Alisol A 24-acetate Stimulates Lipolysis In Adipocytes And Alleviates Hepatic Steatosis In Hepatocytes

Alisma orientalis(Sam.)Juzep is a well-known traditional Chinese medicine with a wide range of pharmaceutical benefits,such as diuretic,anti-inflammatory,anti-tumor,hypoglycemic and hypolipidemic effects.Alisol A 24-acetate(AA-24-a)is one of the main active triterpenes isolated from Alisma orientalis(Sam.)Juzep,and has been determined as the quality control of this crude drug.AA-24-a was demonstrated to possess hypolipidemic and cholesterol lowering activities in vivo.It also improved hepatic lipid metabolism in vitro,but the underlying mechanism is still unclear.Therefore,this study focuses on the effects and potential mechanisms of AA-24-a on the control of lipid metabolism in vitro.First of all,we used 3T3-L1 adipocytes to investigate the effect of AA-24-a on adipocytes lipolysis and its underlying mechanism.Lipolysis was evaluated by measuring the amount of glycerol released to the medium and intracelluar lipid content.We found that AA-24-a promoted lipolysis in mature 3T3-L1 adipocytes.To understand the molecular mechanism,mRNA and protein expression of key lipolysisrelated genes and proteins were detected by q-PCR or Western blotting with or without PKA,ERK and AMPK inhibitors.Results indicated that AA-24-a upregulated phosphorylated HSL at site of Ser660 via activation of PKA signaling pathway.AA-24-a downregulated the mRNA and protein levels of PPAR? and the mRNA level of Perilipin A via activation of ERK pathway,therefore promoted the lipolysis.AA-24-a also upregulated the mRNA and protein levels of ATGL and downregulaed the phosphorylation of AKT at site of Ser473,these also probably contributed to the lipolytic action of AA-24-a.Besides,AA-24-a downregulated the phosphorylation of Perilipin via activation of AMPK.This may lower the FFA produced during the lipolysis.And activation of AMPK maybe related to the effect that AA-24-a significantly increased the expression of PPAR?,CPT1 and PGC-1?.Then,we investigated the effect of AA-24-a on triglyceride accumulation in HL7702 hepatocytes and the molecular mechanism.When HL7702 hepatocytes were treated with sodium oleate,significant triglyceride accumulation was observed.Under the same experimental condition,the addition of AA-24-a significantly suppressed the sodium oleate-induced lipid accumulation in HL7702 hepatocytes.Meanwhile,the expression of C/EBP?,a key molecule involved in lipogenesis,was decreased in AA-24-a-treated hepatocytes.AA-24-a was found to upregulate phosphorylated levels of ACC via activation of AMPK which reduced ACC activity.Thus,AA-24-a can reduce fatty acid synthesis and increase fatty acid oxidation.Moreover,we found that AA-24-a upregulated the mRNA and protein levels of CPT1 and the mRNA level of ACOX1 via activation of PPAR?.This contributed to the oxidation of fatty acid and the inhibition of lipid accumulation in HL7702 hepatocytes.We also found that AA-24-a treatment increased the phosphorylation of AKT at site of Ser473,suggesting that AKT pathway was probably involved in AA-24-a's effect on alleviating hepatic steatosis.In conclusion,AA-24-a attenuated triglyceride accumulation in mature adipocytes through promoting lipolysis via PKA-HSL and ERK-PPAR?-Perilipin A pathways.At the same time,AMPK-Perilipin pathway was also activated by AA-24-a to regulate adipocytes lipolysis.AA-24-a also attenuated triglyceride accumulation in hepatocytes via AMPK-ACC and PPAR?-CPT1 A pathways to prevent hepatic steatosis.This study provided scientific evidence for the utilization of Alisol A 24-acetate and Alisma orientalis(Sam.)Juzep,also provided experimental data for the modernization of traditional Chinese medicine Alisma orientalis(Sam.)Juzep.