Indomethacin Induces Browning and Brown Adipogenesis in Murine 3T3‐L1 White Adipocytes and Human Brown Fat Cells

Abstract

Functional brown adipose tissue, including classical brown adipocytes and brown‐like or beige adipocytes, which develop within white adipose tissue in response to various stimuli, have emerged as novel targets for obesity treatment and prevention. Identifying strategies that increase UCP‐1 expression and induce browning and brown adipogenesis is key to unlocking thermogenic potential. Indomethacin (INDO) is a non‐selective Cox‐1/Cox‐2 inhibitor which is an approved NSAID used to treat and relief inflammatory diseases. It has been reported that INDO induced C3H10T1/2 mesenchymal stem cell differentiation into adipocytes by activating PPARg, which is also important for browning and brown adipogenesis. In this study, INDO was tested at various doses (2, 5, 10, 20, 50 mM) in 3T3‐L1 mouse pre‐adipocytes in a 7‐day differentiation process as well as human brown fat cells in a 21‐day differentiation process. Rosiglitazone (ROSI), a well‐known browning agent and PPARg agonist, was used as a positive control. At the end of differentiation, 3T3‐L1 cells were subjected to thermogenic activation by isoproterenol (ISO), a b‐adrenergic receptor agonist, or the control for 6–24 hours. Protein and mRNA expression of UCP‐1, PPARg, and PGC1a were measured. mRNA analysis of other brown and beige maker genes (PRDM16, Cidea, and CD137), and white marker genes (IGFBP3), as well as mitochondrial related genes (Nrf and Cox4a) were also measured. For mechanistic exploration, PPARg transactivation reporter gene assays were conducted. In addition, an adenovirus mediated PPARg knock down (KD) in 3T3‐L1 cells was generated. We report that similar to ROSI, INDO significantly induced UCP‐1 mRNA expression in 3T3‐L1 cells under basal and ISO stimulated condition compared to the controls (p<0.05 for INDO at 10, 20, 50 μM under basal condition and INDO at 2, 5, 10, 20 μM under ISO stimulated condition). INDO also significantly induced mRNA expression of Nrf and PGC1a (p<0.05). INDO also significantly induced UCP1 and PGC1α protein expression under basal condition (p<0.05 INDO at 10 and 20 μM) while suppressing PPARg protein expression (p<0.05 INDO at 10 and 20 μM). Mechanistically, INDO significantly activated PPARg in 3T3‐L1 cells (p<0.01 for INDO at 20 and 50 μM), which is consistent with the previous report in C3H10T1/2 cells. Moreover, upregulation of UCP1 and other brown markers by INDO were decreased in PPARg KD cells compared to non‐targeting adenovirus infected control cells. Furthermore, we explored INDO's effects on promoting brown adipogenesis in human brown fat cells. We report that INDO significantly induced PPARg, PGC1α, and DIO2 at 20 μM (p<0.05). INDO also significantly induced UCP1 mRNA and protein expression (p<0.01 for INDO at 20 μM for mRNA and p<0.05 for INDO at 2, 5, 10 μM, for protein). Taken together, despite INDO's potential systemic side‐effects, our results reveal a novel capacity for INDO to induce browning and brown adipogenesis through acting as a PPARg agonist.Support or Funding InformationThe work has been partially supported by NIH 1R15AT008733 award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.