The Regulation Mechanism Of NEFA,BHBA,PRL On Insulin Signaling Pathway In Hypertrophied 3T3-L1 Adipocytes

Lipolysis energy supply is a normal physiological phenomenon that fills the gap in dairy cows' postpartum energy supply and demand under the comprehensive control of multiple factors,but high body condition score(BCS)cows are prone to excessive lipolysis after birth,leading to high morbidity and high elimination rate.Prolactin(PRL)in plasma,as well as fat metabolites non-esterified fatty acids(NEFA)and ?-hydroxybutyric acid(BHBA)play an important role in regulating postpartum dairy cows' energy metabolism.In this experiment,a 3T3-L1 preadipocyte strain was used to construct a mast cell model;the effect of adding different concentrations of NEFA,BHBA,and PRL on the regulation of mature adipocytes and mast cell on lipolysis,fat synthesis,and insulin signaling pathways was studied.,To provide a theoretical basis for revealing the mechanism of post-partum lipolysis in high BCS dairy cows.The test is divided into four parts:1.Establishment of a model of hypertrophic fat cells.The 3T3-L1 mature adipocytes differentiated to the 7th day were starved in serum-free medium(containing 1% BSA)for 12 h,and sodium palmitate solution was added(sodium palmitate concentration in the medium was 0,0.1,0.3,0.6,0.9mmol/L)After 24 h incubation,determine the content of triglyceride(TG),the number and area of??lipid droplets in the cells.The results showed that the TG content and lipid droplet area of ??the 0.3 mmol/L sodium palmitate treatment group increased significantly(P<0.05),and there was no significant difference in the number of lipid droplets between the treatment groups(P>0.05),indicating 0.3 mmol/L palmitic acid The sodium treatment group can establish a model of hypertrophic adipocytes.2.NEFA regulates the mechanism of insulin signaling pathway in mast cells.After adding different concentrations of NEFA to the culture medium of mature adipocytes and hypertrophic adipocytes(the NEFA concentration in the medium is 0,0.15,0.3,0.45 mmol/L)for 9 hours,Western Blotting was used to determine the correlation between fat metabolism and insulin signaling pathway.Protein expression.Fatty acid synthase(FAS)and peroxisome proliferator-activated receptor ?2(PPAR?2)were added to the treatment group with the addition of 0.15,0.3,and 0.45 mmol/L NEFA in the mature adipocyte treatment group.)Significantly reduced protein expression(P<0.05);fatty triglyceride lipase(ATGL)protein expression was significantly reduced,and hormone-sensitive lipase(HSL)protein expression was significantly increased(P<0.05);insulin The signaling pathway-related protein glucose transporter 4(GLUT4)protein expression and the insulin receptor substrate P-IRS1(Ser307)/IRS1 ratio were significantly increased,and the protein kinase B P-AKT(Ser473)/AKT ratio was significantly decreased(P<0.05).The results showed that the addition of 0.15,0.3,and 0.45 mmol/LNEFA ??inhibited fat synthesis and insulin signaling pathways in mast cells,as well as the ability to promote lipolysis of fat demand than mature fat cells.3.The mechanism by which BHBA regulates the insulin signaling pathway of mast cells.After adding different concentrations of BHBA(medium BHBA concentration of 0,0.6,1.2,2.4 mmol/L in culture medium)to mature adipocyte and mast cell culture medium for 9 hours,Western Blotting was used to determine fat metabolism and insulin signaling pathway related proteins The amount of expression.Compared with the 0.6,1.2,and 2.4 mmol/LBHBA mature adipocyte treatment group,the protein concentration of PPAR?2,FAS,HSL,and ATGL in the hypertrophic adipocyte treatment group with the same concentration of BHBA was significantly reduced(P<0.05);insulin signaling pathway was related The protein expression levels of the proteins PI3 K and GLUT4 and the ratio of P-AKT/AKT were significantly reduced,and the ratio of P-IRS1/IRS1 was significantly increased(P<0.05).The results showed that the ability of adding 0.6,1.2,and 2.4 mmol/LBHBA to inhibit fat synthesis of mast cells,basal lipolysis and lipolysis demand was higher than that of mature adipocytes,and inhibited insulin signaling pathway of mast cells.4.PRL regulates the mechanism of insulin signaling pathway in mast cells.After adding different concentrations of PRL(PRL concentration in the medium is 0,25,50,75,100,150ng/mL)to mature adipocyte and mast cell culture medium for 6 hours,Western Blotting was used to measure fat metabolism and insulin signalPathway-related protein expression.Compared with the mature adipocyte treatment group supplemented with 25,50,75ng/mLPRL,the FAS and PPAR?2 protein expression levels of the hypertrophic adipocyte treatment group supplemented with the same concentration of PRL were significantly increased(P<0.05);insulin signaling pathway related protein GLUT4 Protein expression and P-AKT/AKT ratio increased significantly,P-IRS1/IRS1 ratio decreased significantly(P<0.05),and the results of hypertrophic adipocytes in the 100 and 150ng/m L treatment groups were the opposite.The results showed that the addition of 25,50,75ng/mLPRL promoted the fat synthesis capacity of mast cells higher than that of mature adipocytes,and promoted the expression of insulin signaling pathway-related proteins in mast cells,but 100,150ng/mLPRL inhibited fat synthesis of mast cells.And the insulin signaling pathway.In summary,NEFA and BHBA inhibit the expression of insulin signaling pathway-related proteins,thereby damaging their insulin signaling pathways.Contrary to the effects of the 100 and 150ng/mLPRL treatment groups,low-concentration PRL promotes the expression of insulin signaling pathway-related proteins and enhances insulin signaling path.