Transcriptional Levels of Autophagy and UPR Markers in the Brain and Liver of Pregnant Rats

Background: Pregnancy is associated with oxidative stress that results in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Prolonged-unalleviated ER stress causes the activation of the autophagy pathway via UPR. Expression of genes encoding glucose-regulated protein 78 (GRP78) and BECLIN1 are induced in UPR and autophagy. Objectives: We studied the mRNA expression of the aforementioned genes in the liver and brain of Nulligravida versus saline and ethanol-treated pregnant rats. Methods: Control pregnant rats were orally treated with normal saline, and test animals received ethanol 250 mg/kg or resveratrol 120 mg/kg from day 1 to day 21 of gestation. Nulligravida rats treated by saline comprised the non-pregnant control group. On day 21, mRNAs encoding GRP78 and BECLIN1 were extracted from the liver and brain tissues and assessed using real-time PCR. Results: Our results showed that the level of transcripts encoding GRP78 and BECLIN1 was higher in the liver of pregnant rats compared to Nulligravida ones. Further, ethanol decreased the mRNA levels of GRP78 and BECLIN1 in the liver of pregnant rats, an effect that was reversed by resveratrol. Levels of GRP78 transcripts were decreased, and those of BECLIN1 remained unchanged in the brain of ethanol exposed pregnant rats. Conclusions: Levels of mRNAs for GRP78 and BECLIN1 are up-regulated during pregnancy. These levels are reduced in the liver of ethanol-treated rats, and resveratrol compensates these effects.

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Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae

Scientific Reports ◽

10.1038/s41598-019-51054-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Antonisamy William James ◽

Chidambaram Ravi ◽

Malathi Srinivasan ◽

Vasanthi Nachiappan

Keyword(s):

Er Stress ◽

Protein Quality ◽

Protein Glycosylation ◽

Lipid Homeostasis ◽

Sterol Synthesis ◽

Peroxisome Biogenesis ◽

Unfolded Protein ◽

Expression Of Genes ◽

Phospholipid Transport ◽

Protein Response

Abstract The endoplasmic reticulum (ER) is a multi functional organelle and plays a crucial role in protein folding and lipid biosynthesis. The SEC59 gene encodes dolichol kinase, required for protein glycosylation in the ER. The mutation of sec59-1 caused a protein N-glycosylation defect mediated ER stress resulting in increased levels of phospholipid, neutral lipid and sterol, whereas growth was reduced. In the sec59-1∆ cell, the N-glycosylation of vacuolar carboxy peptidase-Y (CPY) was significantly reduced; whereas the ER stress marker Kar2p and unfolded protein response (UPR) were significantly increased. Increased levels of Triacylglycerol (TAG), sterol ester (SE), and lipid droplets (LD) could be attributed to up-regulation of DPP1, LRO1, and ARE2 in the sec 59-1∆ cell. Also, the diacylglycerol (DAG), sterol (STE), and free fatty acids (FFA) levels were significantly increased, whereas the genes involved in peroxisome biogenesis and Pex3-EGFP levels were reduced when compared to the wild-type. The microarray data also revealed increased expression of genes involved in phospholipid, TAG, fatty acid, sterol synthesis, and phospholipid transport resulting in dysregulation of lipid homeostasis in the sec59-1∆ cell. We conclude that SEC59 dependent N-glycosylation is required for lipid homeostasis, peroxisome biogenesis, and ER protein quality control.

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Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0039 ◽

2016 ◽

Vol 27 (9) ◽

pp. 1536-1551 ◽

Cited By ~ 72

Author(s):

Michael E. Fusakio ◽

Jeffrey A. Willy ◽

Yongping Wang ◽

Emily T. Mirek ◽

Rana J. T. Al Baghdadi ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Transcription Factor ◽

Er Stress ◽

Unfolded Protein Response ◽

Cholesterol Metabolism ◽

Unfolded Protein ◽

Expression Of Genes ◽

The Unfolded Protein Response ◽

Protein Response

Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins—PERK (PEK/EIF2AK3), IRE1, and ATF6—is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.

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l-Glutamine Represses the Unfolded Protein Response in the Small Intestine of Weanling Piglets

Journal of Nutrition ◽

10.1093/jn/nxz155 ◽

2019 ◽

Vol 149 (11) ◽

pp. 1904-1910 ◽

Cited By ~ 2

Author(s):

Yu He ◽

Xiaoxiao Fan ◽

Ning Liu ◽

Qingqing Song ◽

Jiao Kou ◽

...

Keyword(s):

Small Intestine ◽

Er Stress ◽

Unfolded Protein Response ◽

Initiation Factor ◽

Control Group ◽

Unfolded Protein ◽

Gene And Protein Expression ◽

Weanling Piglets ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT Background Dysfunction of the endoplasmic reticulum (ER) results in apoptosis, inflammation, and enhanced proteolysis in the small intestine of humans and animals. l-Glutamine (Gln) is required for intestinal mucosal homeostasis in piglets. However, a functional role of the ER in the enterocytes of weanling piglets and its contribution to intestinal mucosal integrity remain largely unknown. Objective This study was conducted to test the hypothesis that preweaning administration of Gln alleviates the activation of unfolded protein response (UPR) in the small intestine of weanling piglets. Methods Eighteen sow-reared piglets aged 7 d from 3 litters (6 piglets/litter) were assigned randomly into 1 of 3 treatment groups. Piglets were reared by sows until age 24 d, or were reared by sows and orally administered either l-alanine [1.84 g · kg body weight (BW)−1 · d−1] or Gln (1.52 g · kg BW−1 · d−1) twice daily between 7 and 21 d of age, and then weaned to a corn- and soybean meal-based diet. The small-intestinal samples were collected at 24 d of age for analyses of abundance of proteins related to ER stress and apoptosis, concentrations of inflammatory cytokines, and mRNA abundance for genes implicated in protein degradation. Results Compared with age-matched suckling piglets, weaning stress increased apoptosis and decreased cell proliferation in the jejunum. The abundance of proteins related to ER stress [binding immunoglobulin protein, activating transcription factor 6α, phosphorylated (p)-inositol-requiring kinase 1α, and p-eukaryotic initiation factor 2α] was elevated by 200% to 320%, and that of apoptotic proteins (CCAAT/enhancer-binding protein homologous protein, p-Jun-N-terminal kinase, caspase-12, cleaved caspase-3, and Bcl-2-associated X) was augmented by 100% to 350% in the jejunum of weanling piglets. The protein abundance for IL-1β, TNF-α, and IL-8 was increased by 100% to 230% in the jejunum of weanling piglets. These alterations in gene and protein expression were markedly abrogated by Gln supplementation. The mRNA concentration of F-Box protein 32 in the jejunum of weanling piglets was increased by 70%, compared with the control group, and was not affected by Gln supplementation. Conclusion Our results indicate that preweaning administration of Gln to nursing piglets alleviates the weaning-activated UPR.

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Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response

Biochemical Journal ◽

10.1042/bj20020391 ◽

2002 ◽

Vol 366 (2) ◽

pp. 585-594 ◽

Cited By ~ 346

Author(s):

Tetsuya OKADA ◽

Hiderou YOSHIDA ◽

Rieko AKAZAWA ◽

Manabu NEGISHI ◽

Kazutoshi MORI

Keyword(s):

Endoplasmic Reticulum ◽

Transcription Factor ◽

Protein Kinase ◽

Er Stress ◽

Double Stranded Rna ◽

Unfolded Protein ◽

Genes Encoding ◽

Membrane Bound ◽

Activating Transcription Factor ◽

Protein Response

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249–258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells.

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Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression

AJP Cell Physiology ◽

10.1152/ajpcell.00391.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. C756-C766 ◽

Cited By ~ 49

Author(s):

András Rab ◽

Rafal Bartoszewski ◽

Asta Jurkuvenaite ◽

John Wakefield ◽

James F. Collawn ◽

...

Keyword(s):

Cystic Fibrosis ◽

Er Stress ◽

Mrna Levels ◽

Transmembrane Conductance Regulator ◽

Wild Type ◽

Transmembrane Conductance ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Cystic Fibrosis Transmembrane

The unfolded protein response (UPR) is a cellular recovery mechanism activated by endoplasmic reticulum (ER) stress. The UPR is coordinated with the ER-associated degradation (ERAD) to regulate the protein load at the ER. In the present study, we tested how membrane protein biogenesis is regulated through the UPR in epithelia, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a model. Pharmacological methods such as proteasome inhibition and treatment with brefeldin A and tunicamycin were used to induce ER stress and activate the UPR as monitored by increased levels of spliced XBP1 and BiP mRNA. The results indicate that activation of the UPR is followed by a significant decrease in genomic CFTR mRNA levels without significant changes in the mRNA levels of another membrane protein, the transferrin receptor. We also tested whether overexpression of a wild-type CFTR transgene in epithelia expressing endogenous wild-type CFTR activated the UPR. Although CFTR maturation is inefficient in this setting, the UPR was not activated. However, pharmacological induction of ER stress in these cells also led to decreased endogenous CFTR mRNA levels without affecting recombinant CFTR message levels. These results demonstrate that under ER stress conditions, endogenous CFTR biogenesis is regulated by the UPR through alterations in mRNA levels and posttranslationally by ERAD, whereas recombinant CFTR expression is regulated only by ERAD.

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IRE1 and efferent signaling from the endoplasmic reticulum

Journal of Cell Science ◽

10.1242/jcs.113.21.3697 ◽

2000 ◽

Vol 113 (21) ◽

pp. 3697-3702 ◽

Cited By ~ 4

Author(s):

F. Urano ◽

A. Bertolotti ◽

D. Ron

Keyword(s):

Gene Expression ◽

Endoplasmic Reticulum ◽

Transcription Factor ◽

Er Stress ◽

Signaling Pathway ◽

Unfolded Protein ◽

Genes Encoding ◽

The Unfolded Protein Response ◽

Protein Response ◽

Stress Signaling Pathway

Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response - UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.

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Caffeine and MDMA (ecstasy) exacerbate ER stress triggered by hyperthermia

10.1101/2022.01.14.476356 ◽

2022 ◽

Author(s):

Kathleen A Trychta ◽

Brandon K Harvey

Keyword(s):

Er Stress ◽

Drugs Of Abuse ◽

Drug Effects ◽

Mrna Levels ◽

Club Drugs ◽

Unfolded Protein ◽

Club Drug Use ◽

The Unfolded Protein Response ◽

Protein Response ◽

Related Proteins

Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion which causes ER exodosis, the secretion of ER resident proteins. Club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. Here we examine the secretion of ER resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER resident proteins and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased BiP/Grp78 mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis contributing to cellular toxicity.

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Messenger RNA levels of enzymes involved in glycerolipid synthesis in the brain of the mouse and its alterations in Agpat2-/- and db/db mice

10.21203/rs.3.rs-128675/v1 ◽

2020 ◽

Author(s):

Lila Gonzalez-Hodar ◽

Anil K. Agarwal ◽

Víctor Cortés

Keyword(s):

Mouse Brain ◽

Messenger Rna ◽

Brain Regions ◽

Mrna Levels ◽

Expression Of Genes ◽

Genes Encoding ◽

Glycerolipid Synthesis ◽

Altered Gene ◽

The Brain

Abstract Background and Aims: Expression of genes encoding enzymes involved in glycerolipid and monoacylglycerol pathways in specific brain regions is poorly known and their alterations in insulin resistance (IR) and type 2 diabetes (T2D) remain unreported. We determined the mRNA levels of enzymes involved in glycerolipid synthesis in specific regions of the mouse brain and their changes in two models of severe IR, the lipodystrophic Agpat2−/− and the obese Leprdb/db mice. Methods Cerebral cortex, hypothalamus, hippocampus and cerebellum were dissected from adult Agpat2−/− mice, Leprdb/db mice and their respective wild type littermates. Total RNA was isolated and the relative mRNA abundance of enzymes was determined by RT-qPCR. Results GPAT1, AGPAT1-4, LIPIN1/2, DGAT1/2 and MOGAT1 mRNAs were detected in all studied brain regions, whereas GPAT2, LIPIN3 and MOGAT2 were undetectable. Abundance of GPAT1, AGPAT1, AGPAT2, AGPAT4, LIPIN1, and MOGAT1, was higher in the hypothalamus. AGPAT3 and DGAT1 were higher in cortex and cerebellum, and LIPIN2 and DGAT2 were higher in cortex and hippocampus. In Agpat2−/− mice, LIPIN1 levels were increased in all the brain regions. By contrast, GPAT1 and AGPAT4 in hypothalamus, AGPAT3 in hippocampus and hypothalamus, and MOGAT1 in cortex, hypothalamus and cerebellum were lower in Agpat2−/− mice. Leprdb/db mice showed fewer and milder changes, with increased levels of GPAT1 and LIPIN1 in cerebellum, and AGPAT3 in hypothalamus. Conclusions Enzymes involved in glycerolipids synthesis are differentially expressed across regions of the mouse brain and IR and T2D determine altered gene expression of these enzymes in the mouse brain.

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Aging alters the metabolic flux signature of the ER unfolded protein response in vivo in mice

10.1101/2021.04.14.439896 ◽

2021 ◽

Author(s):

Catherine P Schneider ◽

Lucy Peng ◽

Sam Yuen ◽

John Halstead ◽

Hector Palacios ◽

...

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

De Novo ◽

De Novo Lipogenesis ◽

Mrna Levels ◽

Aged Mice ◽

Unfolded Protein ◽

Protein Response ◽

Young Mice

Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPRER) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a signature of the UPRER in vivo in mouse liver was developed by inducing ER stress and measuring rates of both proteome-wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared to young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, only a significant reduction was seen in the triglyceride fraction. These data indicate that aged mice have an exaggerated response to ER stress, which may indicate that the aging renders the UPRER less effective in resolving proteotoxic stress.

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