Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, College Station, Texas 77843, United States.
Chem Res Toxicol. 2021 Nov 15;34(11):2251-2260. doi: 10.1021/acs.chemrestox.1c00115. Epub 2021 Sep 14.
Phthalates, a plasticizer group, are used extensively in many of the products we use every day. Public health concerns are growing as recent studies have implicated butyl benzyl phthalate (BBP) as an obesogen. However, BBP-induced epigenetic regulation during adipogenesis is still unknown. We investigated if BBP altered miR-34a-5p, a key miRNA involved in obesity, and regulated its downstream pathway. Differentiating 3T3-L1 cells were exposed to various doses of BBP without exogenous adipogenic stimuli, tested for adipogenesis markers (PPARγ and aP2), and stained for lipid accumulation with Oil Red O staining. We then measured the expression of miR-34a-5p and its target genes, Nampt and Sirt1, along with another significant epigenetic modulator, Sirt3. Furthermore, using antagomiR, we examined whether miR-34a-5p knockdown decreased adipogenesis. BBP exposure resulted in augmented expression levels of miR-34a-5p with an associated increase in adipogenesis. BBP significantly decreased the Nampt, Sirt1, and Sirt3 gene expression levels. However, a decrease in the protein expression was observed only for Nampt, indicating that miR-34a-5p under BBP exposure may regulate Sirt1/Sirt3 only at the transcriptional level. Interestingly, in the presence of BBP, knockdown of miR-34a-5p decreased adipogenesis in the differentiating 3T3-L1 cells. Furthermore, miR-34a-5p knockdown increased the Nampt protein expression levels as well as NAD levels, indicating that miR-34a-5p regulates Nampt during BBP exposure. Additionally, the NAD-dependent sirtuin activity decreased in BBP-treated cells and increased in miR-34a-5p knockdown cells with BBP treatment. BBP exposure demonstrated the involvement of epigenetic regulation by altering the expression patterns of miR-34a-5p and its target Nampt, which may perturb the energy homeostasis of the differentiating adipocytes by altering NAD levels and sirtuin activity, resulting in increased adipogenesis.
邻苯二甲酸酯是一种广泛应用于我们日常用品的增塑剂。最近的研究表明丁基苄基邻苯二甲酸酯（BBP）是一种致肥胖物，因此公众对其健康影响的担忧日益增加。然而，BBP 在脂肪生成过程中的表观遗传调控仍不清楚。我们研究了 BBP 是否会改变参与肥胖的关键 miRNA 之一 miR-34a-5p，并调节其下游途径。将分化的 3T3-L1 细胞在没有外源性脂肪生成刺激的情况下暴露于不同剂量的 BBP 中，检测脂肪生成标志物（PPARγ 和 aP2），并用油红 O 染色检测脂质积累。然后，我们测量了 miR-34a-5p 及其靶基因 Nampt 和 Sirt1 的表达，以及另一个重要的表观遗传调节剂 Sirt3。此外，我们使用 antagomiR 检查了 miR-34a-5p 敲低是否会减少脂肪生成。BBP 暴露导致 miR-34a-5p 的表达水平升高，脂肪生成增加。BBP 显著降低了 Nampt、Sirt1 和 Sirt3 的基因表达水平。然而，只有 Nampt 的蛋白表达下降，这表明在 BBP 暴露下，miR-34a-5p 可能仅在转录水平上调节 Sirt1/Sirt3。有趣的是，在 BBP 存在的情况下，miR-34a-5p 的敲低降低了分化的 3T3-L1 细胞中的脂肪生成。此外，miR-34a-5p 敲低增加了 Nampt 蛋白表达水平和 NAD 水平，表明 miR-34a-5p 在 BBP 暴露期间调节 Nampt。此外，BBP 处理细胞中的 NAD 依赖性 Sirtuin 活性降低，而在 miR-34a-5p 敲低细胞中增加。BBP 暴露表明通过改变 miR-34a-5p 和其靶基因 Nampt 的表达模式来进行表观遗传调控，这可能通过改变 NAD 水平和 Sirtuin 活性来破坏分化脂肪细胞的能量平衡，导致脂肪生成增加。
