Butyl Benzyl Phthalate Promotes Adipogenesis in 3T3-L1 Cells via the miRNA-34a-5p Signaling Pathway in the Absence of Exogenous Adipogenic Stimuli,Chemical Research in Toxicology

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Butyl Benzyl Phthalate Promotes Adipogenesis in 3T3-L1 Cells via the miRNA-34a-5p Signaling Pathway in the Absence of Exogenous Adipogenic Stimuli
Chemical Research in Toxicology ( IF 3.7 ) Pub Date : 2021-09-14 , DOI: 10.1021/acs.chemrestox.1c00115
Sunitha Meruvu ₁ , Jian Zhang ₁ , Mahua Choudhury ₁

Affiliation

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.

中文翻译：

邻苯二甲酸丁基苄酯通过 miRNA-34a-5p 信号通路促进 3T3-L1 细胞中的脂肪生成，在没有外源性脂肪刺激的情况下

邻苯二甲酸盐是一种增塑剂，广泛用于我们每天使用的许多产品中。由于最近的研究表明邻苯二甲酸丁苄酯 (BBP) 是一种致肥胖物质，因此公众健康问题日益关注。然而，脂肪生成过程中 BBP 诱导的表观遗传调控仍然未知。我们研究了 BBP 是否改变了 miR-34a-5p（一种参与肥胖的关键 miRNA）并调节其下游通路。将分化的 3T3-L1 细胞暴露于不同剂量的 BBP 而没有外源性脂肪形成刺激，测试脂肪形成标志物（PPARγ 和 aP2），并用油红 O 染色对脂质积累进行染色。然后，我们测量了 miR-34a-5p 及其靶基因 Nampt 和 Sirt1 以及另一种重要的表观遗传调节剂 Sirt3 的表达。此外，使用 antagomiR，我们检查了 miR-34a-5p 敲低是否会减少脂肪生成。BBP 暴露导致 miR-34a-5p 的表达水平增加，并伴随脂肪生成的增加。BBP 显着降低了 Nampt、Sirt1 和 Sirt3 基因的表达水平。然而，仅在 Nampt 中观察到蛋白质表达的降低，表明 BBP 暴露下的 miR-34a-5p 可能仅在转录水平上调节 Sirt1/Sirt3。有趣的是，在存在 BBP 的情况下，miR-34a-5p 的敲低降低了分化 3T3-L1 细胞中的脂肪生成。此外，miR-34a-5p 敲低增加了 Nampt 蛋白表达水平以及 NAD 表明 BBP 暴露下的 miR-34a-5p 可能仅在转录水平调节 Sirt1/Sirt3。有趣的是，在存在 BBP 的情况下，miR-34a-5p 的敲低降低了分化 3T3-L1 细胞中的脂肪生成。此外，miR-34a-5p 敲低增加了 Nampt 蛋白表达水平以及 NAD 表明 BBP 暴露下的 miR-34a-5p 可能仅在转录水平调节 Sirt1/Sirt3。有趣的是，在存在 BBP 的情况下，miR-34a-5p 的敲低降低了分化 3T3-L1 细胞中的脂肪生成。此外，miR-34a-5p 敲低增加了 Nampt 蛋白表达水平以及 NAD⁺水平，表明 miR-34a-5p 在 BBP 暴露期间调节 Nampt。此外，在 BBP 处理的细胞中，NAD ⁺依赖性沉默调节蛋白活性降低，而在 BBP 处理的 miR-34a-5p 敲低细胞中增加。BBP 暴露通过改变 miR-34a-5p 及其靶标 Nampt 的表达模式证明了表观遗传调控的参与，这可能通过改变 NAD ⁺水平和 sirtuin 活性来扰乱分化脂肪细胞的能量稳态，导致脂肪生成增加。

更新日期：2021-11-15

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