Effects Of Neuroendocirne Factors And Mill Key Enzymes Involved To Fatty Acid Metabolism In Bovine Hepatocytes Cultured In Vitro

In this study, bovine hepatocytes were primary cultured in vitro. RT-qPCR andWestern Blotting were used to research the effects of energy metabolism relatedneuroendocrine factors (such as IGF-, ADPN and NPY), and milk fat precursors(AcOH, BHBA and NEFA) on the transcriptional and translational levels of the keyenzymes ACSL1, CS and ACCα for hepatic fatty acid metabolism of dairy cow. Theobjective of this study is to probe the regulation mechanism of IGF-, ADPN andNPY on the metabolism mentioned above, and the feed back regulation mechanismof AcOH, BHBA and NEFA on the bovine hepatic fatty acid metabolism. Thenidentify the regulation mechanism of these factors mentioned above on the milk fatproduction of dairy cow, which may provide rationale and systematic data for thefurther study.According to the published sequences in GenBank, primers and TaqMan probeswere designed and RT-PCR was employed to amplify the partial mRNA of ACSL1,CS and ACCα, and the target sequences were recombinated into cloning vector. Themethod of RT-qPCR was established to detect the mRNA expression of ACSL1, CSand ACCα.In the experiment of the effects of IGF-, ADPN and NPY on thetranscriptional and translational levels of the key enzymes ACSL1, CS and ACCαthat catalysis fatty acid metabolism in hepatocyte of dairy cow. The total RNA andprotein were isolated from hepatocytes, which were treated with differentconcentrations of IGF-Ⅰ (0,10,30,50,100and150ng/mL), ADPN (0,8,16,32,64and128ng/mL) and NPY (0,50,100,200,500and1000pg/mL) after12h. The transcriptional and translational levels of the key lipid metabolism genes weredetermined with the methods RT-qPCR and Western Blotting, respectively. Theresults showed that:①the transcriptional and translational levels of ACSL1and CSwere decreased with increasing concentration of IGF-Ⅰ in culture media, but thelevels of ACCα were increased significantly.②the transcriptional and translationallevels of ACSL1and CS were upregulated with increasing concentration of ADPN inculture media significantly, but the levels of ACCα were downregulated significantly.③the transcriptional and translational levels of ACSL1and CS were decreased withincreasing concentration of NPY in culture media, but the levels of ACCα wereincreased significantly.In the experiment of the effects of AcOH, BHBA and NEFA on thetranscriptional and translational levels of the key enzymes ACSL1, CS and ACCαthat catalysis fatty acid metabolism in hepatocyte of dairy cow. The total RNA andprotein were isolated from hepatocytes, which were treated with differentconcentrations of AcOH (0,0.9,1.8,3.6,7.2and14.4mmol/L), BHBA (0,0.3,0.6,1.2,2.4and4.8mmol/L) and NEFA (0,0.3,0.6,1.2,2.4and4.8mmol/L) after12h.The transcriptional and translational levels of the key lipid metabolism genes weredetermined with the methods RT-qPCR and Western Blotting, respectively. Theresults showed that:①the transcriptional and translational levels of ACSL1and CSwere increased with increasing concentration of AcOH in the limited range in culturemedia, but the levels of ACCα were decreased significantly.②the transcriptionaland translational levels of ACSL1, CS and ACCα were decreased with increasingconcentration of BHBA in culture media significantly.③the transcriptional andtranslational levels of ACSL1and CS were upregulated with increasingconcentration of NEFA in the limited range in culture media, but the levels of ACCαwere downregulated significantly.In conclusion: Firstly, IGF-I and NPY would decrease hepatocytes fatty acidoxidative metabolism of dairy cow and increase the de nove synthesis of fatty acid.Secondly, ADPN would promote fatty acid oxidative metabolism and inhibit the denove synthesis of fatty acid. Thirdly, appropriate plasma concentration of AcOH and NEFA would promote lipid oxidation, but high concentration would inhibit it, andthe de nove synthesis of fatty acid was promoted with the high concentration ofAcOH and NEFA. Fourthly, high concentration of BHBA would inhibit lipidsynthesis and oxidation through decrease the transcriptional and translational levelsof ACSL1, CS and ACCα. Finally, the energy metabolism related neuroendocrinefactors and milk fat precursors would regulate the hepatocytes fatty acid metabolism,which may effect the production and supply of milk fat precursors, then effect themilk fat synthesis.