Allen Volchuk

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.

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.

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

Activating transcription factor 6 (ATF6) is one of three principle endoplasmic reticulum (ER) stress response proteins and becomes activated when ER homeostasis is perturbed. ATF6 functions to increase ER capacity by stimulating transcription of ER-resident chaperone genes such as GRP78. Using an antibody that recognizes active ATF6α-p50, we found that active ATF6α was detected in insulinoma cells and rodent islets even under basal conditions and the levels were further increased by ER stress. To examine the function of ATF6α-p50, we depleted endogenous ATF6α-p50 levels using small interfering RNA in insulinoma cells. Knockdown of endogenous ATF6α-p50 levels by ∼60% resulted in a reduction in the steady-state levels of GRP78 mRNA and protein levels in nonstressed cells. Furthermore, ATF6α knockdown resulted in an apoptotic phenotype. We hypothesized that removal of the ATF6α branch of the unfolded protein response (UPR) would result in ER stress. However, neither the PKR-like endoplasmic reticulum kinase (PERK), nor the inositol requiring enzyme 1 (IRE1) pathways of the UPR were significantly activated in ATF6α knockdown cells, although these cells were more sensitive to ER stress-inducing compounds. Interestingly, phosphorylation of JNK, p38, and c-Jun were elevated in ATF6α knockdown cells and inhibition of JNK or p38 kinases prevented apoptosis. These results suggest that ATF6α may have a role in maintaining β-cell survival even in the absence of ER stress.

Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression.

Pancreatic β-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6β, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.