Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
J Anim Sci. 2010 Jun;88(6):1999-2008. doi: 10.2527/jas.2009-2439. Epub 2010 Feb 12.
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.
我们的目的是使用外植体分离牛基质血管细胞，并确定促进成熟脂肪细胞分化的介质成分，以便研究脂肪生成酶的调节。基质血管细胞从外植体中生长，并在达到汇合后用分化培养基处理 8 天。通过测量放射性标记的乙酸盐掺入脂质、甘油-3-磷酸脱氢酶活性以及脂肪酸结合蛋白-4、PPAR-γ和乙酰辅酶 A 羧化酶-α（ACCalpha）的 mRNA 表达来评估分化。分化 8 天后，含有 10 μg/mL 胰岛素、0.25 μM 地塞米松、0.5 mM 异丁基甲基黄嘌呤、1 mM 辛酸盐和 2%Intralipid（Fisher Scientific，Suwanee，GA）的培养基产生了更大的乙酸盐掺入（P <0.001）和甘油-3-磷酸脱氢酶活性（P <0.001）与其他测试的培养基相比。与未分化的对照细胞相比，这种分化培养基还使脂肪酸结合蛋白-4、PPARγ 和 ACCalpha 的 mRNA 表达分别增加了 180 倍、7 倍和 3 倍（P <0.05）。为了进一步改进分化方案，检查了 Intralipid、罗格列酮和曲格列酮的作用。去除 2%Intralipid 并不能改善任何分化措施。添加罗格列酮（1 μM），一种 PPAR-γ 激动剂，增加了乙酸盐掺入和 ACCalpha mRNA（P <0.01）。添加曲格列酮（5 μM），另一种 PPAR-γ 激动剂，增加了乙酸盐掺入的程度与罗格列酮相似，并产生了最大的 ACCalpha mRNA 表达（P <0.01），但在任何其他分化措施方面并不优于包含罗格列酮的培养基。细胞接种密度会影响达到汇合所需的细胞分裂，增加接种密度（2 x 10(4) 个细胞/cm(2) 与 6.7 x 10(3) 个细胞/cm(2))可使乙酸盐掺入增加 100%（P <0.001）。在反式-10、顺式-12 CLA 的存在下分化基质血管细胞会抑制基质血管细胞向成熟脂肪细胞的分化，减少放射性标记的乙酸盐掺入脂质（P <0.001）、硬脂酰辅酶 A 去饱和酶-1 mRNA（P <0.05）和蛋白丰度（P <0.05）以及 ACCalpha 蛋白丰度（P <0.05）。我们已经开发出一种分化原代牛脂肪细胞的方法，这将使我们能够研究营养和内分泌因素对脂肪生成酶的调节。
