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.     

Chaperones Ameliorate Beta Cell Dysfunction Associated with Human Islet Amyloid Polypeptide Overexpression

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.     

Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.     

The effect of Aβ on IAPP aggregation in the presence of an isolated β-cell membrane

Fibrillar aggregates of the islet amyloid polypeptide (IAPP) and amyloid-β (Aβ) are known to deposit at pancreatic β-cells and neuronal cells and are associated with the cell degenerative diseases type-2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Since IAPP is secreted by β-cells and a membrane-damaging effect of IAPP has been discussed as a reason for β-cell dysfunction and the development of T2DM, studies of the interaction of IAPP with the β-cell membrane are of high relevance for gaining a molecular-level understanding of the underlying mechanism. Recently, it has also been shown that patients suffering from T2DM exhibit an increased risk to develop AD and vice versa, and a molecular link between AD and T2DM has been suggested. In this study, membrane lipids from the rat insulinoma-derived INS-1E β-cell line were isolated, and their interaction with the amyloidogenic peptides IAPP and Aβ and a mixture of both peptides has been studied. To yield insight into the associated peptides' conformational changes and their effect on the membrane integrity during aggregation, we have carried out attenuated total reflection Fourier transform infrared spectroscopy, fluorescence microscopy, and atomic force microscopy experiments. The IAPP-Aβ heterocomplexes formed were shown to adsorb, aggregate, and permeabilize the isolated β-cell membrane significantly slower than pure IAPP, however, at a rate that is much faster than that of pure Aβ. In addition, it could be shown that isolated β-cell membranes cause similar effects on the kinetics of IAPP and IAPP-Aβ fibril formation as anionic heterogeneous model membranes.     

Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle

It has been reported that asprosin is a novel adipokine which is augmented in mice and humans with type 2 diabetes (T2DM). Asprosin stimulates hepatic gluconeogenesis under fasting conditions. However, the roles of asprosin in inflammation, endoplasmic reticulum (ER) stress, and insulin resistance in skeletal muscle has not been studied. In the currents study, elevated levels of asprosin expression were observed in adipocytes under hyperlipidemic conditions. Treatment of C2C12 myocytes with asprosin-induced ER stress markers (phosphorylated inositol-requiring enzyme 1 and eukaryotic initiation factor 2, and CHOP expression) as well as inflammation markers (interleukin-6 expression, phosphorylated IκB, and nuclear translocated nuclear factor-κβ). Finally, asprosin treatment promoted exacerbation of insulin sensitivity as determined by levels of insulin receptor substrate 1 and Akt phosphorylation as well as glucose uptake. Moreover, treatment of asprosin augmented protein kinase C-δ (PKCδ) phosphorylation and nuclear translocation, but suppressed messenger RNA expression of sarcoplasmic reticulum Ca²⁺ ATPase 2b in both C2C12 myocytes and in mouse soleus skeletal muscle. These asprosin-induced effects were markedly decreased in small interfering (si) RNA-mediated PKCδ-knockdown in C2C12 myocytes. These results suggest that asprosin results in impairment of insulin sensitivity in skeletal muscle through PKCδ-associated ER stress/inflammation pathways and may be a valuable strategy for management of insulin resistance and T2DM.     

Fluorescence microscopy studies on islet amyloid polypeptide fibrillation at heterogeneous and cellular membrane interfaces and its inhibition by resveratrol

Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic β-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.     

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.     

Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact

The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.     

Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.     

Inhibition of PTEN Attenuates Endoplasmic Reticulum Stress and Apoptosis via Activation of PI3K/AKT Pathway in Alzheimer’s Disease

Amyloid plaques and neurofibrillary tangles are pathologic hallmarks of Alzheimer’s disease (AD). Endoplasmic reticulum (ER) stress has been implicated in the loss of neurons in AD. The phosphatase and tensin homolog deleted on chromosome ten (PTEN) plays an important role in regulating neuronal survival processes. However, the direct effects of the PTEN on ER stress and apoptosis in AD have not been elucidated. In this study, we demonstrate that the expression of PTEN and ER stress related proteins, GRP78 and CHOP, increased in APP/PS1 transgenic AD mice compared with WT mice. A PTEN inhibitor, dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate (bpv) could decrease apoptosis, induce AKT phosphorylation and inhibit the ER stress response proteins in hippocampus in APP/PS1 transgenic AD model mice. Furthermore, treatment with the specific PI3K inhibitor, LY294002, significantly blocked the anti-apoptotic effects of bpv in AD mice. The expression in GRP78, CHOP and apoptosis levels by bpv was reversed after PI3K inhibitor treatment. Taken together, our results indicate that the neuroprotective role of bpv involves the suppression of ER stress via the activation of the PI3K/AKT signalling pathways in APP/PS1 transgenic AD model mice.     

D4F alleviates the C/EBP homologous protein-mediated apoptosis in glycated high-density lipoprotein-treated macrophages by facilitating autophagy

The present study aimed to investigate the role of D4F, an apolipoprotein A-I mimetic peptide, in macrophage apoptosis induced by the glycated high-density lipoprotein (gly-HDL)-induced endoplasmic reticulum (ER) stress C/EBP homologous protein (CHOP) pathway, and unravel the regulatory role of autophagy in this process. Our results revealed that except for suppressing the accumulation of lipids within RAW264.7 macrophages caused by gly-HDL, D4F inhibited gly-HDL-induced decrease in the cell viability and increase in lactate dehydrogenase leakage and cell apoptosis, which were similar to 4-phenylbutyric acid (PBA, an ER stress inhibitor). Besides, similar to PBA, D4F inhibited gly-HDL-induced ER stress response activation evaluated through the decreased PERK and eIF2α phosphorylation, together with reduced ATF6 nuclear translocation as well as the downregulation of GRP78 and CHOP. Interestingly, D4F facilitated gly-HDL-triggered activation of autophagy, measured as elevated levels of beclin-1, LC3-II, and ATG5 expressions in macrophages. Furthermore, the inhibition effect of D4F on gly-HDL-induced ER stress-CHOP-induced apoptosis of macrophages was restrained after beclin-1 siRNA and 3-methyladenine (3-MA, an inhibitor of autophagy) treatments, while this effect was further reinforced after rapamycin (Rapa, an inducer of autophagy) treatment. Furthermore, administering D4F or Rapa to T2DM mice upregulated LC3-II and attenuated CHOP expression, cell apoptosis, and atherosclerotic lesions. However, the opposite results were obtained when 3-MA was administered to these mice. These results support that D4F effectively protects macrophages against gly-HDL-induced ER stress-CHOP-mediated apoptosis by promoting autophagy.     

Identification of 1,2,3-triazole derivatives that protect pancreatic β cells against endoplasmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression

The C/EBP-homologous protein (CHOP) acts as a mediator of endoplasmic reticulum (ER) stress-induced pancreatic insulin-producing β cell death, a key element in the pathogenesis of diabetes. Chemicals that inhibit the expression of CHOP might therefore protect β cells from ER stress-induced apoptosis and prevent or ameliorate diabetes. Here, we used high-throughput screening to identify a series of 1,2,3-triazole amide derivatives that inhibit ER stress-induced CHOP-luciferase reporter activity. Our SAR studies indicate that compounds with an N,1-diphenyl-5-methyl-1H-1,2,3-triazole-4-carboxamide backbone potently protect β cell against ER stress. Several representative compounds inhibit ER stress-induced up-regulation of CHOP mRNA and protein, without affecting the basal level of CHOP expression. We further show that a 1,2,3-triazole derivative 4e protects β cell function and survival against ER stress in a CHOP-dependent fashion, as it is inactive in CHOP-deficient β cells. Finally, we show that 4e significantly lowers blood glucose levels and increases concomitant β cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of small molecule inhibitors of CHOP expression that prevent ER stress-induced β cell dysfunction and death may provide a new modality for the treatment of diabetes.     

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERβ and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERβ and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERβ antibodies are insensitive for ERβ and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERβ antibody is shown to be highly specific and sensitive for detection of full-length ERβ and its variant forms. Strong and variable staining patterns for endogenous levels of ERβ protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERβ was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERβ antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERβ in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERβ, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.