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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:The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels-carbenoxolone, octanol, proadifen and Gap26-as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.