OB gene expression during porcine preadipocyte differentiation in vitro

The Ob gene is expressed in adipose tissue, but it is not known when the ob gene is expressed during adipocyte differentiation. We used porcine S-V cell cultures to examine ob gene expression at different stages of adipocyte differentiation. Gene expression was detected in S-V cultures by Northern-blot analysis using a 320 bp PCR product of rodent ob cDNA. Preadipocyte number was determined by immunocytochemistry with a preadipocyte marker, the AD-3 monoclonal antibody. Cultures treated with ITS (insulin 5ug/ml, transferrin 5ug/ml, and selenium 5ug/ml) for four to five days following three days with fetal bovine serum (FBS) plus dexamethasone (DEX), expressed significant levels of Ob rnRNA. Significant levels of Ob rnRNA were also found in cultures examined immediately after three days with FBS plus DEX. The lowest level of Ob gene expression was observed in the cultures treated with FBS for three days followed by ITS for four to five days. Seeding with FBS + DEX for three days produced the highest proportion of preadipocytes (AD-3 positive cells; 223±45 cells/mm2) and little to no lipid accretion. Seeding with FBS alone produced a significantly lower proportion of preadipocytes (10.511.4 cells/mm2). Additionally four to five days of ITS treatment following FBS + DEX produced a very high proportion of adipocytes while ITS treatment following FBS alone produced a very low proportion of adipocytes. These data indicate that the Ob gene may be expressed in preadipocytes and DEX may augment Ob expression by preadipocytes and/or adipocytes.