Factors influencing the differentiation of bovine preadipocytes in vitro

Our objectives were to isolate bovine stromal-vascular cells using explants and to determine media components that promote differentiation into mature adipocytes for studies of lipogenic enzyme regulation. Stromal-vascular cells were grown from explants and treated with differentiation media for 8 d after reaching confluence. Differentiation was assessed by measuring radiolabeled acetate incorporation into lipids, glycerol-3-phosphate dehydrogenase activity, and the mRNA expression of fatty acid binding protein-4, PPAR-gamma, and acetyl-CoA carboxylase-alpha (ACC alpha). After 8 d of differentiation, medium containing 10 mu g/mL of insulin, 0.25 mu M dexamethasone, 0.5 mM isobutyl-methylxanthine, 1 mM octanoate, and 2% Intralipid (Fisher Scientific, Suwanee, GA) produced greater acetate incorporation (P < 0.001) and glycerol-3-phosphate dehydrogenase activity (P < 0.001) compared with other media tested. This differentiation medium also increased mRNA expression of fatty acid binding protein-4, PPAR gamma, and ACC alpha by 180-, 7-, and 3-fold, respectively, compared with undifferentiated control cells (P < 0.05). To further improve the differentiation protocol, the effects of Intralipid, rosiglitazone, and troglitazone were examined. Removal of 2% Intralipid did not improve any differentiation measures. Addition of rosiglitazone (1 mu M), a PPAR-gamma agonist, increased acetate incorporation and ACC alpha mRNA (P < 0.01). Addition of troglitazone (5 mu M), another PPAR-gamma agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest expression of ACC alpha mRNA (P < 0.01), but was not superior to medium that included rosiglitazone for any other differentiation measures. Cell-seeding density influences the cell divisions required to reach confluence, and increased plating density (2 x 10(4) cells/cm(2) vs. 6.7 x 10(3) cells/cm(2)) increased acetate incorporation by 100% (P < 0.001). Differentiating stromal-vascular cells in the presence of trans-10, cis-12 CLA inhibited differentiation of stromal-vascular cells into mature adipocytes, reducing radiolabeled acetate incorporation into lipids (P < 0.001), stearoyl-CoA desaturase-1 mRNA (P < 0.05) and protein abundance (P < 0.05), and ACC alpha protein abundance (P < 0.05). We have developed a method to differentiate primary bovine adipocytes, which will allow us to study the regulation of lipogenic enzymes by nutrient and endocrine factors.