Endoplasmic Reticulum Stress Resistance Research Articles

Transcription factor GmAlfin09 regulates endoplasmic reticulum stress in soybean via peroxidase GmPRDX6.

Soybean (Glycine max [L.] Merr.) is a valuable oil crop but is also highly susceptible to environmental stress. Thus, developing approaches to enhance soybean stress resistance is vital to soybean yield improvement. In previous studies, transcription factor Alfin has been shown to serve as an epigenetic regulator of plant growth and development. However, no studies on Alfin have yet been reported in soybean. In this study, the endoplasmic reticulum (ER) stress- and reactive oxygen species (ROS)-related GmAlfin09 was identified. Screening of genes co-expressed with GmAlfin09 unexpectedly led to the identification of soybean peroxidase 6 (GmPRDX6). Further analyses revealed that both GmAlfin09 and GmPRDX6 were responsive to ER stress, with GmPRDX6 localizing to the ER under stress. Promoter binding experiments confirmed the ability of GmAlfin09 to bind to the GmPRDX6 promoter directly. When GmAlfin09 and GmPRDX6 were overexpressed in soybean, enhanced ER stress resistance and decreased ROS levels were observed. Together, these findings suggest that GmAlfin09 promotes the upregulation of GmPRDX6, and GmPRDX6 subsequently localizes to the ER, reduces ROS levels, promotes ER homeostasis, and ensures the normal growth of soybean even under ER stress. This study highlights a vital target gene for future molecular breeding of stress-resistant soybean lines.

Thyroid-stimulating hormone induces insulin resistance in adipocytes via endoplasmic reticulum stress.

Subclinical hypothyroidism (SCH) is closely related to insulin resistance, and thyroid-stimulating hormone (TSH) level is an independent factor for insulin resistance associated with subclinical hypothyroidism. This study aims to explore the effects of TSH levels on insulin signal transduction in adipocytes and to establish the role of endoplasmic reticulum (ER) stress in this process. In this study, the SCH mouse model was established, and 3T3-L1 adipocytes were treated with TSH or tunicamycin (TM), with or without 4-phenylbutyric acid (4-PBA), an inhibitor of ER stress. SCH mice exhibited impaired glucose tolerance, inactivation of the IRS-1/AKT pathway and activation of the IRE1/JNK pathway in adipose tissue, which can all be alleviated by 4-PBA. Supplementation with levothyroxine restored the TSH to normal, alongside alleviated ER stress and insulin resistance in SCH mice, which is characterized by improved glucose tolerance, decreased mRNA expression of IRE1, and decreased phosphorylation of JNK in adipose tissue. In 3T3-L1 adipocytes, TSH induces insulin resistance, leading to a decrease in glucose uptake. This effect is mediated by the downregulation of IRS-1 tyrosine phosphorylation, reduced AKT phosphorylation, and inhibited GLUT4 protein expression. Notably, all these effects can be effectively reversed by 4-PBA. Moreover, TSH induced TNF-α and IL-6 production and upregulated the expression of ER stress markers. Similarly, these changes can be recovered by 4-PBA. These findings indicate that TSH has the capability to induce insulin resistance in adipocytes. The mechanism through which TSH disrupts insulin signal transduction appears to involve the ER stress-JNK pathway.

CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

The naked mole rat (NMR) is the longest-lived rodent, resistant to multiple age-related diseases including neurodegeneration. However, the mechanisms underlying the NMR's resistance to neurodegenerative diseases remain elusive. Here, we isolated oligodendrocyte progenitor cells (OPCs) from NMRs and compared their transcriptome with that of other mammals. Extracellular matrix (ECM) genes best distinguish OPCs of long- and short-lived species. Notably, expression levels of CD44, an ECM-binding protein that has been suggested to contribute to NMR longevity by mediating the effect of hyaluronan (HA), are not only high in OPCs of long-lived species but also positively correlate with longevity in multiple cell types/tissues. We found that CD44 localizes to the endoplasmic reticulum (ER) and enhances basal ATF6 activity. CD44 modifies proteome and membrane properties of the ER and enhances ER stress resistance in a manner dependent on unfolded protein response regulators without the requirement of HA. HA-independent role of CD44 in proteostasis regulation may contribute to mammalian longevity.

CCDC85A is regulated by miR-224-3p and augments cancer cell resistance to endoplasmic reticulum stress.

MicroRNAs (miRNAs) play pivotal roles in the tumor microenvironment. Here, we analyzed miRNAs in tumor stromal fibroblasts. Expression of miR-224-3p in cancer-associated fibroblasts (CAF) from scirrhous gastric cancer patients was lower than in normal fibroblasts (NF). Introduction of a miR-224-3p mimic attenuated migration and invasion of CAF. Coiled-coil domain containing 85A (CCDC85A), whose function in tumors is not understood, was the target gene of miR-224-3p. Immunohistological analysis revealed that CCDC85A is expressed to varying degrees by cancer cells and CAFs in gastric and pancreatic carcinomas. Downregulation of CCDC85A in cancer cells revealed that these cells are vulnerable to endoplasmic reticulum (ER) stress induced by thapsigargin or tunicamycin, which were ameliorated after addback of CCDC85A. Injection of NF-derived exosomes containing miR-224-3p into the xenograft tumor increased tumor shrinkage by cisplatin treatment. Mechanistically, CCDC85A associated with the molecular chaperone GRP78 and GRP94, thereby inhibiting association of these negative regulators of the unfolded protein response (UPR), leading to sustained activation of PERK and downstream eIF2〈 and ATF4 upon ER stress. These data suggest a novel miR-224-3p-mediated function for CCDC85A: protection from ER stress and cisplatin resistance.

Triphenyl phosphate proved more potent than its metabolite diphenyl phosphate in inducing hepatic insulin resistance through endoplasmic reticulum stress

Triphenyl phosphate (TPHP) is a widely used flame retardant and plasticizer and has been detected extensively in environmental media, wildlife and human bodies. Several epidemiological and animal studies have revealed that TPHP exposure is positively associated with glucose homeostasis disruption and diabetes. However, the effects of TPHP on hepatic glucose homeostasis and the underlying mechanisms remain unclear. The present work aimed to investigate the cytotoxicity and glucose metabolism disruption of TPHP and its metabolite diphenyl phosphate (DPHP) within hepatocytes. The cell viability assay undertaken on human normal liver (L02) cells showed that TPHP exhibited more potent hepatotoxicity than DPHP. RNA sequencing (RNA-seq) data showed that TPHP and DPHP presented different modes of toxic action. Insulin resistance is one of the predominant toxicities for TPHP, but not for DPHP. The insulin-stimulated glucose uptake and glycogen synthesis were impaired by TPHP, while DPHP exhibited no significant impairment on these factors. TPHP exposure induced endoplasmic reticulum (ER) stress, and the ER stress antagonist 4-PBA restored the impairment of insulin-stimulated glucose uptake and glycogen synthesis induced by TPHP. TPHP could also induce liver ER stress and insulin resistance in mice. Taken together, the results suggested that TPHP induces more potent insulin resistance through ER stress than its metabolite DPHP.

Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes

Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggesting that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by increasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/Neo vectors containing short hairpin RNA targeting IRE1α were used to silence IRE1α transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1α branch improved NEFA-induced insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3β, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1α kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3β, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3β, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined.

Modulation of endoplasmic reticulum stress via sulforaphane-mediated AMPK upregulation against nonalcoholic fatty liver disease in rats

Nonalcoholic fatty liver disease (NAFLD) is a major health concern. Endoplasmic reticulum (ER) stress, inflammation, and metabolic dysfunctions may be targeted to prevent the progress of nonalcoholic fatty liver disease. Sulforaphane (SFN), a sulfur-containing compound that is abundant in broccoli florets, seeds, and sprouts, has been reported to have beneficial effects on attenuating metabolic diseases. In light of this, the present study was designed to elucidate the mechanisms by which SFN ameliorated ER stress, inflammation, lipid metabolism, and insulin resistance — induced by a high-fat diet and ionizing radiation (IR) in rats. In our study, the rats were randomly divided into five groups: control, HFD, HFD + SFN, HFD + IR, and HFD + IR + SFN groups. After the last administration of SFN, liver and blood samples were taken. As a result, the lipid profile, liver enzymes, glucose, insulin, IL-1β, adipokines (leptin and resistin), and PI3K/AKT protein levels, as well as the mRNA gene expression of ER stress markers (IRE-1, sXBP-1, PERK, ATF4, and CHOP), fatty acid synthase (FAS), peroxisome proliferator–activated receptor-α (PPAR-α). Interestingly, SFN treatment modulated the levels of proinflammatory cytokine including IL-1β, metabolic indices (lipid profile, glucose, insulin, and adipokines), and ER stress markers in HFD and HFD + IR groups. SFN also increases the expression of PPAR-α and AMPK genes in the livers of HFD and HFD + IR groups. Meanwhile, the gene expression of FAS and CHOP was significantly attenuated in the SFN-treated groups. Our results clearly show that SFN inhibits liver toxicity induced by HFD and IR by ameliorating the ER stress events in the liver tissue through the upregulation of AMPK and PPAR-α accompanied by downregulation of FAS and CHOP gene expression.

Engineering the endoplasmic reticulum secretory pathway in Trichoderma reesei for improved cellulase production

The filamentous fungus Trichoderma reesei is an extraordinarily efficient cell factory of industrial cellulase for production of biofuels and other bio-based products because of its excellent potential to secrete cellulolytic enzymes. Engineering the protein secretory pathway may be a powerful means for efficient protein production. However, it is uncertain whether this engineering approach could improve cellulase production in T. reesei. Herein, the endoplasmic reticulum (ER) secretory pathway was engineered for the production of cellulolytic enzymes by multiple strategies, including: (I) overexpression of the key components of protein folding (Pdi1, Ero1 and BiP); (II) overexpression of the glycosylation-related elements (Gpt1 and Gls2); (III) knockout of the ER mannosidase I (Mns1) encoding gene mns1. By utilizing these ER engineering strategies, the secretion of β-glucosidase was remarkably elevated in the engineered strains, ranging from 29.2 % to 112.5 %. Furthermore, it was found that engineering these components also regulated the ER stress resistance. More importantly, the total cellulase production was increased with varying degrees, which reached a maximum of 149.4 %, using the filter paper assay (FPA) as a characterization method. These results demonstrated that engineering the ER secretory pathway can enhance protein secretion, particularly for cellulase production, which shed light for the development of high-efficient cellulolytic enzymes for economically feasible bioethanol production from lignocellulosic biomass.

Insulin Increases Adipose Adiponectin in Pregnancy by Inhibiting Ubiquitination and Degradation: Impact of Obesity.

Circulating adiponectin levels are decreased in pregnant women with obesity or gestational diabetes, and this is believed to contribute to the insulin resistance and increased risk of fetal overgrowth associated with these conditions. However, the molecular mechanisms regulating adiponectin secretion from maternal adipose tissues in pregnancy are poorly understood. We tested the hypothesis that obesity in pregnancy is associated with adipose tissue insulin resistance and increased adiponectin ubiquitination and degradation, caused by inflammation and endoplasmic reticulum (ER) stress. Visceral adipose tissues were collected from lean and obese pregnant humans and mice. Total and ubiquitinated adiponectin, and markers of inflammation, ER stress, and insulin resistance were examined in adipose tissues. The role of insulin, inflammation, and ER stress in mediating adiponectin ubiquitination and degradation was examined using 3T3L-1 adipocytes. Obesity in pregnancy is associated with adipose tissue inflammation, ER stress, insulin resistance, increased adiponectin ubiquitination, and decreased total abundance of adiponectin. Adiponectin ubiquitination was increased in visceral fat of obese pregnant women as compared to lean pregnant women. We further observed that insulin prevents, whereas ER stress and inflammation promote, adiponectin ubiquitination and degradation in differentiated 3T3-L1 adipocytes. We have identified adiponectin ubiquitination as a key mechanism by which obesity diminishes adiponectin secretion in pregnancy. This information will help us better understand the mechanisms controlling maternal insulin resistance and fetal growth in pregnancy and may provide a foundation for the development of strategies aimed at improving adiponectin production in pregnant women with obesity or gestational diabetes.

Leptin sensitizing effect of 1,3-butanediol and its potential mechanism

Leptin is an adipocyte-derived hormone that regulates appetite and energy expenditure via the hypothalamus. Since the majority of obese subjects are leptin resistant, leptin sensitizers, rather than leptin itself, are expected to be anti-obesity drugs. Endoplasmic reticulum (ER) stress in the hypothalamus plays a key role in the pathogenesis of leptin resistance. ATP-deficient cells are vulnerable to ER stress and ATP treatment protects cells against ER stress. Thus, we investigated the therapeutic effects of oral 1,3-butanediol (BD) administration, which increases plasma β-hydroxybutyrate and hypothalamic ATP concentrations, in diet induced obese (DIO) mice with leptin resistance. BD treatment effectively decreased food intake and body weight in DIO mice. In contrast, BD treatment had no effect in leptin deficient ob/ob mice. Co-administration experiment demonstrated that BD treatment sensitizes leptin action in both DIO and ob/ob mice. We also demonstrated that BD treatment attenuates ER stress and leptin resistance at the hypothalamus level. This is the first report to confirm the leptin sensitizing effect of BD treatment in leptin resistant DIO mice. The present study provides collateral evidence suggesting that the effect of BD treatment is mediated by the elevation of hypothalamic ATP concentration. Ketone bodies and hypothalamic ATP are the potential target for the treatment of obesity and its complications.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1–7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1–7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1–7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

Abstract 2558: Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer

Abstract Up to 20% of all cancer deaths in women can be attributed to excess body weight. Endometrial cancer was the first cancer identified with an increased risk among obese populations, and incidence of endometrial cancer is rising steadily due to the obesity epidemic. Obesity is associated with over 60% of endometrial cancer occurring in the United States, but like many cancers, the causes of endometrial cancer are multi-faceted. Increased estrogen abundance in obese women is considered the primary driver, but obesity-linked complications, including hyperinsulinemia, are also known to contribute to endometrial cancer. However, obesity alone is not sufficient for tumorigenesis. Genetic mutations driving endometrial cancer are well characterized, but these mutations alone are not sufficient for EC pathogenesis as they exist in uteri without cancer. In normal cells, the PI3K pathway maintains metabolic homeostasis, but in cancer the PI3K pathway is activated to promote cell survival. There is an urgent need to understand the mechanisms by which obesity and genetic mutation interact to promote EC. Metabolic signaling influences protein production and folding. The unfolded protein response pathway acts to maintain protein quality control, and is activated upon endoplasmic reticulum stress. During endoplasmic reticulum stress, if proteostasis cannot be achieved, the unfolded protein response is activated, resulting in cell death. Obesity results in systemic endoplasmic reticulum stress, with overabundant estrogen and hyperinsulinemia likely as contributing factors. In our preliminary studies, we have identified a role for PI3K pathway mutations in regulating endoplasmic reticulum stress in the endometrial epithelium. By antagonizing endoplasmic reticulum stress, PI3K pathway mutations may promote survival of endometrial epithelium through resistance to unfolded protein response-mediated cell death mechanisms. Through understanding endoplasmic reticulum stress resistance in the endometrial epithelium, we hope to develop preventative interventions for obese women at risk for endometrial cancer. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2558.

From the Editor’s Desk...

From the Editor’s Desk...

Imoxin inhibits tunicamycin-induced endoplasmic reticulum stress and restores insulin signaling in C2C12 myotubes.

Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been implicated in skeletal muscle dysfunction. However, pathological roles of PKR in ER stress in muscle are not fully understood. The current study aimed to investigate the effect of imoxin (IMX), a selective PKR inhibitor, on tunicamycin (TN)-induced promotion of ER stress and suppression of insulin signaling in C2C12 myotubes. Cells were pretreated with 5 µM IMX for 1 h and exposed to 0.5 µg/mL TN for 23 h. A subset of cells was stimulated with 100 nM insulin for the last 15 min. mRNA expression and protein levels involved in ER stress were measured by RT-PCR and Western blotting, respectively. TN significantly augmented PKR phosphorylation by 231%, which was prevented by IMX. In addition, IMX reduced mRNA and protein levels of ER stress-related markers, including CCAAT-enhancer-binding protein homologous protein (CHOP, mRNA: 95% decrease; protein: 98% decrease), activating transcription factor 4 (ATF4, mRNA: 69% decrease; protein: 99% decrease), cleavage of ATF6, and spliced X-box-binding protein 1 (XBP-1s, mRNA: 88% decrease; protein: 79% decrease), which were induced by TN. Furthermore, IMX ameliorated TN-induced suppression of phospho-insulin receptor β (317% increase) and Akt phosphorylation (by 36% at Ser473 and 30% at Thr308) in myotubes, while augmenting insulin-stimulated AS160 phosphorylation and glucose uptake (by ∼30%). These findings suggest that IMX may protect against TN-induced skeletal muscle ER stress and insulin resistance, which are potentially mediated by PKR.

The XBP1‒MARCH5‒MFN2 Axis Confers Endoplasmic Reticulum Stress Resistance by Coordinating Mitochondrial Fission and Mitophagy in Melanoma

Melanoma cells are relatively resistant to endoplasmic reticulum (ER) stress, which contributes to tumor progression under stressful conditions and renders tolerance to ER stress‒inducing therapeutic agents. Mitochondria are tightly interconnected with ER. However, whether mitochondria play a role in regulating ER stress resistance in melanoma remains elusive. In this study, we reported that the XBP1‒MARCH5‒MFN2 axis conferred ER stress resistance by coordinating mitochondrial fission and mitophagy in melanoma. Our integrative bioinformatics first revealed that the downregulation of mitochondrial genes was highly correlated with unfolded protein response activation in melanoma. Then we proved that mitochondrial fission and mitophagy were prominently induced to contribute to ER stress resistance both invitro and invivo by maintaining mitochondrial function. Mechanistically, the activation of IRE1α/ATF6-XBP1 branches of unfolded protein response promoted the transcription of E3 ligase MARCH5 to facilitate the ubiquitination and degradation of MFN2, which thereby triggered mitochondrial fission and mitophagy under ER stress. Together, our findings show a regulatory axis that links mitochondrial fission and mitophagy to the resistance to ER stress. Targeting mitochondrial quality control machinery can be exploited as an approach to reinforce the efficacy of ER stress‒inducing agents against cancer.

FOXA3 induction under endoplasmic reticulum stress contributes to non-alcoholic fatty liver disease

Chronic endoplasmic reticulum (ER) stress in the liver has been shown to play a causative role in non-alcoholic fatty liver disease (NAFLD) progression, yet the underlying molecular mechanisms remain to be elucidated. Forkhead box A3 (FOXA3), a member of the FOX family, plays critical roles in metabolic homeostasis, although its possible functions in ER stress and fatty liver progression are unknown. Adenoviral delivery, siRNA delivery, and genetic knockout mice were used to crease FOXA3 gain- or loss-of-function models. Tunicamycin (TM) and a high-fat diet (HFD) were used to induce acute or chronic ER stress in mice. Chromatin immunoprecipiation (ChIP)-seq, luciferase assay, and adenoviral-mediated downstream gene manipulations were performed to reveal the transcriptional axis involved. Key axis protein levels in livers from healthy donors and patients with NAFLD were assessed via immunohistochemical staining. FOXA3 transcription is specifically induced by XBP1s upon ER stress. FOXA3 exacerbates the excessive lipid accumulation caused by the acute ER-inducer TM, whereas FOXA3 deficiency in hepatocytes and mice alleviates it. Importantly, FOXA3 deficiency in mice reduced diet-induced chronic ER stress, fatty liver, and insulin resistance. In addition, FOXA3 suppression via siRNA or adeno-associated virus delivery ameliorated the fatty liver phenotype in HFD-fed and db/db mice. Mechanistically, ChIP-Seq analysis revealed that FOXA3 directly regulates Period1 (Per1) transcription, which in turn promotes the expression of lipogenic genes, including Srebp1c, thus enhancing lipid synthesis. Of pathophysiological significance, FOXA3, PER1, and SREBP1c levels were increased in livers of obese mice and patients with NAFLD. The present study identified FOXA3 as the bridging molecule that links ER stress and NAFLD progression. Our results highlighted the role of the XBP1s-FOXA3-PER1/Srebp1c transcriptional axis in the development of NAFLD and identified FOXA3 as a potential therapeutic target for fatty liver disease. The molecular mechanisms linking endoplasmic reticulum stress to non-alcoholic fatty liver disease (NAFLD) progression remain undefined. Herein, via invitro and invivo analysis, we identified Forkhead box A3 (FOXA3) as a key bridging molecule. Of pathophysiological significance, FOXA3 protein levels were increased in livers of obese mice and patients with NAFLD, indicating that FOXA3 could be a potential therapeutic target in fatty liver disease.

In vitro treatment of 3\u2009T3-L1 adipocytes with recombinant Calcium/calmodulin-dependent Protein Kinase IV (CaMKIV) limits ER stress and improves insulin sensitivity through inhibition of autophagy via the mTOR/CREB signaling pathway

BackgroundRecently, CaMKIV has been identified as a potential regulator of skeletal muscle glucose metabolism, it can also affect insulin gene expression in pancreas. However, its effects on adipose insulin resistance have yet to be explored. Autophagy has been shown as a potential therapeutic target for ER (endoplasmic reticulum) stress and insulin resistance. The purpose of this study is to investigate the effects of CaMKIV on ER stress, autophagic function and insulin signaling in tunicamycin-treated adipocytes.MethodsIn this study, mature 3 T3-L1 adipocytes were treated with tunicamycin to induce ER stress. Tunicamycin-treated 3 T3-L1 adipocytes were treated with recombinant CaMKIV in the presence or absence of targeted-siRNA mediated down-regulation of CREB and mTOR. The ER stress markers, autophagy activation, mTOR/CREB signaling and insulin sensitivity were analyzed by western blotting or electron microscopy.ResultsTreatment with CaMKIV significantly reversed tunicamycin-induced expression of p-PERK, cleaved-ATF6, Atg7 and LC3II. It also reduced p62 expression. In addition, levels of p-Akt and p-IRS-1 were increased. Moreover, CaMKIV inhibited activated ER stress and insulin resistance in Atg7 siRNA transfected adipocytes. However, the protective effects of CaMKIV on ER stress, insulin signaling, and autophagy function were nullified by suppression of mTOR or CREB in tunicamycin-treated adipocytes.ConclusionThis study proves recombinant CaMKIV inhibits tunicamycin-induced ER stress and insulin resistance by regulating autophagy. The protective effect of CaMKIV in adipocytes is affected at least partly through mTOR/CREB signaling. Our finding may offer novel opportunities for treating obesity and type 2 diabetes.

The unc-51 like autophagy activating kinase 1-autophagy related 13 complex has distinct functions in tunicamycin-treated cells

Endoplasmic reticulum (ER) stress and autophagy are regulated by shared signaling pathways, and their dysfunction is directly related to pathological conditions. This study investigated the function of the unc-51 like autophagy activating kinase 1 (ULK1)-autophagy related 13 (ATG13) complex in ER stress conditions through a knockout (KO) approach. Unlike other autophagy genes, KO of ULK1 or ATG13 attenuated ER stress and promoted mammalian target of rapamycin complex 1 (mTORC1) activation. Compared with wild type (WT) cells, ULK1 and ATG13 KO cells displayed increased viability, while beclin 1, ATG14, and ULK1/2 KO cells did not. Tunicamycin treatment upregulated the expression of ER stress markers (DNA damage inducible transcript 3, heat shock protein family A (Hsp70) member 5, and phosphorylated eukaryotic translation initiation factor 2 alpha kinase 3, eukaryotic translation initiation factor 2 subunit alpha, and endoplasmic reticulum to nucleus signaling 1); however, these were decreased in ULK1 and ATG13 KO cells. Insulin treatment upregulates the phosphorylation of ribosomal protein S6 kinase B1 (RPS6KB1) and AKT serine/threonine kinase 1 (AKT1), which was suppressed by tunicamycin. Notably, ATG13 and ULK1 deficiency ameliorated tunicamycin-induced insulin resistance, with enhanced RPS6KB1 and AKT1 phosphorylation in KO cells compared to WT cells. Although ULK1 and ATG13 are necessary for autophagy induction after tunicamycin-induced ER stress, autophagy does not seem to directly affect tunicamycin-induced cell death, ER stress, or insulin resistance. Our results indicate that loss of the ULK1-ATG13 complex attenuates ER stress and cell death and increases mTORC1 signaling.

DEL-1 ameliorates high-fat diet-induced insulin resistance in mouse skeletal muscle through SIRT1/SERCA2-mediated ER stress suppression

Inflammation and endoplasmic reticulum (ER) stress are associated with the development of insulin resistance and diabetes. Developmental endothelial locus-1 (DEL-1) enhances efferocytosis by macrophage and suppresses inflammatory response. However, effects of DEL-1 on ER stress-mediated insulin resistance in skeletal muscle remain unclear. Here, DEL-1 treatment augmented SIRT1 expression in C2C12 myocytes, thereby increasing SERCA2 expression in a dose-dependent fashion, and attenuated ER stress and insulin resistance under palmitate treatment condition. SIRT1/SERCA2 knockdown abrogated effects of DEL-1 on palmitate-induced insulin resistance as well as ER stress. Pharmacological significance of DEL-1 was confirmed by in vivo experiments. DEL-1 administration suppressed ER stress, insulin resistance, and SIRT1/SERCA2 expression in skeletal muscle of high-fat diet (HFD)-fed mice. Additionally, siRNA transfection-mediated in vivo downregulation of SIRT1 suppressed the effects of DEL-1 on expression of SERCA2, ER stress, and insulin resistance in skeletal muscle of HFD-fed mice. DEL-1 attenuates palmitate-induced and HFD-induced skeletal muscle ER stress and insulin resistance via SIRT1/SERCA2-mediated signaling.

Involvement of Kisspeptin in Androgen-Induced Hypothalamic Endoplasmic Reticulum Stress and Its Rescuing Effect in PCOS Rats

Background: Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and Endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. Methods: An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, We detected Kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. Findings: We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly down-regulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, Kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Interpretation: Our data has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent. Funding Statement: National Basic Research Program of China, National Natural Science Foundation of China (2012CB944902, 2012CB944703, 2013CB967404, 2013CB967401, 2013CB967403, 81571403, 81471429) and State Key laboratory of Reproductive Medicine (SKLRM-K201805). Declaration of Interests: The authors have no conflicts of interest about this work. Ethics Approval Statement: The study was approved by the Ethics Committee of Tongji University and all patients gave informed consent. All procedures in this study were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong, and the methods were used in accordance with the guidelines of the Chinses University of Hong Kong.