Liling Zhang

GRP78, but Not Protein-disulfide Isomerase, Partially Reverses Hyperglycemia-induced Inhibition of Insulin Synthesis and Secretion in Pancreatic β-Cells

Chronic hyperglycemia contributes to pancreatic β-cell dysfunction during the development of type 2 diabetes. Treatment of pancreatic β-cells with prolonged high glucose concentrations has been shown to reduce insulin promoter activity and insulin gene expression. Here, we examined the effect of high glucose on endoplasmic reticulum (ER) stress pathway activation and insulin production in INS-1 832/13 pancreatic β-cells. Treatment of cells with 25 mm glucose for 24-48 h decreased insulin mRNA and protein levels and reduced the proinsulin translation rate, which was accompanied by enhanced unfolded protein response pathway activation (XBP-1 mRNA splicing and increased phospho-eIF2α, CHOP, and active ATF6 levels). Overexpressing the ER chaperone GRP78 partially rescued high glucose-induced suppression of proinsulin levels and improved glucose-stimulated insulin secretion with no effect on insulin 2 mRNA levels. Under these conditions, there was little effect of GRP78 overexpression on ER stress markers. Knockdown of GRP78 expression under basal glucose conditions reduced cellular insulin levels and glucose-stimulated insulin secretion. Thus, GRP78 is essential for insulin biosynthesis, and enhancing chaperone capacity can improve β-cell function in the presence of prolonged hyperglycemia. In contrast, overexpression of the ER chaperone and oxidoreductase protein-disulfide isomerase (PDI) reduced glucose-stimulated insulin secretion and induced ER stress resulting from the accumulation of proinsulin in the ER. These results suggest a role for both GRP78 and PDI in insulin biosynthesis, although an excess of PDI disrupts normal proinsulin processing.

Oleanolic acid enhances insulin secretion in pancreatic β‐cells

We investigated the effect of oleanolic acid, a plant‐derived triterpenoid, on insulin secretion and content in pancreatic β‐cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS‐1 832/13 cells and enhanced acute glucose‐stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin‐4 under glucose‐stimulated conditions, yet neither Ca2+ nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti‐diabetic properties of this natural product.

Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

BackgroundCells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response.ResultsExpression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival.ConclusionsThe inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in β-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.

OASIS/CREB3L1 Induces Expression of Genes Involved in Extracellular Matrix Production But Not Classical Endoplasmic Reticulum Stress Response Genes in Pancreatic β-Cells

Old astrocyte specifically induced substance (OASIS) has previously been shown to be a putative endoplasmic reticulum (ER) stress sensor in astrocytes with a mechanism of activation that is similar to ATF6. In this study we investigated the expression and activation of endogenous and overexpressed OASIS in pancreatic beta-cells. OASIS mRNA expression was detected in pancreatic beta-cell lines and rodent islets, and the expression level was up-regulated by ER stress-inducing compounds. Endogenous OASIS protein, however, is expressed at low levels in pancreatic beta-cell lines and rodent islets, possibly due to abundant levels of the micro-RNA miR-140 present in these cells. In contrast, expression of both full-length and cleaved (active) OASIS was readily detectable in the developing mouse pancreas (embryonic d 15.5). Microarray analysis after expression of an active nuclear-localized version of OASIS in an inducible INS-1 beta-cell line resulted in the up-regulation of many genes implicated in extracellular matrix production and protein transport but not classical ER stress response genes. Consistent with this, expression of active OASIS failed to induce glucose-regulated protein 78 kDa promoter activity in pancreatic beta-cells. These results suggest that the repertoire of genes induced by OASIS is cell type-dependent and that the OASIS protein may have a role in pancreas development.

OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87) and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

SDF2L1 interacts with the ER-associated degradation machinery and retards the degradation of mutant proinsulin in pancreatic β-cells.

Summary Stromal cell-derived factor 2-like 1 (SDF2L1) is an endoplasmic reticulum (ER)-localized protein whose function is undefined. Here we show that SDF2L1 protein levels are increased in response to ER stress-inducing compounds, but not other cell stressors that we tested in insulinoma cell lines. SDF2L1 protein levels were also induced by expression of misfolded proinsulin in insulinoma cells and in islets from diabetic mice. Immunoprecipitation and binding assays demonstrated that SDF2L1 interacts with the ER chaperone GRP78/BiP, the ER-associated degradation (ERAD) machinery and with misfolded proinsulin. Unexpectedly, knockdown of SDF2L1 in INS-1 (insulin 2 C96Y-GFP) cells increased the degradation kinetics of mutant proinsulin, suggesting that SDF2L1 regulates substrate availability for the ERAD system. We suggest that SDF2L1 increases the time that misfolded proteins have to achieve a correctly folded conformation and thus that SDF2L1 can act as a buffer for substrate availability for ERAD in pancreatic &bgr;-cells.

p24 family type 1 transmembrane proteins are required for insulin biosynthesis and secretion in pancreatic β-cells

The p24 protein family have multiple functions in protein transport in the early secretory pathway. In this study we examined the role of p24 proteins in insulin transport. Several members were detected in insulinoma cell lines and rat islets and expression levels positively correlated with insulin abundance, particularly for p24δ1 and p24β1. Knocking down p24δ1 in insulinoma cell lines, which also resulted in the concomitant knock‐down of other family members, impaired glucose‐stimulated insulin secretion, decreased total cellular insulin content and reduced proinsulin biosynthesis. There was no effect on overall protein biosynthesis or ER stress. These results suggest that p24δ1 and possibly other p24 family proteins are required for normal insulin biosynthesis and subsequent secretion in pancreatic β‐cells.

IRE1 inhibition perturbs the unfolded protein response in a pancreatic β-cell line expressing mutant proinsulin, but does not sensitize the cells to apoptosis

BackgroundThe Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic β-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line.ResultsThe IRE1 endoribonuclease inhibitors 4μ8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1.ConclusionsAlthough maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic β-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis.

ATF6β regulates the Wfs1 gene and has a cell survival role in the ER stress response in pancreatic β-cells.

Endoplasmic reticulum (ER) stress is implicated in pancreatic β-cell dysfunction and death resulting in type 2 diabetes. Activating transcription factor 6 (ATF6) is an essential component of the Unfolded Protein Response (UPR) and consists of two isoforms, ATF6α and ATF6β. Here we investigated the role of ATF6β. ATF6β mRNA was detected in pancreatic β-cell lines and rodent and human islets. We also detected ATF6β protein and production of the active form (ATF6βp60) in response to ER stress. Knock-down of ATF6β in INS-1 832/13 insulinoma cells did not affect mRNA induction of several major UPR genes in response to ER stress, suggesting ATF6β is not essential for the basic UPR. Expressing active ATF6βp60 or ATF6αp50 followed by microarray analysis showed that they regulate similar UPR genes, although some genes such as Wfs1 are ATF6β-specific. ATF6β, but not ATF6α, is able to bind the Wfs1 promoter and induce Wfs1 gene and protein expression. Knock-down of ATF6β increased the susceptibility of β-cells to ER stress-induced apoptosis, while overexpression of active ATF6βp60 reduced apoptosis. Thus, ATF6β is not essential for induction of most UPR genes, but is required to maintain cell survival in β-cells undergoing chronic ER stress, which in part relates to its ability to induce Wfs1, a pro-survival gene.

Endoplasmic reticulum redox state is not perturbed by pharmacological or pathological endoplasmic reticulum stress in live pancreatic β-cells.

Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER) causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR) in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor) and mCherry protein driven by a GRP78 promoter (UPR-sensor). Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.