Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus

Diabetes is caused by impaired insulin secretion in pancreatic beta-cells and peripheral insulin resistance. Overload of pancreatic beta-cells leads to beta-cell exhaustion and finally to the development of diabetes. Reduced beta-cell mass is evident in type 2 diabetes, and apoptosis is implicated in this process. One characteristic feature of beta-cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important functions, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various conditions can interfere with ER function and these conditions are called ER stress. Recently, we found that nitric oxide (NO)-induced apoptosis in beta-cells is mediated by the ER-stress pathway. NO causes ER stress and leads to apoptosis through induction of ER stress-associated apoptosis factor CHOP. The Akita mouse with a missense mutation (Cys96Tyr) in the insulin 2 gene has hyperglycemia and a reduced beta-cell mass. This mutation disrupts a disulfide bond between A and B chains of insulin and may induce its conformational change. In the development of diabetes in Akita mice, mRNAs for an ER chaperone Bip and CHOP were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 beta-cells induced CHOP expression and led to apoptosis. Targeted disruption of the CHOP gene did not delay the onset of diabetes in the homozygous Akita mice, but it protected islet cells from apoptosis and delayed the onset of diabetes in the heterozygous Akita mice. We conclude that ER overload in beta-cells causes ER stress and leads to apoptosis via CHOP induction. These results highlight the importance of chronic ER stress in beta-cell apoptosis in type 2 diabetes, and suggest a new target to the management of the disease.     

Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide

The islet in type 2 diabetes is characterized by an approximately 60% beta-cell deficit, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) but not rodent IAPP (rIAPP) forms toxic oligomers and amyloid fibrils in an aqueous environment. We previously reported that overexpression of hIAPP in transgenic rats triggered endoplasmic reticulum (ER) stress-induced apoptosis in beta-cells. In the present study, we sought to establish whether the cytotoxic effects of hIAPP depend on its propensity to oligomerize, rather than as a consequence of protein overexpression. To accomplish this, we established a novel homozygous mouse model overexpressing rIAPP at a comparable expression rate and, on the same background, as a homozygous transgenic hIAPP mouse model previously reported to develop diabetes associated with beta-cell loss. We report that by 10 wk of age hIAPP mice develop diabetes with a deficit in beta-cell mass due to increased beta-cell apoptosis. The rIAPP transgenic mice counterparts do not develop diabetes or have decreased beta-cell mass. Both rIAPP and hIAPP transgenic mice have increased expression of BiP, but only hIAPP transgenic mice have elevated ER stress markers (X-box-binding protein-1, nuclear localized CCAAT/enhancer binding-protein homologous protein, active caspase-12, and accumulation of ubiquitinated proteins). These findings indicate that the beta-cell toxic effects of hIAPP depend on the propensity of IAPP to aggregate, but not on the consequence of protein overexpression.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.     

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic beta-cells

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.     

Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells

Regulation of insulin receptor substrate (IRS)-2 expression is critical to beta-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic beta-cells (INS-1), chronic exposure (>8 h) to 15 mm glucose and/or 5 nm IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTORDelta) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviral-mediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected beta-cells expressing mTORDelta, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased beta-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in beta-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased beta-cell apoptosis. This may be a contributing mechanism as to how beta-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.     

WFS1 is a novel component of the unfolded protein response and maintains homeostasis of the endoplasmic reticulum in pancreatic beta-cells

In Wolfram syndrome, a rare form of juvenile diabetes, pancreatic beta-cell death is not accompanied by an autoimmune response. Although it has been reported that mutations in the WFS1 gene are responsible for the development of this syndrome, the precise molecular mechanisms underlying beta-cell death caused by the WFS1 mutations remain unknown. Here we report that WFS1 is a novel component of the unfolded protein response and has an important function in maintaining homeostasis of the endoplasmic reticulum (ER) in pancreatic beta-cells. WFS1 encodes a transmembrane glyco-protein in the ER. WFS1 mRNA and protein are induced by ER stress. The expression of WFS1 is regulated by inositol requiring 1 and PKR-like ER kinase, central regulators of the unfolded protein response. WFS1 is normally up-regulated during insulin secretion, whereas inactivation of WFS1 in beta-cells causes ER stress and beta-cell dysfunction. These results indicate that the pathogenesis of Wolfram syndrome involves chronic ER stress in pancreatic beta-cells caused by the loss of function of WFS1.