Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains

PDZD2 (PDZ-domain-containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi-PDZ-domain protein. We have shown that PDZD2, which shows extensive homology to pro-interleukin-16 (pro-IL-16), is localized mainly to the endoplasmic reticulum (ER). Pro-IL-16 is cleaved in a caspase-3-dependent mechanism to generate the secreted cytokine IL-16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro-IL-16, suggests that similar post-translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope-tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi-PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains.     

PDZ-domain containing-2 (PDZD2) is a novel factor that affects the growth and differentiation of human fetal pancreatic progenitor cells

Early-trimester human fetal pancreas is a promising potential source of pancreatic progenitor cells. However, the ethical controversy associated with the source of these cells, and technical difficulties associated with their differentiation into insulin-producing cells have limited both their availability and utility. This study aimed to characterize a population of pancreatic progenitor cells (PPCs) isolated from human fetus and describe the effects of a novel factor, PDZ-domain containing-2 (PDZD2), and its secreted form (sPDZD2), on PPC proliferation and differentiation. In particular, we examined and characterized the expression of several stem cell (nestin, ABCG2, c-kit), growth and differentiation markers (GLP-1R, c-met, erbB1), and PDZD2 in PPCs by RT-PCR, Western blot, and immunocytochemistry. We also examined the effects of sPDZD2 on PPC proliferation and differentiation by examining BrdU incorporation, MTT, cell number, and real-time PCR as well as ELISA. PPCs were isolated, cultured and characterized from human fetal pancreas. PDZD2 and sPDZD2 were detected at high levels in both human fetal pancreas and in PPCs. sPDZD2 acted as a potent mitogen on PPCs, and inhibited the differentiation of PPC-derived islet-like cell-clusters (ICCs), evidenced by the downregulation of Isl-1, Pdx-1, and insulin mRNA levels. sPDZD2 treatment also reduced levels of C-peptide in ICCs. These results show that a novel pancreatic developmental factor, PDZD2, is sufficient to promote the proliferation of human fetal PPCs while limiting differentiation of ICCs into islet/endocrine cells. Findings from this study will contribute to the development of improved methods for islet transplantation therapy in the treatment of diabetes.     

20-HETE对小鼠葡萄糖刺激胰岛素分泌反应的影响

为了考察20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acids, 20-HETE)对葡萄糖刺激胰岛素分泌反应的影响,本研究选择CYP4F2转基因小鼠和小鼠胰岛素瘤INS-1E细胞作为研究材料,通过LCMS/MS检测WT和TG小鼠的胰腺20-HETE水平。通过IPGTT测定小鼠葡萄糖耐量,通过ELISA测定小鼠血浆C肽水平来检测胰岛素分泌。通过Western blotting、Real time PCR、免疫组化和免疫荧光来检测小鼠胰腺或INS-1E细胞中Glut2、GSK-3β(Ser9点)和AKT (Ser473点)的磷酸化水平。TG小鼠的20-HETE水平((7.26±2.03) ng/mg蛋白)显著高于WT小鼠((2.14±0.76) ng/mg蛋白)。在用20-HETE合成的选择性抑制剂HET0016处理后,TG小鼠((0.33±0.07) ng/mg蛋白)和WT小鼠((0.27±0.06) ng/mg蛋白)胰腺组织中的20-HETE水平均急剧降低。给予葡萄糖处理30 min后,TG小鼠的血糖水平均显著高于WT小鼠,而血浆C肽水平显著低于WT小鼠(p<0.05)。与WT小鼠相比,TG小鼠的胰腺组织中Glut2 m RNA和蛋白水平显著降低。与WT小鼠相比,CYP4F2转基因小鼠的GSK-3β和AKT磷酸化均显著降低。20-HETE处理可导致INS-1E细胞中AKT/GSK-3β磷酸化水平和Glut2表达水平显著降低(p<0.05)。此外,用17 mmol/L葡萄糖处理INS-1E细胞1 h,20-HETE处理组的胰岛素分泌显著降低。应用GSK-3β选择性抑制剂TWS119预处理INS-1E细胞3 h后,TWS119 (一种GSK-3β选择性抑制剂)预处理显著逆转了Glut2表达水平的降低以及胰岛素分泌的减少。20-HETE主要通过AKT/GSK-3β信号通路来下调Glut2的表达,进而减弱胰岛素分泌,导致胰岛素分泌功能障碍。     

Pro-IL-16 regulation in activated murine CD4+ lymphocytes

Prior DNA microarray studies suggested that IL-16 mRNA levels decrease following T cell activation, a property unique among cytokines. We examined pro-IL-16 mRNA and protein expression in resting and anti-CD3 mAb-activated primary murine CD4(+) T cells. Consistent with the microarray reports, pro-IL-16 mRNA levels fell within 4 h of activation, and this response is inhibited by cyclosporin A. Total cellular pro-IL-16 protein also fell, reaching a nadir at 48 h. Pro-IL-16 comprises a C-terminal cytokine domain and an N-terminal prodomain that are cleaved by caspase-3. Pro-IL-16 expressed in transfected tumor cells was previously shown to translocate to the nucleus and to promote G(0)/G(1) arrest by stabilizing the cyclin-dependent kinase inhibitor p27(Kip1). In the present study, we observed increased S-phase kinase-associated protein 2 mRNA expression in IL-16 null mice, but basal expression and activation-dependent regulation of p27(Kip1) were no different from wild-type mice. Stimulation with anti-CD3 mAb induced transiently greater thymidine incorporation in IL-16-deficient CD4(+) T cells than wild-type controls, but there was no difference in cell survival or in the CFSE dilution profiles. Analysis of CD4(+) T cell proliferation in vivo using BrdU labeling similarly failed to identify a hyperproliferative phenotype in T cells lacking IL-16. These data demonstrate that pro-IL-16 mRNA and protein expression are dynamically regulated during CD4(+) T cell activation by a calcineurin-dependent mechanism, and that pro-IL-16 might influence T cell cycle regulation, although not in a dominant manner.     

Nuclear translocation of the N-terminal prodomain of interleukin-16

Interleukin-16 (IL-16) is a pleiotropic cytokine that functions as a chemoattractant factor, a modulator of T cell activation, and an inhibitor of human immunodeficiency virus (HIV) replication. These diverse functions are exclusively attributed to the secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of proteolytic release of the third PDZ domain of pro-IL-16. Here we show that the N-terminal prodomain of pro-IL-16 translocates into the nucleus following cleavage of the C-terminal segment. The nuclear localization signal of pro-IL-16 consists of a classical bipartite nuclear targeting motif. We also show that the nuclear targeting of the IL-16 prodomain induces a G(0)/G(1) arrest in the cell cycle. Taken together, the high degree of conservation of the prodomain among species, the presence of two PDZ motifs, and the nuclear localization and subsequent inhibitory effect on cell cycle progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.     

Increased DNase I activity in diabetes might be associated with injury of pancreas

DNase I is an endonuclease responsible to destruction of chromatin during apoptosis. However, its role in diabetes is still unclear. With blood samples from our previous study related to type 2 diabetes, we examined the DNase I activity in the serum of these patients and the role of DNase I in the injury of pancreas was further investigated in rats and INS-1 cells. Serum and pancreatic tissues from human and rats were used for the study. Insulin resistance and diabetes were induced by high fat diet and STZ injection, respectively. DNase I activity was determined by radial enzyme-diffusion method. Expressions of DNase I and caspase-3 in pancreas were determined in rat pancreatic tissues and INS-1 cells. Apoptosis of INS-1 cells was determined by both TUNEL assay and Flow Cytometry. There was a significant elevation of DNase I activity in serum of patients with type 2 diabetes and rats with STZ injection. Moreover, increase in DNase I expression was observed in the pancreas of diabetic person and rats. Furthermore, high glucose induced both DNase I and caspase-3 expression and at the same time increased apoptosis rate of INS-1 cells. In conclusion, elevated DNase I in diabetes may be related to pancreatic injury and could be one of the causes that induce diabetes.     

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.     

Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells

Phoenixin (PNX) is a recently discovered neuropeptide which modulates appetite, pain sensation and neurons of the reproductive system in the central nervous system. PNX is also detectable in the circulation and in peripheral tissues. Recent data suggested that PNX blood levels positively correlate with body weight as well as nutritional status suggesting a potential role of this peptide in controlling energy homeostasis. PNX is detectable in endocrine pancreas, however it is unknown whether PNX regulates insulin biosynthesis or secretion. Using insulin producing INS-1E cells and isolated rat pancreatic islets we evaluated therefore, whether PNX controls insulin expression, secretion and cell proliferation. We identified PNX in pancreatic alpha as well as in beta cells. Secretion of PNX from pancreatic islets was stimulated by high glucose. PNX stimulated insulin mRNA expression in INS-1E cells. Furthermore, PNX enhanced glucose-stimulated insulin secretion in INS-1E cells and pancreatic islets in a time-dependent manner. Stimulation of insulin secretion by PNX was dependent upon cAMP/Epac signalling, while potentiation of cell growth and insulin mRNA expression was mediated via ERK1/2- and AKT-pathway. These results indicate that PNX may play a role in controlling glycemia by interacting with pancreatic beta cells.     

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1β secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1β expression, it suppressed the secretion of cleaved IL-1β. In contrast, HGF decreased active IL-1β secretion without affecting pro-IL-1β expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1β expression level and active IL-1β secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.     

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.     

Interleukin-33 Is Biologically Active Independently of Caspase-1 Cleavage

The new interleukin (IL)-1 family cytokine IL-33 is synthesized as a 30-kDa precursor. Like pro-IL-1β, human pro-IL-33 was reported to be cleaved by caspase-1 to generate an 18-kDa fragment, which is sufficient to activate signaling by the IL-33 receptor T1/ST2. However, the proposed caspase-1 cleavage site is poorly conserved between species. In addition, it is not clear whether caspase-1 cleavage of pro-IL-33 occurs in vivo and whether, as for IL-1β, this cleavage is a prerequisite for IL-33 secretion and bioactivity. In this study, we further investigated caspase-1 cleavage of mouse and human pro-IL-33 and assessed the potential bioactivity of the IL-33 precursor. We observed the generation of a 20-kDa IL-33 fragment in cell lysates, which was enhanced by incubation with caspase-1. However, in vitro assays of mouse and human pro-IL-33 indicated that IL-33 is not a direct substrate for this enzyme. Consistently, caspase-1 activation in THP-1 cells induced cleavage of pro-IL-1β but not of pro-IL-33, and activated THP-1 cells released full-length pro-IL-33 into culture supernatants. Finally, addition of full-length pro-IL-33 induced T1/ST2-dependent IL-6 secretion in mast cells. However, we observed in situ processing of pro-IL-33 in mast cell cultures, and it remains to be determined whether full-length pro-IL-33 itself indeed represents the bioactive species. In conclusion, our data indicate that pro-IL-33 is not a direct substrate for caspase-1. In addition, our results clearly show that caspase-1 cleavage is not required for pro-IL-33 secretion and bioactivity, highlighting major differences between IL-1β and IL-33.Interleukin (IL)² -33, the most recently described cytokine of the IL-1 family, is synthesized as a 30-kDa precursor. Human pro-IL-33, like pro-IL-1β, was reported to be cleaved by caspase-1 in vitro to generate an 18-kDa fragment, termed mature IL-33, which is sufficient to activate signaling by the IL-33 receptor T1/ST2 (1).Caspase-1 is an endoproteinase that specifically cleaves Asp-Xaa bonds, where Xaa typically refers to a small, often hydrophobic residue (2–4). Caspase-1 activity absolutely requires the presence of an Asp residue at position −1 of the cleavage site. Consistently, replacement of Asp¹¹⁶ by other amino acids, such as Ala, was previously demonstrated to prevent caspase-1 cleavage of pro-IL-1β (2). Recombinant (r) mature IL-33 starts at Ser¹¹² for human (h) IL-33 and at Ser¹⁰⁹ for mouse (m) IL-33, neither of which corresponds exactly to the position of a potential caspase-1 cleavage site. Indeed, the N-terminal moiety of human pro-IL-33 sequence contains a single Asp at position 110, and the N-terminal portion of mouse pro-IL-33 contains an Asp at positions 88 and 106. In fact, the region located between amino acids 80 and 110 of pro-IL-33 is rather poorly conserved between species (5). In particular, no Asp residues can be consistently found at an identical position across species to hint at the presence of a conserved caspase-1 cleavage site. So far, caspase-1 cleavage of pro-IL-33 has not been investigated in any species other than human.Expression of endogenous IL-33 has been described most extensively in endothelial cells, where essentially nuclear, full-length 30-kDa pro-IL-33 is detected (5–7). To date, only two studies have examined potential effects of caspase-1 activation on the processing and secretion of pro-IL-33 in living cells. In one study, stimulation of murine glial cultures with caspase-1 activators induced secretion of bioactive IL-33 into culture supernatants, but the size of the secreted protein was not assessed (8). It is thus not clear whether caspase-1 cleavage of pro-IL-33 occurs in mouse cells. In a second study, Western blot analysis revealed the presence of a 32-kDa protein and minor 17 and 20 kDa bands reacting with anti-IL-33 antibodies in the supernatants of THP-1 cells upon caspase-1 activation, suggesting secretion of full-length pro-IL-33 and of two potential cleavage products (9). Although this last observation suggests that some pro-IL-33 may be secreted, it not known to what extent IL-33 secretion is dependent on caspase-1 cleavage. Finally, so far all studies reporting T1/ST2-mediated effects of IL-33 were performed using the recombinant mature form of IL-33, whereas potential biological activity of the full-length precursor form has not been tested. It thus remains to be shown whether, as for IL-1β, caspase-1 cleavage is indeed required for IL-33 bioactivity. In the present study, we thus further investigated caspase-1 cleavage of mouse and human pro-IL-33 in vitro and in cultured cells and assessed the potential bioactivity of the IL-33 precursor.     

Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.