Jeng Yie Chan

Cytokine-induced β-cell death is independent of endoplasmic reticulum stress signaling

OBJECTIVE—Cytokines contribute to β-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress–mediated apoptosis has been proposed as a mechanism for β-cell death. We tested whether ER stress was necessary for cytokine-induced β-cell death and also whether ER stress gene activation was present in β-cells of the NOD mouse model of type 1 diabetes. RESEARCH DESIGN AND METHODS—INS-1 β-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin (IL)-1β and γ-interferon (IFN-γ). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression in INS-1 β-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed. RESULTS—Cytokines and palmitate triggered ER stress in β-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK), eukaryotic initiation factor (EIF)2α, and Jun NH2-terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA levels of several ER stress genes were reduced (ATF4, BiP [binding protein], GRP94 [glucose regulated protein 94], p58, and XBP-1 [X-box binding protein 1] splicing) or unchanged (CHOP and Edem1 [ER degradation enhancer, mannosidase α–like 1]). CONCLUSIONS—While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP ER stress–signaling pathway is not necessary for cytokine-induced β-cell death.

TNF-related apoptosis-inducing ligand (TRAIL) protects against diabetes and atherosclerosis in Apoe / mice

Aims/hypothesisTNF-related apoptosis-inducing ligand (TRAIL) is implicated in the regulation of diabetes and is reduced in patients with cardiovascular disease. Although TRAIL receptors are widespread, and TRAIL can promote cell proliferation and apoptosis, it is not known how TRAIL might protect against diabetes and atherosclerosis.MethodsWe examined the development of atherosclerosis and diabetes in Apoe−/−, Trail (also known as Tnfsf10)−/−Apoe−/− and Trail−/− mice that were fed a high-fat diet. Plasma cholesterol, triacylglycerol, glucose and insulin, as well as changes in various metabolic enzymes and regulators were assessed. Glucose and insulin tolerance tests were performed. Pancreatic islets were examined for insulin and beta cell dysfunction (apoptosis and macrophage infiltration).ResultsCompared with Apoe−/− mice, Trail−/−Apoe−/− and Trail−/− mice exhibited several features of diabetes, including increased weight, hyperglycaemia, reduced plasma insulin, impaired glucose tolerance, beta cell dysfunction, reduced islet insulin, macrophage infiltration and increased apoptosis. Trail−/−Apoe−/− mice had increased plasma cholesterol, triacylglycerol, and VLDL- and LDL-cholesterol, and increased expression of genes involved in cholesterol synthesis and lipogenesis. Trail−/−Apoe−/− mice also had increased atherosclerosis, with several features of plaque instability.Conclusions/interpretationWe show for the first time that TRAIL deficiency promotes numerous features of diabetes that are typical of human disease, and are associated with reduced insulin and pancreatic inflammation/apoptosis. TRAIL also regulates cholesterol and triacylglycerol homeostasis in Apoe−/− mice by increasing the expression of genes involved in (1) cholesterol synthesis and absorption, and (2) triacylglycerol production.

Failure of the Adaptive Unfolded Protein Response in Islets of Obese Mice Is Linked With Abnormalities in β-Cell Gene Expression and Progression to Diabetes

The normal β-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with β-cells that are susceptible to failure. Here, we investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for β-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several β-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with β-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of β-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.

The balance between adaptive and apoptotic unfolded protein responses regulates β-cell death under ER stress conditions through XBP1, CHOP and JNK

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been implicated in β-cell death in type 1 and type 2 diabetes. However, the UPR is also a fundamental mechanism required for β-cell adaptation and survival. The mechanisms regulating the transition from adaptive to apoptotic UPR remain to be clarified. Here, we investigated the relationships between XBP1, CHOP and JNK in the transition from adaptive to apoptotic UPR and β-cell death in models of type 1 and type 2 diabetes. XBP1 inhibition potentiated cell death induced by pro-inflammatory cytokines or the saturated fatty acid palmitate in MIN6 β-cells. This response was prevented by CHOP inhibition. IRE1/XBP1 inhibition led to alterations in islets from diabetes-resistant ob/ob mice that resemble those found in diabetes, including increases in cell death and inflammation and antioxidant gene expression. Similarly, IRE1/XBP1 inhibition increased cell death in islets from NOD mice. On the other hand, JNK inhibition: 1) increased adaptive UPR and reduced cell death in islets from diabetic db/db mice, and 2) restored adaptive UPR while protecting against apoptotic UPR gene expression and β-cell death and dysfunction following cytokine exposure. These findings suggest that the balance between XBP1-mediated adaptive and CHOP-dependent apoptotic UPR is critically important for β-cell survival during ER stress. JNK activation regulates the transition from adaptive to apoptotic UPR, thus providing a mechanism for β-cell propensity to cell death rather than ER stress adaptation in type 1 and type 2 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/hypothesisPancreatic 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.MethodsIsolated 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.ResultsWe 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/interpretationThese 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.

Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant–mitochondrial response in mouse beta cells

Aims/hypothesisOxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress.MethodsMIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1−/−, Id3−/− and diabetic db/db mice were used.ResultsID1–4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition.Conclusions/interpretationOur study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant–mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2–small MAF pathway.

Influence of chronic hyperglycemia on the loss of the unfolded protein response in transplanted islets

Chronic hyperglycemia contributes to β-cell dysfunction in diabetes and with islet transplantation, but the mechanisms remain unclear. Recent studies demonstrate that the unfolded protein response (UPR) is critical for β-cell function. Here, we assessed the influence of hyperglycemia on UPR gene expression in transplanted islets. Streptozotocin-induced diabetic or control nondiabetic mice were transplanted under the kidney capsule with syngeneic islets either sufficient or not to normalize hyperglycemia. Twenty-one days after transplantation, islet grafts were excised and RT-PCR was used to assess gene expression. In islet grafts from diabetic mice, expression levels of many UPR genes of the IRE1/ATF6 pathways, which are important for adaptation to endoplasmic reticulum stress, were markedly reduced compared with that in islet grafts from control mice. UPR genes of the PERK pathway were also downregulated. The normalization of glycemia restored the changes in mRNA expression, suggesting that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression. Similar correlations were observed between blood glucose and mRNA levels of transcription factors involved in the maintenance of β-cell phenotype and genes implicated in β-cell function, suggesting convergent regulation of UPR gene expression and β-cell differentiation by hyperglycemia. However, the normalization of glycemia was not accompanied by restoration of antioxidant or pro-inflammatory cytokine mRNA levels, which were increased in islet grafts from diabetic mice. These studies demonstrate that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression in transplanted mouse islets. Failure of the adaptive UPR may contribute to β-cell dedifferentiation and dysfunction in diabetes.

On-target action of anti-tropomyosin drugs regulates glucose metabolism

The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 β-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1’s function with few off-target effects.