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 (ACCalpha). After 8 d of differentiation, medium containing 10 microg/mL of insulin, 0.25 microM dexamethasone, 0.5 mM isobutylmethylxanthine, 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, PPARgamma, and ACCalpha 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 microM), a PPAR-gamma agonist, increased acetate incorporation and ACCalpha mRNA (P < 0.01). Addition of troglitazone (5 microM), another PPAR-gamma agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest expression of ACCalpha 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 ACCalpha 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.
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
A J
AJ
Lengi
B A
BA
Corl
D000085:Acetates / Q000378:metabolism; D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000502:physiology*; D000273:Adipose Tissue / Q000166:cytology / Q000502:physiology*; D000818:Animals; D002417:Cattle / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002839:Chromans / Q000494:pharmacology; D005217:Fat Emulsions, Intravenous / Q000494:pharmacology; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000502:physiology*; D044243:Linoleic Acids, Conjugated / Q000502:physiology*; D008297:Male; D047495:PPAR gamma / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000494:pharmacology; D000077288:Troglitazone
D000085:Acetates; D002839:Chromans; D005217:Fat Emulsions, Intravenous; D050556:Fatty Acid-Binding Proteins; D044243:Linoleic Acids, Conjugated; D047495:PPAR gamma; D012333:RNA, Messenger; D045162:Thiazolidinediones; D000077154:Rosiglitazone; D005993:Glycerolphosphate Dehydrogenase; D000103:Acetyl-CoA Carboxylase; D000077288:Troglitazone