Metabolic Function Research On Bovine FABP4Gene And Analysis Of Varietal Features On Adipocyte Development

The aim of this study was to detect effects of FABP4(fatty acid binding protein4) geneon expressions of differentiation and lipid metabolism related genes in bovine preadipocytes.In addition, varietal differences in the structure of subcutaneous fat tissue, adipogenesis andmitogenesis of preadipocytes between Wagyu and Angus cattle were evaluated in this study.The main contents were as follows:(1) Adenovirus AD-FABP4and effective AD-shRNA-261were constructed,respectively. Primary bovine preadipocytes were acquired via enzyme digestion method.Twenty-four hours after transfecting bovine preadipocytes with AD-FABP4, the mRNAcontent of FABP4increased176%(P <0.01). Moreover, AD-shRNA-261transfectionresulted in a41%decrease (P <0.01). The results show AD-FABP4and AD-shRNA-261could be used in further experiments.(2) Functions of FABP4gene on differentiation and lipid metabolism of bovinepreadipocytes were analyzed via Real-time PCR (polymerase chain reaction). Results showthat as one of the downstream genes of PPARγ (peroxisome proliferator-activated receptor γ)signaling pathway, FABP4failed to influence adipocyte differentiation related geneexpressions, including PPARγ, CEBPA (CCAAT/enhancer-binding protein A), and SREBP1(sterol regulatory element binding protein1)(P>0.05). In addition, FABP4did not influenceexpressions of FAS (fatty acid synthase) and FATP1(fatty acid transporter member1)(P>0.05) but positively regulated gene expressions of ADIPOQ (adiponectin) and LEP (leptin)(P<0.05). FABP4played a key role in lipid metabolism networks of bovine adipocytes.(3) Paraffin sections and H&E (hematoxylin and eosin) staining were processed tocompare the structure of subcutaneous fat tissue between Wagyu and Angus cattle. In addition,varietal differences in adipogenesis and mitogenesis of primary preadipocytes were evaluatedvia Oil Red-O staining, Real-time PCR, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, and Western blotting technology. Histological analysisshowed that Wagyu cattle appeared a shift toward larger mature adipocytes compared toAngus cattle. As assessed by Oil Red-O staining, Wagyu preadipocytes possessed a notabletrend of lower adipogenic ability (P=0.094). The mRNA or protein expression of PPARγ in Wagyu preadipocytes and fat tissue was lower compared to Angus cattle, which played a rolein the reduced adipogenesis in Wagyu subcutaneous fat. Interestingly, significant highermitogenic ability was discovered in Wagyu preadipocytes (P=0.028), which was associatedwith the elevated phosphorylation of extracellular signal-regulated kinase (Erk)-1/2(P=0.013). Moreover, exogenous fibroblast growth factor (FGF)-2enhanced mitogenesis andErk1/2activity of preadipocytes to a greater degree in Angus compared to Wagyu.Comparisons of mRNA expressions for several autocrine growth factors in Wagyu and Anguscattle showed the expression of transforming growth factor (TGF)-β3(P=0.008) and bonemorphogenetic protein (BMP)-2(P=0.043) in Wagyu preadipocytes was lower than that ofAngus cattle, providing potential clues for varietal differences on mitogenesis ofpreadipocytes in these two cattle breeds.