| This study aims to investigate the mechanism of AMPKa/SIRT1 signalling pathway in hyperlipidemia dairy cows with lipid metabolism disorder which will lay a theoretical basis for elucidating the pathogenesis of hyperlipidemia in dairy cows and finding therapeutic targets.The blood of the cows was collected from the jugular vein,and the serum was separated.After testing the serum triglyceride(TG)and total cholesterol(TC)content,12 cows were selected and divided into the control group and the high fat group,6 cows in each group.The perirenal adipose tissue sample was collected under aseptic conditions after killing dairy cows by routine method.The contents of glucose,liver function,lipid metabolism indexes such as TG and TC in serum and adipose tissue were detected by automatic biochemical analyzer.HE staining and oil red O staining were used to observe the pathological changes and lipid accumulation of adipose tissue.Serum nonesterified fatty acids(NEFA)and β-hydroxybutyric acid(BHBA)were detected by enzyme-linked immunosorbent assay(ELISA).Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative genes expression of lipid metabolism and SIRT1/AMPKa signaling pathway.Western blotting was used to detect the expression levels of key proteins in SIRT1/AMPK alpha signaling pathway,SREBP-lc,PPAR gamma 2 and other important lipid metabolism proteins.The adipocytes of newborn calves were isolated and cultured aseptically.The morphological changes was observed during the primary and differentiation process.The TG content and the expression of PPARy2 and SREBP-1c mRNA were detected during adipocyte differentiation.The CCK8 method was used to determine the optimal action time of NEFA and BHBA treatment.Cells were collected at the optimal time point by adding different concentrations of NEFA or BHBA solutions(0,0.6,1.2,2.4 mM)after differentiation and maturation.The effects of NEFA and BHBA on lipid metabolism in adipocytes were detected by automatic biochemical analyzer.The effects of NEFA and BHBA on SIRT1/AMPKa signaling pathway,lipid metabolism related genes,SREBP-lc and PPARγ2 protein expression levels in adipocytes were detected by qRT-PCR and Western blotting.ELISA was used to detect the effects of NEFA and BHBA on ATP and AMP contents and AMPKa2 activity in adipocytes.Results:1.Compared with the control group,serum INS levels in high-fat dairy cows were significantly lower(P<0.05),NEFA and BHBA levels were significantly increased(P<0.05),and lipid metabolism indicators TC and TG levels in adipose tissue were significantly increased(P<0.05).2.HE staining showed that the cells in the high-fat group were swollen,the volume became larger,and some cell lipid droplets increased.Oil red O staining showed that the lipid droplets of the high-fat group merged to form vacuolar fat cells,the cell boundaries were blurred,the cell outline was unclear,and the volume became large.3.Compared with the control group,the relative expression of SIRT1 and LKB1 mRNA in the adipose tissue of the high fat group was significantly decreased(P<0.05).The mRNA expression level of ACSL,a key enzyme gene in lipid synthesis,was significantly increased(P<0.05),and the relative mRNA expression of key enzymes SREBP-lc and PPARy2 in lipid synthesis regulation was significantly increased(P<0.05).The relative mRNA expression of ACO,a key enzyme gene for lipolysis,was significantly decreased(P<0.05).The results of protein expression showed that the ratio of p-LKB1/LKB1 and p-AMPKa2/AMPKa2 in the high-fat group was significantly lower than that in the control group(P<0.05),and the expression of lipid-oxidized protein CPT1 was significantly decreased(P<0.01),the expression levels of lipid oxidation and protein ACO and HSL were significantly decreased(P<0.05),and the expression level of lipoprotein FAS was significantly increased(P<0.05).The expression levels of lipid regulatory proteins PPARy and SREBP-lc were significantly increased(P<0.05).4.After the primary cultured adipocytes were induced to differentiate and mature,the optimal time for NEFA and BHBA were determination by CCK8 method was 12 h and 9 h,respectively.Compared with the control group,the levels of TC and TG in the 1.2,2.4 mM NEFA treatment group and 1.2 and 2.4 mM BHBA treatment groups were significantly increased(P<0.05).5.The relative mRNA expressions of AMPKa2,LKB1 and SIRT1 decreased significantly with the increase of NEFA and BHBA concentration(P<0.05).The expression was increased when AICAR was added.The relative expression of lipid synthesis genes ACCa and FAS in 2.4 mM treatment groups was decreased significantly(P<0.05),and the expression of HSL and CPT1 was significantly decreased in the 2.4 mM treatment groups(P<0.05).When AICAR was added,the expression of HSL and CPT1 were decreased.The expression level of PPARy2 mRNA and SREBP-lc was significantly increased in the 1.2 and 2.4 mM treatment groups(P<0.05),and the expression level was decreased when AICAR was added(P<0.05).The results of protein expression test showed that the expression of AMPKa2,LKB1,SIRT1 decreased significantly with the increase of NEFA and BHBA concentration in 2.4 mM treatment group(P<0.05),the expression of PPARγ 2 and SREBP-lc increased significantly with the increase of NEFA and BHBA concentration in 1.2 and 2.4 mM treatment group(P<0.05),and the expression of both decreased with the addition of AICAR(P>0.05).6.The AMP content in adipocytes was significantly lower in the 1.2 and 2.4 mM treatment groups(P<0.05),The AMP content in the AICAR treatment group was higher than that in the control group(P>0.05);the ATP expression trend was opposite.The AMP/ATP ratio was significantly lower in the 1.2 and 2.4 mM treatment groups than in the control group(P<0.05),indicating that the AMPKa activity was significantly decreased(P<0.05).Conclusion:Lipid metabolism disorder exists in hyperlipidemic dairy cows,which can be regulated by inhibiting SIRT1/AMPKα signaling pathway,promoting lipid synthesis,inhibiting lipid oxidation and further causing lipid deposition. |
PDF Full Text Request