Effects Of SIRT3 On Lipid Metabolism In Primary Hepatocytes Of Dairy Cows

Silent information regulator 3(SIRT3 or Sirtuin3)is the main deacetylase in mitochondria,which plays a key role in regulating lipid metabolism in both human and rodents.However,the role and its mechanism of SIRT3 in lipid metabolism in ruminants remain unclear.In this study,effects of SIRT3 on lipid metabolism of primary hepatocytes of dairy cows were investigated by transfecting with si-RNA and SIRT3 overexpressed adenovirus and adding different concentrations of non-esterified fatty acids(NEFAs).The methods and results were as follows:1.Calf primary hepatocytes were isolated and cultured by a two-step collagenase perfusion method.Results showed that the cell shape was irregular,the nucleus round andclear,and the boundary was clearly visible.The cells were in good condition and could be used in subsequent experiments.2.Western blot,qPCR and oil red O staining were used to detect effects of different concentrations of NEFAs on SIRT3 expression and lipid accretion.It was found that 0.3 mM NEFAs promoted SIRT3 expression(P < 0.001),but 0.6 mM and 1.2 mNEFAs inhibited SIRT3 expression(P < 0.001).Also,0.6 mM and 1.2 mM NEFAs treatment resulted in fat deposition in hepatocytes in a dose-dependent manner.3.The effects of SIRT3 overexpression,silence and high concentration of NEFAs and SIRT3 overexpression on lipid metabolism related genes FAS,ACC-1,CPT-I,CTP-II,ACO,MTP,ApoB and ApoE were detected by qPCR.The results showed that SIRT3 overexpression inhibited the expression of FAS,ACC-1(P < 0.05),and promoted the expression of CPT-I,CTP-II,ACO and MTP,ApoB and ApoE(P < 0.001);SIRT3 silence showed no statistical analysis in the expression of FAS,ACC-1(P > 0.05),but inhibited the expression of CPT-I,CTP-II,ACO,MTP,ApoB and ApoE(P < 0.01).A high concentration of NEFAs promoted the expression of FAS and ACC-1(P < 0.01),but inhibited the expression of CPT-I,CTP-II,ACO,MTP,ApoB and ApoE(P < 0.05),while SIRT3 overexpression could counteract the effect of NEFAs(P < 0.01).4.Effects of SIRT3 overexpression,silence and high concentration of NEFAs and SIRT3 overexpression on intracellular TG were detected by biochemical kits.The results showed that SIRT3 overexpression decreased intracellular contents of TG(P < 0.05);SIRT3 silence slightly increased intracellular contents of TG(P > 0.05);high concentration of NEFAs significantly increased intracellular contents of TG(P < 0.001),while SIRT3 could block the effect of NEFAs(P < 0.01).5.ELISA was used to detect effects of SIRT3 overexpression,silence and high concentration of NEFAs and SIRT3 overexpression on contents of VLDL in cell supernatants.The results showed that over-expression of SIRT3 increased contents of VLDL in cell supernatants(P < 0.01);silence of SIRT3 decreased contents of VLDL in cell supernatants(P < 0.05);high concentration of NEFAs could reduce contents of VLDL in cell supernatants(P < 0.05),while SIRT3 could counteract the effect of NEFAs(P < 0.01).In conclusion,SIRT3 reduced the accumulation of lipid in cells by inhibiting the expression of genes related to lipid synthesis and promoting the expression of genes related to lipid decomposition.The results of this study can lay a theoretical foundation for exploring new targets for prevention and treatment of energy metabolic disorders in dairy cows.