Effect of Neudesin Neurotrophic Factor on Differentiation of Bovine Preadipocytes and Myoblasts in a Co-Culture System

SIMPLE SUMMARY: Marbling beef refers to the red and white beef formed by depositing fat between muscle fibers, and “marbling” is an important factor affecting beef quality. Therefore, we established a co-culture system of adipocytes and myocytes to mimic the microenvironment of marbling beef in vivo. However, Neudesin neurotrophic factor (NENF) was not detected in the co-culture system, but was detected in both adipocytes and myoblasts cultured separately. Further studies revealed that NENF knockdown inhibits adipogenesis and promotes myogenesis in separately cultured preadipocytes and myoblasts. However, because the monoculture system does not include the interaction of bovine adipocytes and myoblasts in the formation of marbling beef, in this study we investigated the effect of recombinant protein NENF on the differentiation of adipocytes and myoblasts in the co-culture system. The addition of NENF inhibited the formation of lipid droplets in co-cultured adipocytes but had no significant effect on myotube formation. These results were different from, and even conflicted with, those in the monocultures, which suggested that regulation of NENF expression in the same cell type changed along with the cell microenvironment and the molecular mechanism of marbling beef formation cannot be fully revealed through studies on the monoculture system. ABSTRACT: 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.