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Alisol B Upregulates the cAMP/PKA Signaling Axis to PromoteThermogenesis in Adipocytes

CHEN Kunling^1,2,3, CHEN Lin^1,2,3, QIU Jiannan^1,2,3, DOU Xiaobing^1,2,3, CHEN Siyi^1,2,3, LU Dezhao^1,2,3, ZHOU Liping^1,2,3*

(¹School of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China; ²Zhejiang-Hong Kong Joint Laboratory forIntegrated Traditional Chinese Medicine Treatment of the Liver and Spleen, Hangzhou 310053, China; ³Institute of Lipid Metabolism,Zhejiang Chinese Medical University, Hangzhou 310053, China)

Abstract:

This study investigates the role of Alisol B in promoting thermogenesis in adipocytes and itspotential mechanisms. 3T3-L1 MBX preadipocytes were differentiated into mature adipocytes using the Brown’scocktail method. The CCK-8 assay was used to assess the effects of different concentrations of Alisol B on thesurvival rate of 3T3-L1 MBX cells. Oil red O staining and Raman spectroscopy were used to visualize lipiddroplets in cells and observe their formation within adipocytes. Quantitative reverse transcription polymerasechain reaction was used to detect the expression of genes associated with lipogenesis, lipolysis, key thermogenic genes, and the cAMP/PKA (protein kinase A) signaling axis. ELISA (enzyme-linked immunosorbent assay) wasused to measure intracellular cAMP (cyclic adenosine monophosphate) levels. Western blot analysis was performed to detect the protein expression of key thermogenic genes and genes associated with the cAMP/PKA signaling axis. The role of the cAMP/PKA signaling axis in the thermogenic effects of Alisol B on adipocytes wasvalidated using the PKA inhibitor H-89. The results demonstrated that Alisol B exhibited no evidence of toxicitytowards 3T3-L1 MBX cells at concentrations ranging from 0 to 10 μmol/L. Treatment with Alisol B significantly reduced lipid accumulation in adipocytes, decreased the expression of genes associated with lipogenesis(srebp1c, fas, acc), increased the expression of genes associated with lipolysis (hsl, atgl, cpt1), the expression ofkey thermogenic genes (ucp1, prdm16, pgc-1α) and their corresponding proteins, with these effects being dosedependent. The Alisol B treatment resulted in an upregulation of the cAMP/PKA signalling axis, the use of theH-89 inhibitor reversed the thermogenic-promoting effect of Alisol B on preadipocytes. The results of this studysuggest that Alisol B promotes thermogenesis in preadipocytes, inhibits lipogenesis, and enhances lipolysis byupregulating the cAMP/PKA signalling axis.

CSTR: 32200.14.cjcb.2026.10.0004