In this study, we successfully established a co-culture system of bovine preadipocytes and myoblasts to explore the effect of exogenous addition of Neudesin neurotrophic factor (NENF) recombinant protein on the differentiation of adipocytes and myoblasts in co-culture. The optimal concentration of NENF recombinant protein was 100 pg/mL. NENF promoted the differentiation of bovine preadipocytes and inhibited the differentiation of bovine myoblasts when the cells were cultured separately. After adding NENF recombinant protein to the co-culture system, the accumulation of lipid droplets in bovine preadipocytes decreased, but the differentiation of bovine myoblasts did not change significantly. The results of real-time quantitative PCR (RT-qPCR) and Western blot showed that the expression levels of adipogenesis-related factors such as PPARγ, FABP4 and FASN were significantly down-regulated at the mRNA and protein levels in adipocytes, while myogenic marker genes such as MYOD1, MYOG and MYHC had no significant changes at the mRNA or protein levels in myoblasts. This differs from, and potentially conflicts with, the monoculture system, where NENF expression in each cell type changed with the cell microenvironment. Consequently, the molecular mechanism of marbling beef formation cannot be fully revealed using monocultures of adipocytes or myocytes.
Keywords: NENF; bovine; co-culture; myoblasts; preadipocytes.