gene for T1D and the activation of a specific downstream proapoptotic pathway in β-cells.