Eukaryotic Initiation Factor 2α Kinase Research Articles

Knockdown of EIF2AK2-OAS1 axis reduces ATP production inducing AMPK phosphorylation to inhibit the malignant behavior of gastric cancer cells.

Energy metabolism has always been a hot topic in cancer progression and targeted therapy, and exploring the role of genes in energy metabolic pathways in cancer cells has become key to address this issue. Eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) plays regulatory roles in cancer and disorders of energy metabolism. Indeed, the role of EIF2AK2 in energy metabolism has been underestimated. The aim of this study is to reveal the expression specificity of EIF2AK2 in gastric cancer (GC) progression and metastasis, and to demonstrate the role of EIF2AK2 in energy metabolism, cytoskeleton, proliferation, death and metastasis pathways in GC cells. Mechanistically, EIF2AK2 overexpression promoted cytoskeleton remodeling and ATP production, mediated cell proliferation and metastasis, upregulated OAS1 expression, decreases p-AMPK expression and inhibited apoptosis in GC cells. Conversely, knockdown of EIF2AK2 resulted in the opposite effect. However, overexpression of OAS1 mediated the upregulation of mitochondrial membrane potential and promoted ATP production and NAD+/NADH ratio, but knockdown of OAS1 inhibited the above effects. In addition, knockdown of OAS1 had no effect on EIF2AK2 expression, but inhibited AMPK and upregulated p-AMPK expression. In conclusion, our study identified EIF2AK2 and OAS1 as previously undescribed regulators of energy metabolism in GC cells. We hypothesized that EIF2AK2-OAS1 axis may regulate energy metabolism and inhibit cellular malignant behavior in cancer cells by affecting ATP production to induce AMPK phosphorylation, suggesting EIF2AK2 as a potential therapeutic target for cancer cell progression.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Eukaryotic translation initiation factor 2α kinase 2 in pancreatic cancer: An approach towards managing clinical prognosis and molecular immunological characterization.

Most patients with pancreatic cancer are already in the late stages of the disease when they are diagnosed, and pancreatic cancer is a deadly disease with a poor prognosis. With the advancement of research, immunotherapy has become a new focus in the treatment of tumors. To the best of our knowledge, there is currently no reliable diagnostic or prognostic marker for pancreatic cancer; therefore, the present study investigated the potential of eukaryotic translation initiation factor 2α kinase 2 (EIF2AK2) as a predictive and diagnostic marker for pancreatic cancer. Immunohistochemical staining of clinical samples independently verified that EIF2AK2 expression was significantly higher in clinically operated pancreatic cancer tissues than in adjacent pancreatic tissues., and EIF2AK2 expression and differentially expressed genes (DEGs) were identified using downloadable RNA sequencing data from The Cancer Genome Atlas and Genomic Tumor Expression Atlas. In addition, Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses and immune cell infiltration were used for functional enrichment analysis of EIF2AK2-associated DEGs. The clinical importance of EIF2AK2 was also determined using Kaplan-Meier survival, Cox regression and time-dependent survival receiver operating characteristic curve analyses, and a predictive nomogram model was generated. Finally, the functional role of EIF2AK2 was assessed in PANC-1 cells using a short hairpin RNA-EIF2AK2 knockdown approach, including CCK-8, wound healing assay, cell cycle and apoptosis assays. The findings suggested that EIF2AK2 may have potential as a diagnostic and prognostic biomarker for patients with pancreatic cancer. Furthermore, EIF2AK2 may provide a new therapeutic target for patients with pancreatic cancer.

Astrocytic deletion of protein kinase R-like ER kinase (PERK) does not affect learning and memory in aged mice but worsens outcome from experimental stroke.

Aging is associated with cognitive decline and is the main risk factor for a myriad of conditions including neurodegeneration and stroke. Concomitant with aging is the progressive accumulation of misfolded proteins and loss of proteostasis. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). The UPR is mediated, in part, by the eukaryotic initiation factor 2α (eIF2α) kinase protein kinase R-like ER kinase (PERK). Phosphorylation of eIF2α reduces protein translation as an adaptive mechanism but this also opposes synaptic plasticity. PERK, and other eIF2α kinases, have been widely studied in neurons where they modulate both cognitive function and response to injury. The impact of astrocytic PERK signaling in cognitive processes was previously unknown. To examine this, we deleted PERK from astrocytes (AstroPERKKO ) and examined the impact on cognitive functions in middle-aged and old mice of both sexes. Additionally, we tested the outcome following experimental stroke using the transient middle cerebral artery occlusion (MCAO) model. Tests of short-term and long-term learning and memory as well as of cognitive flexibility in middle-aged and old mice revealed that astrocytic PERK does not regulate these processes. Following MCAO, AstroPERKKO had increased morbidity and mortality. Collectively, our data demonstrate that astrocytic PERK has limited impact on cognitive function and has a more prominent role in the response to neural injury.

Abstract 638: EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of disorders, ranging from non-progressive bland steatosis to non-alcoholic hepatitis (NASH), cirrhosis, and malignant transformation into hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) exerts a key role in lipid homeostasis and play a promising therapeutic candidate for the treatment of obesity, diabetes, and NASH. Small molecules like EPB-53 that are activators of heme-regulated inhibitor (HRI) of eukaryotic initiation factor 2α (eIF2α) kinase have been synthesized to induce FGF21. Previous study has shown that oral administration of EPB-53 alleviates NASH through increased FGF21 to prevent hepatic steatosis. However, it is unknown whether EPB-53 can prevent NASH-HCC transition. Aim: To investigate the effect of EPB-53 on NASH-HCC transition and the therapeutic mechanism, focusing on the lipid metabolic reprogramming and tumor immune microenvironment (TIM). Methods: NASH-HCC transition model was established in two-week-old male FGF21 knockout (KO) mice and wild type controls (C57BL/6J mice) by diethylnitrosamine (DEN) administration at 40 mg/kg body weight (i.p.). Four weeks after DEN administration, the mice were fed with western-style high fat diet (WSHFD, 60% kcal% fat and 19% kcal% fructose) for 3 months, and gavaged daily with either vehicle, 2% w/v, (2-hydroxypropyl)-β-cyclodextrin or EPB-53 (300 mg/kg/day) in vehicle for 4 weeks before sacrifice. The tumor incidence, lipid metabolism, and carcinogenesis signaling pathways were determined. Mass cytometry (CyTOF) analysis was performed to detect the cell subpopulations in the NASH microenvironment and NASH-HCC TIM. Results: Severe NASH with tumor cell detection, aberrant lipid components and HIPPO-YAP signaling were found in liver tissues of FGF21-KO mice compared to the WT controls. The upregulated sphingosine Kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in liver subsequently induced YAP dephosphorylation and nuclear localization. EPB-53 treatment prevents NASH-HCC transition evident as decreased HCC incidence in WT mice but not in FGFKO mice. Continuously increased FGF21 production in WT mice contributes to down-regulation of SPHK1-S1P-HIPPO-YAP signaling. Interestingly, EPB-53 treatment can alleviate the compromised function of macrophages/Kupffer cells which render a TIM in the NASH-HCC mice. Conclusion: 1) Lack of FGF21 played a key role in lipid metabolic disorder and SPHK1-S1P-HIPPO signaling to promote NASH-HCC transition; 2) Lack of FGF21 rendered a TIM in NASH liver contributing to the NASH-HCC transition; 3) EPB-53 treatment can not only alleviate NASH, but also prevent NASH-HCC transition via lipid metabolic reprogramming, down-regulation of the SPHK1-S1P-HIPPO axis, and modulation of TIM. Citation Format: Min Tan, Eugenia Pujol, Santiago Vazquez, Jingwen Zhang, Jun Yan, Robert C. Martin, Manuel Vazquez-Carrera, Yan Li. EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 638.

Is ATP the Only Nucleoside Triphosphate among ATP, CTP, GTP, and UTP to Have a Role in Kinase Catalysis of Heme-Regulated Inhibitor toward eIF2α during Lung Cancer Development?

The heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase, also known as heme-regulated inhibitor (HRI), detects misfolded proteins and induces cytoprotective response to stress, mainly caused by heme-shortage. The nucleoside triphosphate ATP serves as the main donor of phosphate for the phosphorylation of eIF2α by HRI in human cells. However, the other main nucleoside triphosphates (CTP, GTP, UTP) are also present at relatively high concentrations, especially in human tumor cells. Therefore, in this short communication we evaluate the role of four substrates (namely ATP, CTP, GTP, and UTP) on human HRI kinase activity. Additionally, for the first time, we perform a detailed kinetics study of the HRI G202S mutant, whose presence in the human lung is associated with cancer development. Here, the role of all four tested nucleoside triphosphates during cancer development is discussed from the point of view of the HRI activity. The results showed that the kcat value of GTP was lower than that of ATP but was significantly higher than those of CTP and UTP. Additionally, the kcat value of GTP for G202S was approximately 20% higher than that for wild-type, while the kcat values of ATP, CTP, and UTP for G202S were lower than those for wild-type.

The double-stranded RNA-dependent protein kinase PKR negatively regulates the protein expression of IFN-β induced by RIG-I signaling.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-β expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-β mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-β, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-β expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-β protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-β in RIG-I-mediated antiviral immune responses.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

Shrimp Lipid Prevents Endoplasmic Reticulum-Mediated Endothelial Cell Damage.

Shrimp contains a fat that benefits cardiovascular function and may help in the prevention of diseases. The stress of essential cellular organelle endoplasmic reticulum (ER) is linked to endothelial dysfunction and damage. This research aimed at investigating the effect of shrimp lipid (SL) on endothelial cells in response to ER stress, as well as the underlying mechanisms. Human endothelial cells were pretreated with SL (250 and 500 μg/mL) for 24 h, and treated with 0.16 μg/mL of Thapsigargin (Tg) for 24 h. The apoptosis and necrosis were detected by Hoechst 33342/propidium iodide (PI) co-staining. Cellular signaling pathways and ER stress markers were evaluated by Western blot analysis and immunofluorescence. SL protected against ER-induced endothelial cell apoptosis. According to the results, the viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1%, but SL (250 μg/mL) pretreatment increased cell viability to 77.26 ± 3.9%, and SL (500 μg/mL) increased to 72.42 ± 4.3%. SL suppressed the increase in ER stress regulator glucose-regulated protein 78 (GRP78) and attenuated the RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK) and inositol-requiring ER-to-nucleus signaling protein 1 (IRE1) pathways. SL could inhibit cell damage by reducing the ER-related apoptosis protein, C/EBPα-homologous protein (CHOP), induced by ER stress. Taken together, we found the protective effect and mechanism of SL in protecting ER stress-induced endothelial cell apoptosis through suppression of the ER stress pathway. The findings may support the potential use of SL as an approach with a protective effect on endothelial cells.

Eukaryotic Initiation Factor 2α Kinases Regulate Virulence Functions, Stage Conversion, and the Stress Response in Entamoeba invadens.

ABSTRACTEntamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long served as a surrogate to study stage conversion. In the host, Entamoeba species must manage stress, including nutrient deprivation and host immune pressure. In many systems, the stress response is characterized by downregulation of translation, which is initiated by the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). In mammalian cells, this phosphorylation is carried out by a family of eIF2α kinases. A canonical eIF2α translational control system exists in Entamoeba species; however, no eIF2α kinases have been characterized. In this study, we identified two eIF2α kinases in E. invadens, EiIF2K-A and EiIF2K-B. Their identity as eIF2α kinases was validated using a heterologous yeast system. We used an RNA interference (RNAi) trigger-mediated silencing system to reduce expression of EiIF2K-A, which also reduced expression of EiIF2K-B. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2α and increased sensitivity to oxidative stress. Diminished kinase expression also correlated with an increased rate of encystation, a decreased rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that EiIF2K-A and EiIF2K-B are authentic eIF2α kinases that may regulate the Entamoeba stress response.IMPORTANCE Entamoeba histolytica is a human pathogen that causes dysentery and affects millions of people worldwide. This parasite possesses a two-stage life cycle: an environmentally stable cyst and the pathogenic trophozoite. Cysts are ingested from contaminated food and water; thus, this parasite in prevalent in underdeveloped countries. Current therapies commonly cause adverse side effects; therefore, new treatments are needed. In the host, Entamoeba experiences stress brought on, in part, by the host immune system. Understanding stage conversion and the stress response of this pathogen may lead to new drug therapies. Using the model organism E. invadens, we identified two kinases similar to those involved in stress and stage conversion in other systems. We determined that these kinases may regulate the oxidative stress response, stage conversion, and virulence. This work is significant, as it will inform future studies on the life cycle and pathogenicity of Entamoeba species.

Long-Term Evolution of Patients with the Wolcott Rallison Syndrome: Case Series of 4 Patients and Review of Literatures

Introduction: Wolcott-Rallison syndrome is a rare autosomal recessive disorder characterized by neonatal diabetes in consanguineous families. associated with liver dysfunction, epiphyseal dysplasia, and. growth retardation. It is caused by mutations in the gene encoding eukaryotic translation initiation factor 2α kinase 3 (EIF2AK3).&#x0D; We report a long-term evolution of 4 patients with Wolcott Rallison syndrome.

Does chronic dietary exposure to the mycotoxin deoxynivalenol affect the porcine hepatic transcriptome when an acute-phase response is initiated through first or second-pass LPS challenge of the liver?

The sensitivity of pigs to deoxynivalenol (DON) might be increased by systemic inflammation (SI), which also has consequences for hepatic integrity. Liver lesions and a dys-regulated gene network might hamper hepatic handling and elimination of DON whereby the way of initiation of hepatic inflammation might play an additional role. First and second-pass exposure of the liver with LPS for triggering a SI was achieved by LPS infusion via pre- or post-hepatic venous route, respectively. Each infusion group was pre-conditioned either with a control diet (0.12 mg DON/kg diet) or with a DON-contaminated diet (4.59 mg DON/kg diet) for 4 wk. Liver transcriptome was evaluated at 195 min after starting infusions. DON exposure alone failed to modulate the mRNA expression significantly. However, pre- and post-hepatic LPS challenges prompted transcriptional responses in immune and metabolic levels. The mRNAs for B-cell lymphoma 2-like protein 11 as a key factor in apoptosis and IFN-γ released by T cells were clearly up-regulated in DON-fed group infused with LPS post-hepatically. On the other hand, mRNAs for nucleotide binding oligomerization domain containing 2, IFN-α and eukaryotic translation initiation factor 2α kinase 3 as ribosomal stress sensors were exclusively up-regulated in control pigs with pre-hepatic LPS infusion. These diverse effects were traced back to differences in TLR4 signalling.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Asymmetrical dimethylarginine induces dysfunction of insulin signal transduction via endoplasmic reticulum stress in the liver of diabetic rats

AimsEndoplasmic reticulum stress (ERS) as an emerging factor is involved in insulin resistance (IR), which is the pathological basis of diabetes mellitus. Accumulation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase is associated with IR, but the underlying mechanisms have not been elucidated. This study was to reveal the important role of ADMA in IR and determine whether endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats and hepatocytes. Materials and methodsDiabetic rat model was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg). Phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) was detected to evaluate IR. The protein kinase PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, x-box binding protein-1 (XBP-1) splicing, glucose-regulated protein 78 (GRP78) and C/EBP homologues protein (CHOP) expressions were measured to assess ERS. Key findingsEndogenous ADMA content was significantly increased and positively correlated with either IR as evidenced by increased IRS1 at serine and reduced Akt phosphorylation or ERS as indicated by upregulations of PERK and eIF2α phosphorylation, XBP-1 splicing, GRP78 and CHOP expressions in the liver of diabetic rats compared with control rats. Exogenous ADMA directly caused IR and ERS in dose- and time-dependent manners in primary mouse hepatocytes. Pretreatment with ERS inhibitor 4-phenylbutyrate or ADMA antagonist L-arginine not only improved ADMA-associated or -induced hepatic IR but also attenuated ADMA-associated or -induced ERS in diabetic rats or hepatocytes. SignificanceThese findings indicate that endogenous ADMA accumulation contributes to hepatic IR via ERS in diabetic rats.

Effect of cis- and trans-Monounsaturated Fatty Acids on Palmitate Toxicity and on Palmitate-induced Accumulation of Ceramides and Diglycerides.

Dietary trans fatty acids (TFAs) have been implicated in serious health risks, yet little is known about their cellular effects and metabolism. We aim to undertake an in vitro comparison of two representative TFAs (elaidate and vaccenate) to the best-characterized endogenous cis-unsaturated FA (oleate). The present study addresses the possible protective action of TFAs on palmitate-treated RINm5F insulinoma cells with special regards to apoptosis, endoplasmic reticulum stress and the underlying ceramide and diglyceride (DG) accumulation. Both TFAs significantly improved cell viability and reduced apoptosis in palmitate-treated cells. They mildly attenuated palmitate-induced XBP-1 mRNA cleavage and phosphorylation of eukaryotic initiation factor 2α (eIF2α) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but they were markedly less potent than oleate. Accordingly, all the three unsaturated FAs markedly reduced cellular palmitate incorporation and prevented harmful ceramide and DG accumulation. However, more elaidate or vaccenate than oleate was inserted into ceramides and DGs. Our results revealed a protective effect of TFAs in short-term palmitate toxicity, yet they also provide important in vitro evidence and even a potential mechanism for unfavorable long-term health effects of TFAs compared to oleate.

PERK-Mediated eIF2α Phosphorylation Contributes to The Protection of Dopaminergic Neurons from Chronic Heat Stress in Drosophila.

Environmental high-temperature heat exposure is linked to physiological stress such as disturbed protein homeostasis caused by endoplasmic reticulum (ER) stress. Abnormal proteostasis in neuronal cells is a common pathological factor of Parkinson’s disease (PD). Chronic heat stress is thought to induce neuronal cell death during the onset and progression of PD, but the exact role and mechanism of ER stress and the activation of the unfolded protein response (UPR) remains unclear. Here, we showed that chronic heat exposure induces ER stress mediated by the PKR-like eukaryotic initiation factor 2α kinase (PERK)/eIF2α phosphorylation signaling pathway in Drosophila neurons. Chronic heat-induced eIF2α phosphorylation was regulated by PERK activation and required for neuroprotection from chronic heat stress. Moreover, the attenuated protein synthesis by eIF2α phosphorylation was a critical factor for neuronal cell survival during chronic heat stress. We further showed that genetic downregulation of PERK, specifically in dopaminergic (DA) neurons, impaired motor activity and led to DA neuron loss. Therefore, our findings provide in vivo evidence demonstrating that chronic heat exposure may be a critical risk factor in the onset of PD, and eIF2α phosphorylation mediated by PERK may contribute to the protection of DA neurons against chronic heat stress in Drosophila.

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

Penehyclidine hydrochloride defends against LPS-induced ALI in rats by mitigating endoplasmic reticulum stress and promoting the Hes1/Notch1 pathway

Penehyclidine hydrochloride (PHC) is a novel anticholinergic drug applied broadly in surgeries as a preanesthetic medication. A substantial amount of research indicates that PHC has lung defensive properties. Considering that endoplasmic reticulum (ER) stress exerts a crucial function in cell apoptosis associated with the lipopolysaccharides (LPS)-induced acute lung injury (ALI) model, we aimed to determine whether regulation of ER stress in the LPS-induced ALI model was associated with the lung defensive role of PHC. Adult male SD rats were administered LPS (5 mg/kg, intratracheally) followed by PHC (1.0 mg/kg, intravenously) for 24 h. The NR8383 alveolar macrophages were randomly separated into Sham, LPS (100 ng/mL), and PHC (1, 2.5, or 5 μg/mL) + LPS groups. PHC (1, 2.5, or 5 μg/mL) + LPS groups were treated with PHC alone for 1 h after LPS exposure. Posttreatment with PHC relieved LPS-induced pulmonary impairment and blocked LPS-mediated lung apoptosis, indicated by the downregulation of the lung apoptotic indicators malondialdehyde and superoxide dismutase in serum at 24 h after LPS-induced ALI. PHC (1–5 μg/mL) did not influence the activity of cultivated NR8383 alveolar macrophages in vitro. However, postconditioning with PHC dosage-dependently reduced LPS-mediated cell apoptosis. Additionally, many studies have indicated that PHC administration inhibits ER stress and initiates hairy and enhancer of split 1 (Hes1)/(Notch1) signaling by decreasing phosphorylated α subunit of eukaryotic initiation factor 2α (p-eIF2α)/eukaryotic translation initiation factor 2α (eIF2α) and Phospho-protein kinase R-like ER kinase (p-PERK)/ protein kinase R-like ER kinase (PERK) proportions; inhibiting C/EBP-homologous protein (CHOP), activating transcription factor 4 (ATF4), caspase-3, and Bcl2-associated x (Bax) activity; and enhancing notch1 intracellular domain (NICD), Notch1, B-cell lymphoma-2 (Bcl-2), and Hes1 activity in vivo and in vitro. In addition, the defensive functions of PHC on LPS-activated NR8383 alveolar macrophages were abrogated through the Notch1 pathway antagonist [(3,5-difluorophenacetyl)-1-alanyl] -phenylglycine-butyl ester (DAPT). In conclusion, PHC alleviates LPS-induced ALI by ameliorating ER stress-mediated apoptosis and promoting Hes1/Notch1 signaling in vivo and in vitro.

Oral administration of a new HRI activator as a new strategy to improve high-fat-diet-induced glucose intolerance, hepatic steatosis, and hypertriglyceridaemia through FGF21.

FGF21 has emerged as a therapeutic strategy for treating type 2 diabetes mellitus due to its antidiabetic effects, and this has led to the development of long-acting analogues of FGF21. However, these compounds have some limitations, including a need to be administered by s.c. injection and their prolonged pharmacodynamic effect compared with native FGF21, which might be responsible for their reported side effects. We have previously demonstrated that i.p. administration of haem-regulated eukaryotic translation initiation factor 2α kinase (HRI) activators increases hepatic and circulating levels of FGF21. In this study, we examined the effects of p.o. administration of a new HRI activator, EPB-53, on high-fat diet (HFD)-induced glucose intolerance, hepatic steatosis, and hypertriglyceridaemia, and compared them with those of metformin. EPB-53 administration for the last 2weeks, to mice fed a HFD for 10weeks, reduced body weight gain, improved glucose intolerance, and prevented hepatic steatosis and hypertriglyceridaemia, whereas metformin only ameliorated glucose intolerance. Moreover, EPB-53, similar to the reported effects of FGF21, reduced lipogenesis in cultured human hepatocytes and in the liver of mice fed a HFD. Administration of EPB-53 to Fgf21-knockout mice had no effects, demonstrating that its efficacy is dependent on this hormone. Overall, the findings of this study demonstrate that p.o. administration of HRI activators, by increasing FGF21, is a promising strategy for the treatment of type 2 diabetes mellitus and non-alcoholic fatty liver disease.