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

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.

Aging increases CCN1 expression leading to muscle senescence

Using microarray analysis, we found that aging sarcopenia is associated with a sharp increase in the mRNA of the matricellular protein CCN1 (Cyr61/CTGF/Nov). CCN1 mRNA was upregulated 113-fold in muscle of aged vs. young rats. CCN1 protein was increased in aging muscle in both rats (2.8-fold) and mice (3.8-fold). When muscle progenitor cells (MPCs) were treated with recombinant CCN1, cell proliferation was decreased but there was no change in the myogenic marker myoD. However, the CCN1-treated MPCs did express a senescence marker (SA-βgal). Interestingly, we found CCN1 increased p53, p16Ink4A, and pRP (hypophosphorylated retinoblastoma protein) protein levels, all of which can arrest cell growth in MPCs. When MPCs were treated with aged rodent serum CCN1 mRNA increased by sevenfold and protein increased by threefold suggesting the presence of a circulating regulator. Therefore, we looked for a circulating regulator. Wnt-3a, a stimulator of CCN1 expression, was increased in serum from elderly humans (2.6-fold) and aged rodents (2.0-fold) compared with young controls. We transduced C2C12 myoblasts with wnt-3a and found that CCN1 protein was increased in a time- and dose-dependent manner. We conclude that in aging muscle, the circulating factor wnt-3a acts to increase CCN1 expression, prompting muscle senescence by activating cell arrest proteins.