| 20154153 |
Lengi AJ, Corl BA: Factors influencing the differentiation of bovine preadipocytes in vitro. J Anim Sci. 2010 Feb 12.
Our objectives were to isolate bovine stromal-vascular cells using explants, and 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 (G3PDH) activity, and the mRNA expression of fatty acid binding protein-4 (aP2), PPAR-gamma, and acetyl-CoA carboxylase (ACC)- alpha. After 8 d of differentiation, medium containing 10 mug/mL insulin, 0.25 muM dexamethasone (DEX), 0.5 mM isobutylmethylxanthine (IBMX), 1 mM octanoate, and 2% Intralipid produced greater acetate incorporation (P < 0.001) and G3PDH activity (P < 0.001) compared to other media tested. This differentiation medium also increased mRNA expression of aP2, PPARgamma, and ACCalpha 180-, 7-, and 3-fold, respectively, compared to 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 muM), a PPAR-gamma agonist, increased acetate incorporation and ACCalpha mRNA (P < 0.01). Addition of troglitazone (5 muM), another PPAR-gamma agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest levels of ACCalpha mRNA expression (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 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 ACCalpha protein abundance (P < 0.05). We have developed a method to differentiate primary bovine adipocytes that will allow us to study the regulation of lipogenic enzymes by nutrient and endocrine factors. |
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