| Adipocyte is the basic unit of storing excess energy in body, including triacylglycerol(TAG) for in case of mobilization and consumption of energy. Hepatocyte is an important part of the body’s metabolism, which is limited in the oxidizability and storage of lipids. Apoptosis is a series of active and orderly death process controlled by a series of apoptotic proteases and apoptosis-related genes. Although the adipocyte and hetapocyte also can store a certain amount of lipids, but it was not clear that the similarities and differences between adipocyte and hetapocyte in the process of lipid accumulation. In this study, we used the methods of gene cloning, primary adipocyte and hepatocyte culture. For comparison the apoptosis characterization and its molecular mechanism of adipocyte and hepatocyte, we have detected the cell viability, lipid content, apoptosis rate, the expression of lipogenesis and apoptosis related genes and protein and caspase activity.The results were as follows:(1) Ten caspase family members of grass carp were cloned, including caspase-a(KX231773), caspase-b(KX231774), caspase-2, caspase-3a(KP145001), caspase-3b(KP145002), caspase-6(KX231775), caspase-7(KX231776), caspase-8(KP145003),caspase-9(KP164812) and caspase-xa. Sequence analysis showed that caspase family of grass carp has a large subunit(P20) and a small subunit(P10) and they also contain a typical conserved sequence: QACRG.(2) The results showed that the cellular lipid content and the expression level of lipogenesis related genes increased at first and then decreased during the differentiation of grass carp adipocytes(P<0.05). But the apoptosis rate of adipocytes decreased significant with the adipocyte differentiation(P<0.05). Real-time PCR analysis showed that the expression levels of caspase-3a, caspase-3b, caspase-8, caspase-9 and AIF gene were significantly decreased, but the Bcl-2/Bax ratio of adipocytes were increased gradually(P<0.05). Meanwhile, caspase-3, caspase-8, caspase-9 activity also decreased significant(P<0.05), indicating that grass carp adipocytes are less sensitive to apoptotic stimuli. It was more difficult to apoptosis comared to the early differentiation of adipocytes.(3) The results showed that the cell viability and intracellular lipid accumulation of hepatocytes were significantly promoted after treatment with 0.6mM and 0.4mM oleic acid(P<0.05). The rate of hepatocytes apoptosiswas the lowest when treated with 0.4mM andhighest with 1mM oleic acid(P<0.05). The mRNA expression levels of caspase-3b and caspase-9 were also significantly reduced and the mRNA ration of Bcl-2/Bax was elevated after treated with 0.4mM oleic acid(P<0.05). Consistently, 0.4m M oleic acid significantly reduced caspase-3b, caspase-9 and AIF mRNA levels and increased the mRNA ratio of Bcl-2/Bax(P<0.05) and these genes showed opposite expression pattern in hepatocytes treated with 0.8mM oleic acid(P<0.05). It could be concluded that a certain concentration of fatty acids can enhance grass carp hepatocytes viability, promote intracellular lipid accumulation and inhibit the apoptosis. But high concentration of fatty acids inhibited hepatocytes viability and induced apoptosis. The influence of fatty acid might be related to its effect on the expression of the genes related to lipid metabolism and apoptosis.In conclusion, although the process of adipocyte differentiation was similar to fatty liver cells that induced by fatty acid.The apoptotic activity of adipocyte, a lipid storage pool, was decreased gradually as the differentiation of cells.Hepatocyte is the major place of metabolism and a certain concentration of fatty acids can enhance the viability and inhibit the apoptosis of cell. But excess free fatty acids were easy to cause the toxicity of hepatocytes and induced apoptosis.Although the adipocyte and hepatocyte can accumulate a certain amount of lipid,but it is different of the apoptosis characteristics. |
PDF Full Text Request