leupeptins and Non-alcoholic-Fatty-Liver-Disease

Non-alcoholic fatty liver disease closely contributes to the development of obesity and insulin resistance. Even though pioglitazone has been reported to effectively lessen hepatic steatosis in human studies, its molecular mechanism remains unclear. This study is designed to investigate the regulation of cytosolic lipolysis, β-oxidation and autophagy by pioglitazone in a mice model of high fat diet (HFD) and cell model incubated with palmitic acid. Our results revealed hepatic steatosis was apparently induced by HFD and it was significantly reversed by pioglitazone. The serum insulin and hepatic triglyceride content was significantly decreased by co-administered pioglitazone with HFD. Hepatic expression of cytosolic-lipolysis related proteins (ATGL, HSL), β-oxidation (CPT-1A) and autophagy-related proteins (ATG7, LC3, LAL) was significantly enhanced by pioglitazone. Knockdown PPARα/PPARγ in AML12 cells significantly and proportionally reduced the expressions of ATGL, CPT-1A and LC3II, which was induced by pioglitazone. Furthermore, facilitation of the autophagic flux by pioglitazone was obviously blocked by lysosomal inhibitor, leupeptin, to demonstrate accumulation of the LC3II and intracellular lipid in AML12 cells. Our results demonstrated that pioglitazone attenuating the hepatic steatosis may be mediated by enhancing cytosolic lipolysis, β-oxidation and autophagy in a PPARα and PPARγ dependent manner.

Topics: Animals; Autophagy; Cell Line; Diet, High-Fat; Disease Models, Animal; Humans; Insulin; Leupeptins; Lipolysis; Male; Mice; Non-alcoholic Fatty Liver Disease; Oxidation-Reduction; Palmitic Acid; Pioglitazone; PPAR alpha; PPAR gamma; Triglycerides

Nonalcoholic fatty liver disease (NAFLD) is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA)-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD.

Topics: Base Sequence; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; DNA Primers; Fatty Acids, Nonesterified; Gene Silencing; Humans; Leupeptins; Non-alcoholic Fatty Liver Disease; Real-Time Polymerase Chain Reaction