Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss)

Abstract

Here, we describe optimal conditions for the culture of rainbow trout (Oncorhynchus mykiss) pre-adipocytes obtained from adipose tissue and their differentiation into mature adipocytes, in order to study the endocrine control of adipogenesis. Pre-adipocytes were isolated by collagenase digestion and cultured on laminin or 1% gelatin substrate. The expression of proliferating cell nuclear antigen was used as a marker of cell proliferation on various days of culture. Insulin growth factor-I stimulated cell proliferation especially on days 5 and 7 of culture. Tumor necrosis factor alpha (TNFalpha) slightly enhanced cell proliferation only at a low dose. We verified the differentiation of cells grown in specific medium into mature adipocytes by oil red O (ORO) staining. Quantification of ORO showed an increase in triglycerides throughout culture. Immunofluorescence staining of cells at day 11 revealed the expression of CCAAT/enhancer-binding protein and peroxisome proliferator-activator receptor gamma, suggesting that these transcriptional factors are involved in adipocyte differentiation in trout. We also examined the effect of TNFalpha on the differentiation of these adipocytes in primary culture. TNFalpha inhibited the differentiation of these cells, as indicated by a decrease in glycerol-3-phosphate dehydrogenase activity, an established marker of adipocyte differentiation. In conclusion, the culture system described here for trout pre-adipocytes is a powerful tool to study the endocrine regulation of adipogenesis in this species.