Combined transcriptome and proteome profiling of the pancreatic ß-cell response to palmitate unveils key pathways of ß-cell lipotoxicity.

BACKGROUND: Prolonged exposure to elevated free fatty acids induces ß-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of ß-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of ß-cell failure observed in type 2 diabetes. In order to map the ß-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate. RESULTS: Crossing transcriptome and proteome of palmitate-treated ß-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and ß-oxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to ß-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate. CONCLUSIONS: This is the first study to combine transcriptomic and sensitive time course proteomic profiling of palmitate-exposed ß-cells. Our results provide comprehensive insight into gene and protein expression changes, corroborating and expanding beyond previous findings. The identification of critical drivers and pathways of the ß-cell lipotoxic response points to novel therapeutic targets for type 2 diabetes.

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.

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.

Thrombospondin 1 protects pancreatic ß-cells from lipotoxicity via the PERK-NRF2 pathway.

The failure of ß-cells has a central role in the pathogenesis of type 2 diabetes, and the identification of novel approaches to improve functional ß-cell mass is essential to prevent/revert the disease. Here we show a critical novel role for thrombospondin 1 (THBS1) in ß-cell survival during lipotoxic stress in rat, mouse and human models. THBS1 acts from within the endoplasmic reticulum to activate PERK and NRF2 and induce a protective antioxidant defense response against palmitate. Prolonged palmitate exposure causes THBS1 degradation, oxidative stress, activation of JNK and upregulation of PUMA, culminating in ß-cell death. These findings shed light on the mechanisms leading to ß-cell failure during metabolic stress and point to THBS1 as an interesting therapeutic target to prevent oxidative stress in type 2 diabetes.

Phenylpropenoic Acid Glucoside from Rooibos Protects Pancreatic Beta Cells against Cell Death Induced by Acute Injury.

OBJECTIVE: Previous studies demonstrated that a phenylpropenoic acid glucoside (PPAG) from rooibos (Aspalathus linearis) extract had anti-hyperglycemic activity and significant protective effects on the pancreatic beta cell mass in a chronic diet-induced diabetes model. The present study evaluated the cytoprotective effect of the phytochemical on beta cells exposed to acute cell stress. METHODS: Synthetically prepared PPAG was administered orally in mice treated with a single dose of streptozotocin to acutely induce beta cell death and hyperglycemia. Its effect was assessed on beta cell mass, proliferation and apoptotic cell death. Its cytoprotective effect was also studied in vitro on INS-1E beta cells and on human pancreatic islet cells. RESULTS: Treatment with the phytochemical PPAG protected beta cells during the first days after the insult against apoptotic cell death, as evidenced by TUNEL staining, and prevented loss of expression of anti-apoptotic protein BCL2 in vivo. In vitro, PPAG protected INS-1E beta cells from streptozotocin-induced apoptosis and necrosis in a BCL2-dependent and independent way, respectively, depending on glucose concentration. PPAG also protected human pancreatic islet cells against the cytotoxic action of the fatty acid palmitate. CONCLUSIONS: These findings show the potential use of PPAG as phytomedicine which protects the beta cell mass exposed to acute diabetogenic stress.

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly.

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic ß-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in ß-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to ß-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities.

Nova1 is a master regulator of alternative splicing in pancreatic beta cells.

Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.

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.

GLIS3, a susceptibility gene for type 1 and type 2 diabetes, modulates pancreatic beta cell apoptosis via regulation of a splice variant of the BH3-only protein Bim.

Mutations in human Gli-similar (GLIS) 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD) in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1ß + interferon-γ) or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial) pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS.

Death protein 5 and p53-upregulated modulator of apoptosis mediate the endoplasmic reticulum stress-mitochondrial dialog triggering lipotoxic rodent and human ß-cell apoptosis.

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic ß-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in ß-cells by saturated fatty acids. Here we show that palmitate-induced ß-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human ß-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)-dependent c-Jun NH2-terminal kinase and PKR-like ER kinase (PERK)-induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)-regulated AKT inhibition and FoxO3a activation. DP5(-/-) mice are protected from high fat diet-induced loss of glucose tolerance and have twofold greater pancreatic ß-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes ß-cell death in diabetes.

DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non-diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non-diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in ß-cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles.

PURPOSE: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. METHODS: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). RESULTS: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. CONCLUSIONS: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Use of RNA interference to investigate cytokine signal transduction in pancreatic beta cells.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by immune infiltration of the pancreatic islets resulting in an inflammatory reaction named insulitis and subsequent beta cell apoptosis. During the course of insulitis beta cell death is probably caused by direct contact with activated macrophages and T-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines, nitric oxide, and free oxygen radicals. In vitro exposure of beta cells to the cytokines interleukin(IL)-1ß + interferon(IFN)-γ or to tumor necrosis factor(TNF)-α + IFN-γ induces beta cell dysfunction and ultimately apoptosis. The transcription factors NF-κB and STAT1 are key regulators of cytokine-induced beta cell death. However, little is known about the gene networks regulated by these (or other) transcription factors that trigger beta cell apoptosis. The recent development of RNA interference (RNAi) technology offers a unique opportunity to decipher the cytokine-activated molecular pathways responsible for beta cell death. Use of RNAi has been hampered by technical difficulties in transfecting primary beta cells, but in recent years we have succeeded in developing reliable and reproducible protocols for RNAi in beta cells. This chapter details the methods and settings used to achieve efficient and nontoxic transfection of small interfering RNA in immortal and primary beta cells.

Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance.

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

Oleate-induced beta cell dysfunction and apoptosis: a proteomic approach to glucolipotoxicity by an unsaturated fatty acid.

High levels of fatty acids contribute to loss of functional beta cell mass in type 2 diabetes, in particular in combination with high glucose levels. The aim of this study was to elucidate the role of the unsaturated free fatty acid oleate in glucolipotoxicity and to unravel the molecular pathways involved. INS-1E cells were exposed to 0.5 mM oleate, combined or not with 25 mM glucose, for 24 h. Protein profiling of INS-1E cells was done by 2D-DIGE, covering pH ranges 4-7 and 6-9 (n = 4). Identification of differentially expressed proteins (P < 0.05) was based on MALDI-TOF analysis using Peptide Mass Fingerprint (PMF) and fragmentation (MS/MS) of the most intense peaks of PMF and proteomic results were confirmed by functional assays. Oleate impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 53 and 54 differentially expressed proteins for oleate and the combination of oleate and high glucose, respectively. Exposure to oleate down-regulated chaperones, hampered insulin processing and ubiquitin-related proteasomal degradation, and induced perturbations in vesicle transport and budding. In combination with high glucose, shunting of excess amounts of glucose toward reactive oxygen species production worsened beta cell death. The present findings provide new insights in oleate-induced beta cell dysfunction and identify target proteins for preservation of functional beta cell mass in type 2 diabetes.

p53 up-regulated modulator of apoptosis (PUMA) activation contributes to pancreatic beta-cell apoptosis induced by proinflammatory cytokines and endoplasmic reticulum stress.

Type 1 diabetes is an autoimmune disorder characterized by chronic inflammation and pancreatic beta-cell loss. Here, we demonstrate that the proinflammatory cytokine interleukin-1beta, combined with interferon-gamma, induces the expression of the Bcl-2 homology 3 (BH3)-only activator PUMA (p53 up-regulated modulator of apoptosis) in beta-cells. Transcriptional activation of PUMA is regulated by nuclear factor-kappaB and endoplasmic reticulum stress but is independent of p53. PUMA activation leads to mitochondrial Bax translocation, cytochrome c release, and caspase-3 cleavage resulting in beta-cell demise. The antiapoptotic Bcl-XL protein is localized mainly at the mitochondria of the beta-cells and antagonizes PUMA action, but Bcl-XL is inactivated by the BH3-only sensitizer DP5/Hrk in cytokine-exposed beta-cells. Moreover, a pharmacological mimic of the BH3-only sensitizer Bad, which inhibits Bcl-XL and Bcl-2, induces PUMA-dependent beta-cell death and potentiates cytokine-induced apoptosis. Our data support a hierarchical activation of BH3-only proteins controlling the intrinsic pathway of beta-cell apoptosis in the context of inflammation and type 1 diabetes.

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.