thapsigargin and Fatty-Liver

Topics: Animals; Autophagosomes; Autophagy; Berberine; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Fatty Liver; Liver; Liver Transplantation; Male; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Oxidative Stress; Rats; Rats, Wistar; Thapsigargin

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.

Topics: Animals; Brefeldin A; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fatty Liver; Glycosylation; Heat-Shock Proteins; Liver; Regulatory Factor X Transcription Factors; Thapsigargin; Transcription Factors; Tunicamycin; Unfolded Protein Response; Up-Regulation; Zebrafish; Zebrafish Proteins

Endoplasmic reticulum stress (ERS) has been found in non-alcoholic fatty liver disease. The study was to further explore the mechanistic relationship between ERS and lipid accumulation. To induce ERS, the hepatoblastoma cell line HepG2 and the normal human L02 cell line were exposed to Tg for 48 h. RT-PCR and Western blot were performed to evaluate glucose-regulated protein (GRP-78) expression as a marker of ERS. ER ultrastructure was assessed by electron microscopy. Triglyceride content was examined by Oil Red O staining and quantitative intracellular triglyceride assay. The hepatic nuclear sterol regulatory element-binding protein (SREBP-1c), liver X receptor (LXRs), fatty acid synthase (FAS), and acetyl-coA carboxylase (ACC1) expressions were examined by real-time PCR and Western blot. 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was used to inhibit S1P serine protease inhibitor, and SREBP-1c cleavage was evaluated under ERS. SREBP-1c was knockdown and its effect on lipid metabolism was observed. Tg treatment upregulated GRP-78 expression and severely damaged the ER structure in L02 and HepG2 cells. ERS increased triglyceride deposition and enhanced the expression of SREBP-1c, FAS, and ACC1, but have no influence on LXR. AEBSF pretreatment abolished Tg-induced SREBP-1c cleavage. Moreover, SREBP-1c silencing reduced triglycerides and downregulated FAS expression. Pharmacological ERS induced by Tg leads to lipid accumulation through upregulation of SREBP-1c in L02 and HepG2 cells.

Topics: Acetyl-CoA Carboxylase; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fatty Acid Synthases; Fatty Liver; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Lipid Metabolism; Liver; Liver Neoplasms; Liver X Receptors; Orphan Nuclear Receptors; Proteolysis; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Sulfones; Thapsigargin; Triglycerides; Up-Regulation

Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis. Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c. Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents. Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice. To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector. GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes. Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice. These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation. Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes. These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fatty Liver; Gene Expression; Glucose; Heat-Shock Proteins; Hepatocytes; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Lipid Metabolism; Liver; Male; Mice; Mice, Obese; Models, Biological; Molecular Chaperones; Nuclear Proteins; Obesity; Rats; Rats, Wistar; Rats, Zucker; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Thapsigargin; Transcription Factors

Adiponectin receptors play a key role in steatosis and inflammation; however, very little is known about regulation of adiponectin receptors in liver. Here, we examined the effects of palmitate loading, endoplasmic reticulum (ER) stress, and the hypolipidemic agent fenofibrate on adiponectin receptor R2 (AdipoR2) levels and AMP-activated protein kinase (AMPK) in human hepatoma Huh7 cells and in Huh.8 cells, a model of hepatitis C-induced steatosis. Palmitate treatment reduced AdipoR2 protein and basal AMPK phosphorylation in Huh7 cells. Fenofibrate treatment preserved AdipoR2 and phosphorylated AMPK (pAMPK) levels in palmitate-treated cells accompanied by reduced triglyceride (TG) accumulation and less activation of ER stress markers CCAAT/enhancer binding (C/EBPbeta) and eukaryotic translation initiation factor 2 alpha. ER stress agents thapsigargin and tunicamycin suppressed AdipoR2 and pAMPK levels in Huh7 cells, while fenofibrate and the chemical chaperone 4-phenylbutyrate (PBA) prevented these changes. AdipoR2 levels were lower in Huh.8 cells and fenofibrate treatment increased AdipoR2 while reducing activation of c-Jun N-terminal kinase and C/EBPbeta expression without changing TG levels. Taken together, these results suggest that fatty acids and ER stress reduce AdipoR2 protein and pAMPK levels, while fenofibrate and PBA might be important therapeutic agents to correct lipid- and ER stress-mediated loss of AdipoR2 and pAMPK associated with nonalcoholic steatohepatitis.

Topics: AMP-Activated Protein Kinases; Blotting, Western; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Fatty Acids; Fatty Liver; Fenofibrate; Hepacivirus; Humans; Hypolipidemic Agents; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Phenylbutyrates; Phosphorylation; Receptors, Adiponectin; Thapsigargin; Triglycerides; Tunicamycin