Metformin attenuates lipid accumulation in hepatocytes of blunt snout bream (Megalobrama amblycephala) via activation of AMP-activated protein kinase

Currently, there is a trend to use high-fat diets in intensive aquaculture that is accompanied with incidence of fatty liver when dietary lipid level surpasses an upper limit. So, it is necessary to develop appropriate strategies to reduce the risk of fatty liver in commercial fish farming. Studies in mammals have revealed a correlation between fatty liver and AMP-activated protein kinase (AMPK) activity, which has been recognized as a key modulator of lipid metabolism. Considering the frequent occurrence of fatty liver in blunt snout bream farming, an in-vitro study was designed to evaluate the efficiency of metformin, as a stimulator of AMPK, in activation of AMPK and its subsequent effects on lipid metabolism in primary hepatocytes. Fish hepatocytes were seeded at a density of 1 x 10(6) ml(-1) in 6-well tissue culture plates and treated with three different media including: 1) Leibovitz's L-15 medium [L15] as control, 2) high-fat medium [L15 + 400 mu M oleic acid], and 3) metformin medium [L15 + 400 mu M oleic acid + 200 mu M metformin]. After 48 h of culture, the cells and supernatant were collected for analysis. The results showed significant (P < .05) enhancement of cell triglyceride and total cholesterol concentrations in the high-fat group over control, and metformin addition significantly reduced the values. Also, the high-fat group exhibited significantly higher aspartate aminotransferase activity than both control and metformin groups. The lowest AMPK and phospho-AMPK protein expression was found in the high-fat group while metformin addition significantly up-regulated their expression levels. Mitochondrial and peroxisomal oxidation rates in the high-fat group were significantly lower than control while similar oxidation rates were observed for metformin treated and control groups. The high-fat group showed significantly lower CPT I activity than control, and metformin inclusion increased the activity. Expression of genes associated with lipid metabolism such as PPAR alpha, CPT I, AOX, PGC-1 alpha and TFAM was suppressed in the high-fat group, and metformin supplementation up-regulated their expression levels. The opposite trend was true for the expression of ACC2 gene. Also, the results showed down-regulation of FAS and SREBP-1C genes in the high-fat medium group, and metformin addition resulted in further reduction of their expression level. The lowest activities of mitochondrial complexes (I-III) were found in the high-fat group and metformin prevented high-fat-induced reduction of mitochondrial complexes activity. Notably increased concentrations of reactive oxygen species and malondialdehyde were found in the high-fat group, and metformin treatment reduced their concentrations. Moreover, metformin group exhibited higher glutathione peroxidase activity than the high-fat group. The findings in this study showed clearly that metformin activated AMPK in blunt snout bream hepatocytes, which contributed to enhanced lipid metabolism and attenuated lipid deposition in the cells incubated with high-fat medium.