RESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Estrés del Retículo Endoplásmico , Metabolismo de los Lípidos , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Regulación hacia Arriba , Acetil-CoA Carboxilasa/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/ultraestructura , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Ácido Graso Sintasas/metabolismo , Hígado Graso/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/metabolismo , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/ultraestructura , Receptores X del Hígado , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Sulfonas/farmacología , Tapsigargina/farmacología , Triglicéridos/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.