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

ABSTRACT:

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.

SUBMITTER: Lou HX

PROVIDER: S-EPMC8063434 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. METHOD:Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. RESULTS:We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a-induced AMPK pathway activation and reversed AA-24-a-induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor, also attenuated AA-24-a-induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. CONCLUSIONS:These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKβ-AMPK-p38 MAPK/AS160 pathway.

Project description:In obesity, high levels of tumor necrosis factor ? (TNF?) stimulate lipolysis in adipocytes, leading to hyperlipidemia and insulin resistance. Thiazolidinediones (TZDs), the insulin-sensitizing drugs, antagonize TNF?-induced lipolysis in adipocytes, thereby increasing insulin sensitivity in diabetes patients. The cellular target of TZDs is peroxisome proliferator-activated receptor ? (PPAR?), a nuclear receptor that controls many adipocyte functions. As a transcription factor, PPAR? is closely modulated by coregulators, which include coactivators and corepressors. Previous studies have revealed that in macrophages, the insulin-sensitizing effect of PPAR? may involve suppression of proinflammatory gene expression by recruiting the corepressor complex that contains corepressors and histone deacetylases (HDACs). Therefore, we investigated whether the corepressor complex is involved in TZD-mediated suppression of TNF?-induced lipolysis in 3T3-L1 adipocytes. Trichostatin A (TSA), a pan HDAC inhibitor (HDACI) that inhibits class I and II HDACs, was used to examine the involvement of HDACs in the actions of TZDs. TSA alone increased basal lipolysis and attenuated TZD-mediated suppression of TNF?-induced lipolysis. Increased basal lipolysis may in part result from class I HDAC inhibition because selective class I HDACI treatment had similar results. However, attenuation of TZD-mediated TNF? antagonism may be specific to TSA and related hydroxamate-based HDACI rather than to HDAC inhibition. Consistently, corepressor depletion did not affect TZD-mediated suppression. Interestingly, TSA treatment greatly reduced PPAR? levels in differentiated adipocytes. Finally, extracellular signal-related kinase 1/2 (ERK1/2) mediated TNF?-induced lipolysis, and TZDs suppressed TNF?-induced ERK phosphorylation. We determined that TSA increased basal ERK phosphorylation, and attenuated TZD-mediated suppression of TNF?-induced ERK phosphorylation, consistent with TSA's effects on lipolysis. These studies suggest that TSA, through down-regulating PPAR?, attenuates TZD-mediated suppression of TNF?-induced ERK phosphorylation and lipolysis in adipocytes.