PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.
PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes.
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DOI：
10.1186/s11658-017-0037-1
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发表时间：
2017
影响因子：
8.3
通讯作者：
Veiga-Lopez A
中科院分区：
文献类型：
--
作者：
Pu Y;Veiga-Lopez A
虽然3T3-L1前脂肪细胞系代表了体外脂肪形成研究的信息模型，但通常需要原代培养细胞来了解特定的人类或动物代谢表型。大型哺乳动物体外培养的前脂肪细胞表明，原代培养的细胞需要与3T3-L1细胞系不同的特定成脂分化条件。这些条件也是物种特有的，需要优化步骤。然而，利用啮齿类动物的替代物种分化原代前脂肪细胞的有效方案很少。绵羊是胎儿生物学和健康与疾病研究的发育起源的合适动物模型。在这项工作中，我们提出了第一个详细的程序，以有效地区分原代胎羊和成年羊前脂肪细胞。胚胎和成年羊脂肪和皮肤组织的采集，前脂肪细胞和成纤维细胞的分离，增殖，以及新的脂肪生成分化方案的标准化和优化。使用商业细胞系（3T3-L1和NIH-3T3）进行验证。油红O染色和基因表达验证成脂分化。采用方差分析和Fisher精确检验来确定统计显著性。我们优化的成脂分化方法包括将成脂鸡尾酒暴露时间从2天延长至8天，提高胰岛素浓度，并补充过氧化物酶体增殖物激活受体γ (PPARγ)激动剂罗格列酮。该方案对胎儿和成人前脂肪细胞都进行了优化。我们的方案使胚胎和成年羊前脂肪细胞成功分化成脂肪。这项工作表明，与3T3-L1细胞系相比，胎羊前脂肪细胞在分化末期需要更长时间的暴露于分化混合物中，并且需要IMBX、地塞米松和/或PPARγ激动剂罗格列酮。它们在分化过程中也需要更高的胰岛素浓度来促进脂质积累，与人类原发性前脂肪细胞相似，绵羊的脂肪分化也需要补充PPARγ激动剂。这项工作强调了物种特异性差异对脂肪生成分化的要求，以及开发标准化方法来研究比较脂肪细胞生物学的必要性。
Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes. Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher’s exact test were used to determine statistical significance. Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes. Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.