Abstract
Defective autophagy has been linked to lipotoxicity in several cellular models. We aimed to investigate autophagy in lipid-stimulated hepatoma (Huh7) cells and tested whether 4-phenyl butyric acid (4-PBA), a chemical chaperone, has a beneficial role in hepatic fat accumulation and lipotoxicity. We report that long-term (24 h) exposure of hepatocytes to palmitate block autophagic flux that leads to lipid accumulation and cell death. Western blotting analysis showed increased accumulation of SQSTM1/p62, and decreased expression of Beclin1 and Atg7 in palmitate-treated cells. Autophagy inhibition by 3-methyladenine (3-MA) in palmitate-treated cells neither increased SQSTMI/p62 accumulation nor cell death, thus suggesting complete blockade of autophagy by palmitate. 4-PBA reduced lipid accumulation and cell death that were associated with restoration of autophagy. siRNA-mediated knockdown of Atg7 and presence of autophagy inhibitors, 3-MA and chloroquine, resulted in the decrease in lipid-lowering effect of 4-PBA, suggesting that 4-PBA mediates its lipid-lowering effect via autophagy. Apoptotic parameters, including altered Bcl2:Bax ratio and PARP1 cleavage induced by palmitate, were improved by 4-PBA. Our results indicate that palmitate impairs autophagy and increases lipid accumulation in Huh7 cells, whereas 4-PBA plays a protective role in lipid accumulation and lipotoxicity through activation of autophagy.
Highlights
Defective autophagy has been linked to lipotoxicity in several cellular models
Impaired autophagy in the liver results in protein inclusions in cytosol, such as misfolded proteins and excess accumulation of damaged organelles, which lead to various pathological conditions, including liver disorders [29]
Therapeutic strategies to increase autophagic function may provide a new approach to prevent the lipotoxicity and development of Nonalcoholic fatty liver disease (NAFLD). In view of these facts, we aimed to assess the autophagy in FFA-treated human liver cells and mouse hepatocytes (AML-12 cells), and attention was drawn to the antilipophagic effects of a chemical chaperone, 4-phenyl butyric acid (4-PBA), on restoration of autophagy and clearance of lipid droplet (LD) accumulation in lipid-stimulated hepatocytes (Huh7 and AML-12 cells)
Summary
Defective autophagy has been linked to lipotoxicity in several cellular models. We aimed to investigate autophagy in lipid-stimulated hepatoma (Huh7) cells and tested whether 4-phenyl butyric acid (4-PBA), a chemical chaperone, has a beneficial role in hepatic fat accumulation and lipotoxicity. The primary cause of the accumulation of lipids and subsequent formation of LDs in liver cells is not yet clear, it may arise from increased supply of lipids, de novo lipogenesis, impaired lipoprotein synthesis, or secretion or reduced fatty acid oxidation [14]. Further evidence for the role of autophagy in hepatocyte lipid accumulation is that mice with a specific knockout of Atg developed massively enlarged livers mainly due to increased TG and cholesterol content [15]. These findings suggest that impaired autophagy or defective lipophagy may underlie the development of lipid-associated disease such as NAFLD. We have reported increased levels of cytosolic calcium in palmitate-treated immortalized hepatoma cells [9]
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