Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells

Aims/hypothesis  

Pro-inflammatory cytokines such as IL-1β, IFN-γ and TNF-α may contribute to pancreatic beta cell destruction in type 1 diabetes. A mechanism requiring nitric oxide, which is generated by inducible nitric oxide synthase (iNOS), in cytokine-induced endoplasmic reticulum (ER) stress and apoptosis has been proposed. Here, we tested the role of nitric oxide in cytokine-induced ER stress and the subsequent unfolded protein response (UPR) in beta cells.     

The role for endoplasmic reticulum stress in diabetes mellitus

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of diabetes, contributing to pancreatic beta-cell loss and insulin resistance. Components of the unfolded protein response (UPR) play a dual role in beta-cells, acting as beneficial regulators under physiological conditions or as triggers of beta-cell dysfunction and apoptosis under situations of chronic stress. Novel findings suggest that "what makes a beta-cell a beta-cell", i.e., its enormous capacity to synthesize and secrete insulin, is also its Achilles heel, rendering it vulnerable to chronic high glucose and fatty acid exposure, agents that contribute to beta-cell failure in type 2 diabetes. In this review, we address the transition from physiology to pathology, namely how and why the physiological UPR evolves to a proapoptotic ER stress response and which defenses are triggered by beta-cells against these challenges. ER stress may also link obesity and insulin resistance in type 2 diabetes. High fat feeding and obesity induce ER stress in liver, which suppresses insulin signaling via c-Jun N-terminal kinase activation. In vitro data suggest that ER stress may also contribute to cytokine-induced beta-cell death. Thus, the cytokines IL-1beta and interferon-gamma, putative mediators of beta-cell loss in type 1 diabetes, induce severe ER stress through, respectively, NO-mediated depletion of ER calcium and inhibition of ER chaperones, thus hampering beta-cell defenses and amplifying the proapoptotic pathways. A better understanding of the pathways regulating ER stress in beta-cells may be instrumental for the design of novel therapies to prevent beta-cell loss in diabetes.     

Cross-talk between the unfolded protein response and nuclear factor-κB signalling pathways regulates cytokine-mediated beta cell death in MIN6 cells and isolated mouse islets

Aims/hypothesis

Pancreatic beta cell destruction in type 1 diabetes may be mediated by cytokines such as IL-1β, IFN-γ and TNF-α. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NFκB) signalling are activated by cytokines, but their significance in beta cells remains unclear. Here, we investigated the role of cytokine-induced ER stress and NFκB signalling in beta cell destruction.

Methods

Isolated mouse islets and MIN6 beta cells were incubated with IL-1β, IFN-γ and TNF-α. The chemical chaperone 4-phenylbutyric acid (PBA) was used to inhibit ER stress. Protein production and gene expression were assessed by western blot and real-time RT-PCR.

Results

We found in beta cells that inhibition of cytokine-induced ER stress with PBA unexpectedly potentiated cell death and NFκB-regulated gene expression. These responses were dependent on NFκB activation and were associated with a prolonged decrease in the inhibitor of κB-α (IκBα) protein, resulting from increased IκBα protein degradation. Cytokine-mediated NFκB-regulated gene expression was also potentiated after pre-induction of ER stress with thapsigargin, but not tunicamycin. Both PBA and thapsigargin treatments led to preferential upregulation of ER degradation genes over ER-resident chaperones as part of the adaptive unfolded protein response (UPR). In contrast, tunicamycin activated a balanced adaptive UPR in association with the maintenance of Xbp1 splicing.

Conclusions/interpretation

These data suggest a novel mechanism by which cytokine-mediated ER stress interacts with NFκB signalling in beta cells, by regulating IκBα degradation. The cross-talk between the UPR and NFκB signalling pathways may be important in the regulation of cytokine-mediated beta cell death.     

Role of endoplasmic reticulum stress in cystic fibrosis-related airway inflammatory responses

Chronic airway infection and inflammation are hallmarks of cystic fibrosis (CF) pulmonary disease. The altered airway environment resulting from infection and inflammation can affect the innate defense of the airway epithelia. Luminal bacterial and inflammatory stimuli trigger an adaptation in human airway epithelia, characterized by a hyperinflammatory response to inflammatory mediators, which is mediated by an expansion of the endoplasmic reticulum (ER) and its Ca(2+) stores. Recent studies demonstrated that a form of ER stress, the unfolded protein response (UPR), is activated in airway epithelia by bacterial infection-induced airway inflammation. UPR-dependent signaling is responsible for the ER Ca(2+) store expansion-mediated amplification of airway inflammatory responses. These studies highlight the functional importance of the UPR in airway inflammation and suggest that targeting the UPR may be a therapeutic strategy for airway diseases typified by chronic inflammation. This article reviews the contribution of airway epithelia to airway inflammatory responses, discusses how expansion of the ER Ca(2+) stores in inflamed airway epithelia contributes to airway inflammation, describes the functional role of the UPR in these processes, and discusses how UPR activation might be relevant for CF airways inflammatory disease.     

Mild endoplasmic reticulum stress augments the proinflammatory effect of IL-1β in pancreatic rat β-cells via the IRE1α/XBP1s pathway

The prevalence of obesity and type 1 diabetes in children is increasing worldwide. Insulin resistance and augmented circulating free fatty acids associated with obesity may cause pancreatic β-cell endoplasmic reticulum (ER) stress. We tested the hypothesis that mild ER stress predisposes β-cells to an exacerbated inflammatory response when exposed to IL-1β or TNF-α, cytokines that contribute to the pathogenesis of type 1 diabetes. INS-1E cells or primary rat β-cells were exposed to a low dose of the ER stressor cyclopiazonic acid (CPA) or free fatty acids, followed by low-dose IL-1β or TNF-α. ER stress signaling was inhibited by small interfering RNA. Cells were evaluated for proinflammatory gene expression by RT-PCR and ELISA, gene reporter activity, p65 activation by immunofluorescence, and apoptosis. CPA pretreatment enhanced IL-1β- induced, but not TNF-α-induced, expression of chemokine (C-C motif) ligand 2, chemokine (C-X-C motif) ligand 1, inducible nitric oxide synthase, and Fas via augmented nuclear factor κB (NF-κB) activation. X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1, but not CCAAT/enhancer binding protein homologous protein, knockdown prevented the CPA-induced exacerbation of NF-κB-dependent genes and decreased IL-1β-induced NF-κB promoter activity. XBP1 modulated NF-κB activity via forkhead box O1 inhibition. In conclusion, rat β-cells facing mild ER stress are sensitized to IL-1β, generating a more intense and protracted inflammatory response through inositol-requiring enzyme 1/XBP1 activation. These observations link β-cell ER stress to the triggering of exacerbated local inflammation.     

Autophagy deficiency in beta cells leads to compromised unfolded protein response and progression from obesity to diabetes in mice

Aims/hypothesis

The unfolded protein response (UPR) in endoplasmic reticulum (ER) and autophagy are known to be related. We investigated the role of autophagy in UPR of pancreatic beta cells and the susceptibility of autophagy-deficient beta cells to the ER stress that is implicated in the development of diabetes.

Methods

Rat insulin promoter (RIP)-Cre ⁺;autophagy-related 7 (Atg7)^F/W mice were bred with ob/w mice to derive RIP-Cre ⁺;Atg7 ^F/F-ob/ob mice and to induce ER stress in vivo. GFP-LC3 ⁺-ob/ob mice were generated to examine in vivo autophagic activity. Real-time RT-PCR was performed to study the expression of the genes of the UPR machinery. Proteolysis was assessed by determining release of incorporated radioactive leucine.

Results

Production of UPR machinery was reduced in autophagy-deficient beta cells, which was associated with diminished production of p85?? and p85?? regulatory subunits of phosphoinositide 3-kinase. Because of compromised UPR machinery, autophagy-deficient beta cells were susceptible to ER stressors in vitro. When mice with beta cell-specific autophagy deficiency, which have mild hyperglycaemia, were bred with ob/ob mice to induce ER stress in vivo, severe diabetes developed, which was accompanied by an increase in beta cell death and accumulation of reactive oxygen species. The increased demand for UPR present in obesity was unmet in autophagy-deficient beta cells. Autophagy level and autophagic activity were enhanced by lipid, while proteolysis was reduced.

Conclusions/interpretation

These results suggest that autophagy is important for intact UPR machinery and appropriate UPR in response to lipid injury that increases demand for UPR. Autophagy deficiency in pancreatic beta cells may contribute to the progression from obesity to diabetes.     

Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes

Aims/hypothesis

Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.

Methods

Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.

Results

Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate.

Conclusions/interpretation

Saturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.     

Stress hypERactivation in the β-cell

In pancreatic β-cells, the endoplasmic reticulum (ER) is the crucial site for insulin biosynthesis, as this is where the protein-folding machinery for secretory proteins is localized. Perturbations to ER function of the β-cell, such as a high demand for insulin secretion, can lead to an imbalance in protein homeostasis and lead to ER stress. This stress can be mitigated by an adaptive, cellular response, the unfolded protein response (UPR). UPR activation is vital to the survival of β-cells, as these cells represent one of the most susceptible tissues for ER stress, due to their highly secretory function. However, in some cases, this response is not sufficient to relieve stress, leading to apoptosis and contributing to the pathogenesis of diabetes. Recent evidence shows that ER stress plays a significant role in both type 1 and type 2 diabetes. In this review, we outline the mechanisms of ER stress-mediated β-cell death and focus on the role of ER stress in various forms of diabetes, particularly a genetic form of diabetes called Wolfram syndrome.     

Curative and beta cell regenerative effects of alpha1-antitrypsin treatment in autoimmune diabetic NOD mice

Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing beta cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to beta cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional beta cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with alpha1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to beta cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of beta cells expands in AAT-treated diabetic NOD mice.     

Pancreatic adenocarcinoma patients with localised chronic severe pancreatitis show an increased number of single beta cells, without alterations in fractional insulin area

Aims/hypothesis  Recent histological analysis of pancreases obtained from patients with long-standing type 1 diabetes identified chronic islet inflammation and limited evidence suggestive of beta cell replication. Studies in rodent models also suggest that beta cell replication can be induced by certain inflammatory cytokines and by gastrin. We therefore tested the hypothesis that beta cell replication is observed in non-autoimmune human pancreatic disorders in which localised inflammation or elevated gastrin levels are present. Methods  Resected operative pancreatic specimens were obtained from patients diagnosed with primary adenocarcinoma (with or without chronic severe pancreatitis) or gastrinoma. Additional pancreatic tissue was obtained from autopsy control patients. Immunohistochemistry was used to assess fractional insulin area, beta cell number and replication rate and differentiation factors relevant to beta cell development. Results  Fractional insulin area was similar among groups. Patients with pancreatic adenocarcinoma and localised chronic severe pancreatitis displayed significant increases in the number of single beta cells, as well as increased beta cell replication rate and levels of neurogenic differentiation 1 in islets. Patients with gastrinoma demonstrated significant increases in the number of single beta cells, but the beta cell replication rate and islet differentiation factor levels were similar to those in the control group. Conclusions/interpretation  These findings indicate that chronic severe pancreatic inflammation can be associated with significant effects on beta cell number or replication rate, depending on the distribution of the cells. This information may prove useful for attempts seeking to design therapies aimed at inducing beta cell replication as a means of reversing diabetes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Alcohol abuse, endoplasmic reticulum stress and pancreatitis

Alcohol abuse is a common cause of both acute and chronic pancreatitis. There is a wide spectrum of pancreatic manifestations in heavy drinkers from no apparent disease in most individuals to acute inflammatory and necrotizing pancreatitis in a minority of individuals with some progressing to chronic pancreatitis characterized by replacement of the gland by fibrosis and chronic inflammation. Both smoking and African-American ethnicity are associated with increased risk of alcoholic pancreatitis. In this review we describe how our recent studies demonstrate that ethanol feeding in rodents causes oxidative stress in the endoplasmic reticulum (ER) of the digestive enzyme synthesizing acinar cell of the exocrine pancreas. This ER stress is attenuated by a robust unfolded protein response (UPR) involving X-box binding protein-1 (XBP1) in the acinar cell. When the UPR activation is prevented by genetic reduction in XBP1, ethanol feeding causes significant pathological responses in the pancreas. These results suggest that the reason most individuals who drink alcohol heavily do not get significant pancreatic disease is because the pancreas mounts an adaptive UPR to attenuate the ER stress that ethanol causes. We hypothesize that disease in the pancreas results when the UPR is insufficiently robust to alleviate the ER stress caused by alcohol abuse.     

The anti‐interleukin‐1 in type 1 diabetes action trial—background and rationale

Type 1 diabetes (T1D) is caused by an inflammatory destruction of pancreatic beta‐cells. Pro‐inflammatory cytokines, in particular interleukin‐1 (IL‐1), have been suggested to be effector molecules based on the observations that pro‐inflammatory cytokines cause beta‐cell apoptosis in vitro and aggravate diabetes in vivo, and that inhibition of the action of these cytokines reduce diabetes incidence in animal models of type 1 diabetes and islet graft destruction. This review presents the rationale for and design of a recently launched double‐blind, multicenter, randomized clinical trial that investigates the effect of interleukin‐1 antagonism on beta‐cell function in subjects with T1D of recent‐onset. Copyright © 2009 John Wiley & Sons, Ltd.