Ob gene expression during differentiation of preadipocytes in rat stromal-vascular cultures

Alterations in ob gene expression have been observed in response to adipose tissue expansion and the development of obesity. However, little is known regarding specific factors involved in the acute or chronic regulation of the ob gene. Primary cultures of stromal-vascular (S-V) cells may provide a useful in vitro system for the study of ob gene regulation during adipocyte development. In these studies rat S-V cell cultures were used to examine ob gene expression at different stages of adipocyte differentiation and in response to hormonal manipulation. Stromal-vascular cells from epididymal fat pads of 100 g Sprague-Dawley rats were plated for 3 days in 10% fetal bovine scrum and subsequently exposed to scrum-free (insulin, transferrin, thyroxine, selenium: lllS) media for 1 or 9 days or to ITTS media containing either 0.85 or 850 nM insulin for 11 days. Gene expression was detected in S-V cultures by Northern-blot analysis using a 320 bp PCR product of rodent ob cDNA. Significant levels of Ob mRNA were found in cultures examined after 9 or 11 days in serum-free media containing high levels of insulin. Ob gene expression was not detectable in cultures examined after only one day treatment with ITTS or in those examined after 11 days exposure to the lower level of insulin. These data suggest that insulin may be one factor involved in the regulation of Ob gene expression in differentiating adipose cells. They further support the utility of primary S-V cultures as a system for studying additional factors involved in the regulation of the ob gene.