Anti-adipogenic effect of cryptotanshinone in 3T3-L1 preadipocytes via binding to glucagon-like peptide 1 receptor and its signaling

초록

Background: Obesity and its related metabolic disorders pose significant global health challenges, necessitating the development of novel therapeutic strategies targeting adipose tissue biology and energy metabolism. Cryptotanshinone (CT), a bioactive compound derived from Salvia miltiorrhiza, has recently been investigated for its anti-adipogenic and metabolic regulatory effects. The molecular mechanism by which CT inhibits adipogenesis remains unknown and requires further elucidation. Methods: In this study, we elucidated the molecular mechanisms by which CT modulates adipogenesis by focusing on its interaction with the glucagon-like peptide-1 receptor (GLP-1R). Molecular docking and surface plasmon resonance investigations were employed to examine CT's direct binding affinity to GLP-1R specifically targeting potential allosteric binding sites. Expression analyses were conducted to evaluate changes in GLP-1R and downstream signalling components. Functional assays, including cAMP measurements, Western blotting, and gene expression studies, were performed. Additionally, pharmacological inhibitor and siRNA-mediated knockdown of GLP-1R were performed to assess the dependency of CT's effects on GLP-1R signaling. Results: Our findings demonstrated that CT treatment upregulates GLP-1R expression early in adipogenesis, preceding the induction of the transcription factor CCAAT/enhancer-binding protein beta (C/EBP(3), and shifts G protein coupling by increasing G alpha q and G alpha s while reducing G alpha i, leading to elevated cAMP levels and activation of the Protein Kinase A (PKA) pathway. This cascade robustly activates p38-mitogen-activated protein kinase and AMP-activated protein kinase phosphorylation (AMPK), resulting in suppressed expression of key adipogenic markers, Peroxisome proliferator-activated receptor gamma (PPAR gamma), C/EBP(3, Adipocyte protein 2 (aP2). Importantly, the antiadipogenic effects were repealed by GLP-1R inhibition, confirming its GLP-1R dependent mechanism of action. These findings reveal a novel mechanism by which CT regulates adipogenesis via GLP-1R signaling, highlighting its potential as a small-molecule therapeutic agent for obesity. Conclusions: CT suppresses adipogenesis by acting as a small-molecule modulator of GLP-1R, altering G protein coupling by inducing cAMP, PKA, p38-MAPK, and AMPK signaling. These findings establish CT's GLP1R-dependent anti-adipogenic mechanism and highlight its potential as a therapeutic candidate for obesity.

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