RESUMO
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.
Assuntos
Carcinoma Hepatocelular/metabolismo , Estresse do Retículo Endoplasmático , Metabolismo dos Lipídeos , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Regulação para Cima , Acetil-CoA Carboxilase/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Ácido Graxo Sintases/metabolismo , Fígado Gorduroso/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/ultraestrutura , Receptores X do Fígado , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Sulfonas/farmacologia , Tapsigargina/farmacologia , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Accumulation of saturated fatty acids in the liver can cause nonalcoholic fatty liver disease (NAFLD). This study investigated saturated fatty acid induction of endoplasmic reticulum (ER) stress and apoptosis in human liver cells and the underlying causal mechanism. Human liver L02 and HepG2 cell lines were exposed to the saturated fatty acid sodium palmitate. MTT assay was used for cell viability, flow cytometry and Hoechst 33258 staining for apoptosis, RT-PCR for mRNA expression, and Western blot for protein expression. Silence of PRK-like ER kinase (PERK) expression in liver cells was through transient transfection of PERK shRNA. Treatment of L02 and HepG2 cells with sodium palmitate reduced cell viability through induction of apoptosis. Sodium palmitate also induced ER stress in the cells, indicated by upregulation of PERK phosphorylation and expression of BiP, ATF4, and CHOP proteins. Sodium palmitate had little effect on activating XBP-1, a common target of the other two canonical sensors of ER stress, ATF6, and IRE1. Knockdown of PERK gene expression suppressed the PERK/ATF4/CHOP signaling pathway during sodium palmitate-induced ER stress and significantly inhibited sodium palmitate-induced apoptosis in L02 and HepG2 cells. Saturated fatty acid-induced ER stress and apoptosis in these human liver cells were enacted through the PERK/ATF4/CHOP signaling pathway. Future study is warranted to investigate the role of these proteins in mediating saturated fatty acid-induced NAFLD in animal models.