Caffeic acid phenethyl ester inhibits adipogenic differentiation in association with INSR suppression and AMPK activation

Background Adipogenic differentiation of preadipocytes is a tightly regulated process central to adipose tissue development and dysfunction. Caffeic acid phenethyl ester (CAPE), a major bioactive compound in propolis, has been reported to modulate adipogenesis, yet its upstream molecular targets and mechanisms of action remain unclear.Methods The impact of CAPE on adipogenic differentiation was investigated in 3T3-L1 preadipocytes using Oil Red O and BODIPY 493/503 staining, Western blot, and RT-qPCR. AICAR and Compound C were used to activate or inhibit AMP-activated protein kinase (AMPK), respectively. Potential targets and regulatory interactions involved in CAPE-mediated signaling were explored through in silico prediction, molecular docking, and protein-protein interaction analysis. Functional validation was performed using insulin receptor (INSR) overexpression and analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation.Results CAPE inhibited lipid accumulation and suppressed the expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and fatty acid synthase (FAS) in a dose-dependent manner. Pharmacological activation of AMPK reproduced the anti-adipogenic effects of CAPE, while AMPK inhibition or INSR overexpression reversed these effects. CAPE treatment enhanced AMPK and ACC phosphorylation, whereas INSR overexpression attenuated this activation and abolished CAPE's inhibitory effect on adipogenesis.Conclusion CAPE suppresses adipogenic differentiation by modulating upstream regulators of the AMPK signaling pathway. These findings provide mechanistic insight into the anti-adipogenic actions of CAPE and highlight its therapeutic potential for metabolic disorders involving adipose tissue dysfunction.