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Updated: Oct 20, 2025
An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function
Published on: May 4, 2021
Butyl Benzyl Phthalate Promotes Adipogenesis in 3T3-L1 Cells via the miRNA-34a-5p Signaling Pathway in the Absence of
Sunitha Meruvu1, Jian Zhang1, Mahua Choudhury1
1Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, College Station, Texas 77843, United States.
Abstract:
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.
