Alisol A 24-acetate stimulates lipolysis in 3 T3-L1 adipocytes.

BACKGROUND: Alisol A 24-acetate (AA-24-a), one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., exhibits multiple biological activities including hypolipidemic activity. However, its effect on lipid metabolism in adipocytes remains unclear. The present study aimed to clarify the effect of AA-24-a on adipocyte lipolysis and to determine its potential mechanism of action using 3 T3-L1 cells. METHODS: We assayed the release of glycerol into culture medium of 3 T3-L1 cells under treatment with AA-24-a. Protein and mRNA expression and phosphorylation levels of the main lipases and kinases involved in lipolysis regulation were determined by quantitative polymerase chain reaction and western blotting. Specific inhibitors of protein kinase A (PKA; H89) and extracellular signal-regulated kinase (ERK; PD98059), which are key enzymes in relevant signaling pathways, were used to examine their roles in AA-24-a-stimulated lipolysis. RESULTS: AA-24-a significantly stimulated neutral lipolysis in fully differentiated adipocytes. To determine the underlying mechanism, we assessed the changes in mRNA and protein levels of key lipolysis-related genes in the presence or absence of H89 and PD98059. Both inhibitors reduced AA-24-a-induced lipolysis. Moreover, pretreatment with H89 attenuated AA-24-a-induced phosphorylation of hormone-sensitive lipase at Ser660, while pretreatment with PD98059 attenuated AA-24-a-induced downregulation of peroxisome proliferator-activated receptor-γ and perilipin A. CONCLUSIONS: Our results indicate that AA-24-a promoted neutral lipolysis in 3 T3-L1 adipocytes by activating PKA-mediated phosphorylation of hormone-sensitive lipase and ERK- mediated downregulation of expression of perilipin A.

Extract of Paecilomyces hepiali mycelia induces lipolysis through PKA-mediated phosphorylation of hormone-sensitive lipase and ERK-mediated downregulation of perilipin in 3T3-L1 adipocytes.

BACKGROUND: Cordyceps sinensis has been used for centuries in China as one of the most valued herbal medicine and tonic food. Paecilomyces hepiali, a fungal strain isolated from natural C. sinensis, has been used widely as a substitute of C. sinensis in medicine and health food. P. hepiali has been reported to have various pharmaceutical benefits, including triglyceride-lowing activity. However, its effects on triglyceride metabolism in adipocytes remain unknown. The purpose of the present study was to evaluate the effect of P. hepiali mycelia on adipocyte lipolysis and to clarify the underlying mechanisms. METHODS: The fully differentiated 3T3-L1 adipocytes were treated with methanol extract of Paecilomyces hepiali mycelia (PHME). Contents of glycerol released into the culture medium and intracellular triglyceride were measured as indices of lipolysis using glycerol assay kit and Oil red O staining, respectively. Then, effects of PHME on the main lipases or kinases involved in lipolysis regulation were investigated. Protein expression of adipose triglyceride lipase (ATGL) and perilipin, as well as phosphorylation of hormone-sensitive lipase (HSL), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinases (MAPKs) were determined by western blotting. Moreover, nucleosides, important constituents of PHME, were analyzed using high performance liquid chromatography (HPLC). RESULTS: Treatment with PHME led to a significant increase in glycerol release thereby reduced intracellular triglyceride accumulation in fully differentiated adipocytes. PHME upregulated protein kinase (PK) A-mediated phosphorylation of HSL at serine residues of 563 and 660. Meanwhile, PHME treatment also upregulated phosphorylation of extracellular signal-regulated kinase (ERK), and downregulated the protein level of perilipin. Pretreatment with the PKA inhibitor, H89, blunted the PHME-induced lipolysis and the phosphorylation of HSL (Ser 563 and 660). Moreover, pretreatment with ERK inhibitor, PD98059, weakened the PHME-caused glycerol release and downregulation of perilipin expression. HPLC analysis indicated there were adenosine, cordycepin, uridine and vernine in PHME. CONCLUSIONS: Our results showed that PHME significantly induced lipolysis in 3T3-L1 adipocytes, which is mainly mediated by activation of HSL through PKA pathway and by downregulation of perilipin through activation of ERK pathway.