The Role And Mechanism Of Hhip In Regulating Adipogenesis Of Preadipocytes

The amount of adipocytes directly determines the content of body fat.the growth and development of adipocytes in vitro include many processes such as proliferation of preadipocytes,initiation of differentiation and terminal differentiation.The deposition of fat in meat animals directly affects carcass quality and meat flavor.Hhip(Hedgehog interacting protein)belongs to the Hedgehog(Hh)signaling pathway,which normally competes with the membrane receptor Patched1 for Hh ligand binding,thereby inhibiting the Hh pathway.A number of studies have shown that Hh signaling pathway could regulate lipid metabolism.However,as an important signaling molecule of Hh signaling,Hhip has not yet reported the biological function of lipid deposition.In order to verify the biological function of Hhip,real-time qPCR and Western blot were used to detect the expression patterns of Hhip and Hh pathway members in the subcutaneous adipose tissue of Guanzhong black pig and the expression pattern in 3T3-L1 cell differentiation;Flow cytometry,EdU staining and CCK8 were used to detect the effects of Hhip on the proliferation of Porcine subcutaneous preadipocytes.Oil Red O staining and Bodipy staining were used to discover the effects of Hhip on preadipocytes differentiation.The main results obtained are as follows:1.Hhip mRNA was differentially expressed in subcutaneous adipose tissue of 3 and 180-day-old pigs;preadipocytes were induced differentiation in vitro,the expression of Hhip mRNA and protein gradually increased,reached the highest peak on the 6 th day of differentiation,and then gradually decreased.2.After treatment of rHhip on porcine subcutaneous preadipocytes,cell cycle related genes Cyclin B,Cyclin D and Cyclin E mRNA levels decreased,Cyclin D and Cyclin E protein levels decreased,Besides the number of cells in S phase decreased.Overall,Hhip suppresses preadipocytes proliferation.3.The treatment with rHhip significantly increased the expression of PPARγ and Glut4,inhibited the expression of Gli family in Hh pathway,and promoted adipocyte differentiation and lipid accumulation.4.Addition of Hh activator PM could weaken the promotion of rHhip on preadipocytes differentiation and reverse the inhibitory effect of Hhip on expression of Gli,indicating Hhip promotes preadipocytes differentiation via suppressing the classical Hh-Gli pathway.