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.
Lengi
A J
AJ
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
Corl
B A
BA
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