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L Bouraoui, Physiology, Faculty of Biology, University of Barcelona, Barcelona, Spain
J Gutierrez, Physiology, Faculty of Biology, University of Barcelona, Barcelona, Spain
I Navarro, Physiology, Faculty of Biology, University of Barcelona, Barcelona, 08028, Spain

Correspondence: Isabel Navarro, Email: mnavarro{at}ub.edu

Abstract

Here we describe optimal conditions for the culture of rainbow trout (Oncorhynchus mykiss) preadipocytes obtained from adipose tissue and their differentiation into mature adipocytes, in order to study the endocrine control of adipogenesis. Preadipocytes were isolated by collagenase digestion and cultured on laminin or 1% gelatin substrate. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker of cell proliferation on various days of culture. Insulin growth factor-I (IGF-I) stimulated cell proliferation especially on days 5 and 7 of culture. Tumor necrosis factor (TNF ) 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 (C/EBPs) and peroxisome proliferator-activator receptor (PPAR), suggesting that these transcriptional factors are involved in adipocyte differentiation in trout. We also examined the effect of tumor necrosis factor- (TNF) on the differentiation of these adipocytes in primary culture. TNFinhibited the differentiation of these cells, as indicated by a decrease in glycerol-3-phosphate dehydrogenase activity (GPDH), an established marker of adipocyte differentiation. In conclusion, the culture system described here for trout preadipocytes is a powerful tool to study the endocrine regulation of adipogenesis in this species.

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