Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.
ABSTRACT: Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1?, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.
Project description:We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Müller glial cells.Cultured human retinal Müller cells (MIO-M1) were treated with highly oxidized glycated LDL (HOG-LDL, 200 mg protein/L) or native LDL (N-LDL, 200 mg protein/L) for up to 24 hours with or without pretreatment with N-acetyl-cysteine (NAC, a blocker of oxidative stress) and 4-phenylbutyrate (4-PBA, a blocker of endoplasmic reticulum [ER] stress). Effects of HOG-LDL on cell viability, apoptosis, oxidative stress, and ER stress were assessed by cell viability, TUNEL, and Western blot assays. In separate experiments, Müller cells were treated with 7-ketocholesterol (7-KC, 5-20 ?M) or 4-hydroxynonenal (4-HNE, 5-40 ?M) for up to 24 hours. The same markers were measured.HOG-LDL induced apoptosis (decreased cell viability, increased TUNEL staining, increased expression of cleaved PARP, cleaved caspase-3, and BAX; decreased Bcl-2), oxidative stress (increased NOX4 and antioxidant enzymes, catalase, and superoxide dismutase 2), and ER stress (increased phospho-eIF2?, KDEL, ATF6, and CHOP). Pretreatment with NAC or 4-PBA partially attenuated apoptosis. In addition. NAC attenuated activation of ER stress. Similar to HOG-LDL, 7KC, and 4HNE also induced apoptosis, oxidative stress, and ER stress.Our data suggest that extravasated, modified lipoproteins may be implicated in apoptotic Müller cell death, acting at least partially via enhanced levels of oxidative and ER stresses. They support our main hypothesis that, in addition to hyperglycemia, extravasated and oxidized LDL is an important insult to the diabetic retina.
Project description:This study was designed to explore the protective effect of D4F, an apoA-I mimetic peptide, on oxidized LDL (ox-LDL)-induced endoplasmic reticulum (ER) stress-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) pathway-mediated apoptosis in macrophages. Our results showed that treating apoE knockout mice with D4F decreased the serum ox-LDL level and apoptosis in atherosclerotic lesions with concomitant downregulation of cluster of differentiation 36 (CD36) and inhibition of ER stress. In vitro, D4F inhibited macrophage-derived foam cell formation. Furthermore, like ER stress inhibitor 4-phenylbutyric acid (PBA), D4F inhibited ox-LDL- or tunicamycin (TM, an ER stress inducer)-induced reduction in cell viability and increase in lactate dehydrogenase leakage, caspase-3 activation, and apoptosis. Additionally, like PBA, D4F inhibited ox-LDL- or TM-induced activation of ER stress response as assessed by the reduced nuclear translocation of activating transcription factor 6 and the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2?, as well as the downregulation of glucose-regulated protein 78 and CHOP. Moreover, D4F mitigated ox-LDL uptake by macrophages and CD36 upregulation induced by ox-LDL or TM. These data indicate that D4F can alleviate the formation and apoptosis of macrophage-derived foam cells by suppressing CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway.
Project description:Oxidized HDL (ox-HDL), unlike native HDL that exerts antiatherogenic effects, plays a proatherogenic role. However, the underlying mechanisms are not completely understood. This study was designed to explore the inductive effect of ox-HDL on endoplasmic reticulum (ER) stress-CCAAT-enhancer-binding protein homologous protein (CHOP)-mediated macrophage apoptosis and its upstream mechanisms. Our results showed that ox-HDL could be ingested by macrophages, causing intracellular lipid accumulation. As with tunicamycin (an ER stress inducer), ox-HDL induced macrophage apoptosis with concomitant activation of ER stress pathway, including nuclear translocation of activating transcription factor 6, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2?, and upregulation of glucose-regulated protein 78 and CHOP, which were inhibited by 4-phenylbutyric acid (PBA, an ER stress inhibitor) and CHOP gene silencing. Additionally, diphenyleneiodonium (DPI, an oxidative stress inhibitor), probucol (a reactive oxygen species scavenger), and toll-like receptor 4 (TLR4) silencing reduced ox-HDL-induced macrophage apoptosis, oxidative stress, and CHOP upregulation. Moreover, HDL isolated from patients with metabolic syndrome induced macrophage apoptosis, oxidative stress, and CHOP upregulation, which were blocked by PBA and DPI. These data indicate that ox-HDL may activate ER stress-CHOP-induced apoptotic pathway in macrophages via enhanced oxidative stress and that this pathway may be mediated by TLR4.
Project description:The progression from insulin resistance to type 2 diabetes is caused by the failure of pancreatic beta cells to produce sufficient levels of insulin to meet the metabolic demand. Recent studies indicate that nutrient fluctuations and insulin resistance increase proinsulin synthesis in beta cells beyond the capacity for folding of nascent polypeptides within the endoplasmic reticulum (ER) lumen, thereby disrupting ER homeostasis and triggering the unfolded protein response (UPR). Chronic ER stress promotes apoptosis, at least in part through the UPR-induced transcription factor C/EBP homologous protein (CHOP). We assessed the effect of Chop deletion in multiple mouse models of type 2 diabetes and found that Chop-/- mice had improved glycemic control and expanded beta cell mass in all conditions analyzed. In both genetic and diet-induced models of insulin resistance, CHOP deficiency improved beta cell ultrastructure and promoted cell survival. In addition, we found that isolated islets from Chop-/- mice displayed increased expression of UPR and oxidative stress response genes and reduced levels of oxidative damage. These findings suggest that CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis in beta cells under conditions of increased insulin demand.
Project description:Endoplasmic reticulum stress (ERS) in adipocytes can modulate adipokines secretion. The aim of this study was to explore the protective effect of high-density lipoprotein (HDL) on oxidized low-density lipoprotein (ox-LDL)-induced ERS-C/EBP homologous protein (CHOP) pathway-mediated adipokine secretion. Our results showed that serum adipokines, including visfatin, resistin and TNF-?, correlated inversely with serum HDL cholesterol level in patients with abdominal obesity. In vitro, like ERS inhibitor 4-phenylbutyric acid (PBA), HDL inhibited ox-LDL- or tunicamycin (TM, an ERS inducer)-induced increase in visfatin and resistin secretion. Moreover, HDL inhibited ox-LDL-induced free cholesterol (FC) accumulation in whole cell lysate and in the endoplasmic reticulum. Additionally, like PBA, HDL inhibited ox-LDL- or TM-induced activation of ERS response as assessed by the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2? and reduced nuclear translocation of activating transcription factor 6 as well as the downregulation of Bip and CHOP. Furthermore, HDL increased scavenger receptor class B type I (SR-BI) expression and SR-BI siRNA treatment abolished the inhibitory effects of HDL on ox-LDL-induced FC accumulation and CHOP upregulation. These data indicate that HDL may suppress ox-LDL-induced FC accumulation in adipocytes through upregulation of SR-BI, subsequently preventing ox-LDL-induced ER stress-CHOP pathway-mediated adipocyte inflammation.
Project description:Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein-LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 ?mol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100-200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25-200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.
Project description:The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo.ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet.Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet.We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.
Project description:p58IPK is an endoplasmic reticulum (ER)-resident chaperone playing a critical role in facilitating protein folding and protein homeostasis. Previously, we have demonstrated that p58IPK is expressed broadly in retinal neurons including retinal ganglion cells (RGCs) and loss of p58IPK results in age-related RGC degeneration. In the present study, we investigate the role of p58IPK in neuroprotection by in vitro and in vivo studies using primary RGC culture and two well-established disease-relevant RGC injury models: retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. Our results demonstrate that in both in vivo models, p58IPK -/- mice exhibit significantly increased RGC loss compared to wild type (WT) mice. In vitro, p58IPK-deficient RGCs show reduced viability and are more susceptible to cell death induced by the ER stress inducer tunicamycin (TM). Overexpression of p58IPK by adeno-associated virus (AAV) significantly diminishes TM-induced cell death in both WT and p58IPK -/- RGCs. Interestingly, we find that loss of p58IPK leads to reduced mRNA expression, but not the protein level, of mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor that resides in the ER. Treatment with recombinant MANF protein protects R28 retinal neural cells and mouse retinal explants from TM-induced cell death. Taken together, our study suggests that p58IPK functions as an endogenous neuroprotectant for RGCs. The mechanisms underlying p58IPK's neuroprotective action and the potential interactions between p58IPK and MANF warrant future investigation.
Project description:During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C ? (PKC?) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKC? exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKC? as shown by its phosphorylation and nuclear translocation. PKC? activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKC? participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1?/JNK pathway. Finally, the role of PKC? in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKC? with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKC? as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.
Project description:Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes.