# Nobiletin Attenuates Adipogenesis and Promotes Browning in 3T3-L1 Adipocytes Through Exosomal miRNA-Mediated AMPK Activation

## TL;DR

Nobiletin, a compound from citrus fruits, reduces fat cell formation and increases energy expenditure in fat cells by activating AMPK through exosomal miRNAs.

## Contribution

This study identifies a novel exosome–miRNA–AMPK axis as a mechanism for nobiletin's anti-obesity effects.

## Key findings

- Nobiletin reduces lipid accumulation and downregulates key adipogenic factors in 3T3-L1 adipocytes.
- Nobiletin activates AMPK signaling and enhances thermogenic markers like UCP-1 and PGC-1α.
- Nobiletin alters exosomal miRNA profiles, with miR-181d-5p and miR-221-3p upregulated and others downregulated.

## Abstract

Nobiletin, a citrus-derived polymethoxylated flavone, has been reported to exert anti-obesity effects, but its molecular mechanisms remain poorly understood. This study aimed to investigate whether nobiletin suppresses adipogenesis and promotes browning in 3T3-L1 adipocytes by modulating exosomal microRNAs (miRNAs) and AMPK signaling. To this end, we treated 3T3-L1 adipocytes with various concentrations of nobiletin and evaluated gene and protein expression by RT-qPCR and Western blotting. Nobiletin significantly reduced intracellular lipid accumulation at 50 μM (p < 0.001) and downregulated key adipogenic transcription factors, PPARγ, C/EBPα, and SREBP-1c, and suppressed the lipogenic enzyme FAS, while activating the AMPK/ACC signaling pathway. Concomitantly, it enhanced the expression of thermogenic markers UCP-1, PRDM16, and PGC-1α, indicating a metabolic shift toward energy expenditure. Exosomal RNA-seq revealed 10 differentially expressed miRNAs, of which miR-181d-5p (3.1-fold) and miR-221-3p (2.4-fold) were upregulated, whereas miR-205-5p (−2.9-fold), miR-331-3p (−3.2-fold), miR-130b-3p (−2.6-fold), miR-143-5p (−2.9-fold), miR-183-3p (−2.8-fold), miR-196b-5p (−2.4-fold), miR-26b-3p (−2.2-fold), and miR-378d (−2.7-fold) were verified by RT-qPCR after nobiletin treatment (50 μM). These miRNAs are functionally associated with adipogenic and thermogenic pathways, supporting a regulatory role of the exosomal miRNA network in nobiletin’s action. Collectively, our results identify a novel exosome–miRNA–AMPK axis underlying the anti-adipogenic and browning-inducing activities of nobiletin, highlighting its potential as a therapeutic phytochemical for obesity prevention.