RESUMO
AIM: Free fatty acid-induced lipotoxicity plays a crucial role in the progression of nonalcoholic fatty liver disease (NAFLD). In the present study we investigated the effects of a high-fat diet and free fatty acids on the autophagic process in hepatocytes in vivo and in vitro and the underlying mechanisms. METHODS: LC3-II expression, a hallmark of autophagic flux, was detected in liver specimens from patients with non-alcoholic steatohepatitis (NASH) as well as in the livers of C57BL/6 mice fed a high-fat diet (HFD) up to 16 weeks. LC3-II expression was also analyzed in human SMMC-7721 and HepG2 hepatoma cells exposed to palmitic acid (PA), a saturated fatty acid. PA-induced apoptosis was detected by Annexin V staining and specific cleavage of PARP in the presence and absence of different agents. RESULTS: LC3-II expression was markedly increased in human NASH and in liver tissues of HFD-fed mice. Treatment of SMMC-7721 cells with PA increased LC3-II expression in time- and dose-dependent manners, whereas the unsaturated fatty acid oleic acid had no effect. Inhibition of autophagy with 3MA sensitized SMMC-7721 cells to PA-induced apoptosis, whereas activation of autophagy by rapamycin attenuated PA-induced PARP cleavage. The autophagy-associated proteins Beclin1 and Atg5 were essential for PA-induced autophagy in SMMC-7721 cells. Moreover, pretreatment with SP600125, an inhibitor of JNK, effectively abrogated PA-mediated autophagy and apoptosis. Specific knockdown of JNK2, but not JNK1, in SMMC-7721 cells significantly suppressed PA-induced autophagy and enhanced its pro-apoptotic activity; whereas specific knockdown of JNK1 had the converse effect. Similar results were obtained when HepG2 cells were tested. CONCLUSION: JNK1 promotes PA-induced lipoapoptosis, whereas JNK2 activates pro-survival autophagy and inhibits PA lipotoxicity. Our results suggest that modulation of autophagy may have therapeutic benefits in the treatment of lipid-related metabolic diseases.
Assuntos
Autofagia/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Ácido Palmítico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Dieta Hiperlipídica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
OBJECTIVE: To establish a novel approach for quick and high throughput verification of human gene imprinting. METHODS: By use of a pair of dye-labeled probes, 5' nuclease assay was combined with reverse transcriptase-PCR(RT-PCR) to genotype a coding single nucleotide polymorphism (cSNP), rs705(C/T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lymphoblast cell lines. RESULTS: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphisms. Pedigree analysis verified the paternal origin of expressed allele, which is in consistency with previous report. CONCLUSION: Coding SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach has also a potentiality to discover differential allele expression of non-imprinted genes in order to find gene cis-acting functional polymorphism.