The Effect and Molecular Mechanism of Fto Gene Knockout on Cadmium-Induced Injury in Pancreatic β-Cells.

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Peroxisome proliferator-activated receptor (PPAR)gamma ligands are known to have anti-inflammatory properties that include the inhibition of cytokine signaling, transcription factor activation, and inflammatory gene expression. We have recently observed that increased expression of heat shock protein (HSP)70 correlates with, but is not required for, the anti-inflammatory actions of PPARgamma ligands on cytokine signaling. In this study, we provide evidence that the inhibitory actions of PPARgamma ligands on cytokine signaling are associated with endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation in pancreatic beta-cells. 15-Deoxy-Delta(12,14)-prostaglandin J(2), at concentrations that inhibit cytokine signaling, stimulates phosphorylation of eukaryotic initiation factor-2alpha, and this event is followed by a rapid inhibition of protein translation. Under conditions of impaired translation, PPARgamma ligands stimulate the expression of a number of ER stress-responsive genes, such as GADD 153, BiP, and HSP70. Importantly, ER stress activation in response to PPARgamma ligands or known UPR activators results in the attenuation of IL-1 and IFN-gamma signaling. These findings indicate that PPARgamma ligands induce ER stress, that ER stress activation is associated with an attenuation of cytokine signaling in beta-cells, and that the attenuation of responsiveness to extracellular stimuli appears to be a novel protective action of the UPR in cells undergoing ER stress.

STUDY QUESTIONDoes metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro?SUMMARY ANSWERMetformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs.WHAT IS KNOWN ALREADYPolycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice.STUDY DESIGN, SIZE, DURATIONCumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA.PARTICIPANTS/MATERIALS, SETTING, METHODSThe levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion.MAIN RESULTS AND THE ROLE OF CHANCEThe expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK.LIMITATIONS, REASONS FOR CAUTIONThe number of PCOS patients in this study was small.WIDER IMPLICATIONS OF THE FINDINGSThis study provides further evidence for metformin as a PCOS treatment.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose.TRIAL REGISTRATION NUMBERN/A.

STUDY QUESTION Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? SUMMARY ANSWER Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. WHAT IS KNOWN ALREADY Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. STUDY DESIGN, SIZE, DURATION Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. PARTICIPANTS/MATERIALS, SETTING, METHODS The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. MAIN RESULTS AND THE ROLE OF CHANCE The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. LIMITATIONS, REASONS FOR CAUTION The number of PCOS patients in this study was small. WIDER IMPLICATIONS OF THE FINDINGS This study provides further evidence for metformin as a PCOS treatment. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. TRIAL REGISTRATION NUMBER N/A.

From adaption to death: endoplasmic reticulum stress as a novel target of selective neurodegeneration?

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IkappaB kinase and nuclear factor kappaB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

SY44-03: Targeting the unfolded protein response in cancer

Gene Therapy Strategies to Restore ER Proteostasis in Disease.

Diabetes mellitus comprises a cohort of genetic and metabolic diseases which are characterized by the hallmark symptom of hyperglycemia. Diabetic subtypes are based on their pathogenetic origins: the most prevalent subtypes are the autoimmune-mediated type 1 diabetes mellitus (T1DM) and the metabolic disease of type 2 diabetes mellitus (T2DM). Genetic factors are major contributory aspects to diabetes development, particularly in T2DM where there is close to 80% concordance rates between monozygotic twins. However, the functional state of the pancreatic β cell is of paramount importance to the development of diabetes. Perturbations that lead to β cell dysfunction impair insulin production and secretion and precede diabetes onset. The endoplasmic reticulum (ER) is a subcellular organelle network of tubes and cisternae with multifaceted roles in cellular metabolism. Alterations to ER function such as those begotten by the accumulation of misfolded and unfolded ER client proteins upset the ER homeostatic balance, leading to a condition termed ER stress. Subsequent sensing of ER stress by three ER transmembrane proteins, initiates an adaptive reaction to alleviate ER stress: this is known as the unfolded protein response (UPR). Divergent cascades of the UPR attempt to mitigate ER stress and restore ER homeostasis: Failing that, the UPR initiates pro-apoptotic pathways. The demand of insulin production on the β cell necessitates the presence of a highly functional ER. However, the consequence of dependence on the ER for insulin synthesis and secretion portends disaster for the functional state of the β cell. Disturbances to the ER that elicit ER stress and UPR activation causes β cell dysfunction and may lead to apoptosis. There are numerous well-characterized models of ER stress-mediated diabetes, including genetic mutations in UPR transducers and insulin. Recently, polymorphisms in Wolfram syndrome 1 (WFS1), an ER transmembrane protein involved in the UPR, were suggested to contribute to T2DM risk. In this thesis, one of the highlighted WFS1 polymorphism, H611R, was examined to identify its contribution to β cell function and viability, and hence, diabetes risk. It was revealed that augmentation of WFS1 expression increased insulin secretion and cellular content. In addition, WFS1 protected β cells against ER stress-mediated dysfunction, with a more pronounced effect in the WFS1-R611 protective allele. Subsequent gene expression analysis identified netrin-1 as a WFS1-induced survival factor. As a contributory factor to diabetes progression, ER stress and UPR are potential drug and biomarker targets. In this dissertation, a novel UPR-regulating microRNA (miRNA) family was uncovered in ER stressed, WFS1-deficient islets. These miRNAs, the miR-29 family, are induced in WFS1 -/- islets as a possible adaptive alteration to chronic ER stress conditions, and indirectly decreases the expression of UPR transducers, while directly targeting downstream ER stress-related pro-apoptotic factors.…

Endoplasmic reticulum (ER) is a cellular compartment responsible for biosynthesis and folding of newly synthesized proteins destined for secretion, such as insulin. ER stress plays a role in the pathogenesis of diabetes and is associated with pancreatic β-cell damage. The aim of this study was to examine the effect of KIOM-79, a mixture of plant extracts, on streptozotocin (STZ)-induced ER stress in pancreatic RINm5F β-cells. STZ induced characteristics of ER stress; the release of Ca2+ from ER, increased ER staining, induction of glucose-regulated protein-78, phosphorylation of PKR-like ER kinase, and eukaryotic initiation factor-2α, as well as cleavage of activating transcription factor-6, whereas KIOM-79 inhibited these changes. Moreover, KIOM-79 inhibited STZ-induced apoptotic cell death. STZ induced CCAAT/enhancer-binding protein-homologous protein (CHOP) and cleavage of caspase 12, which are ER stress-induced apoptosis regulatory proteins; however, KIOM-79 suppressed these effects. KIOM-79 suppressed apoptosis induced by STZ treatment in CHOP siRNA-transfected cells. Furthermore, KIOM-79 restored cell viability decreased by STZ treatment via ER-stressed apoptosis. The pancreatic β-cells damaged by STZ had decreased levels of insulin, and KIOM-79 restored the levels. Taken together, these results suggest that KIOM-79 had cytoprotective effects against STZ-induced apoptosis by interrupting the ER stress pathway.

Pancreatic Stress and Regeneration

Proinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca(2+) transporting ATPase sarco/endoplasmic reticulum Ca(2+) pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field. Rat and mouse insulin-producing cells, human pancreatic islets and human EndoC-βH1 cells were exposed to the cytokines IL-1β, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells. NO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis. These observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.