Abstract
BackgroundAlisol 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.MethodEffects 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.ResultsWe 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.ConclusionsThese results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKβ-AMPK-p38 MAPK/AS160 pathway.
Highlights
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
These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the Calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-adenosine monophosphateactivated protein kinase (AMPK)-p38 p38 mitogen-activated protein kinase (MAPK)/AKT substrate of 160 kDa (AS160) pathway
We used antibodies against the following proteins: AMPKα, phospho-AMPKα (Thr172), acetyl-CoA carboxylase (ACC), phosphoACC (Ser79), AS160, phospho-AS160 (Ser588), phospho-p38 MAPK (Thr180/Tyr182), IRS-1, phosphoAKT (Ser473) and phospho-AKT (Thr308), which were purchased from Cell Signaling Technology; glucose transporter 4 (GLUT4) and phosphor-Insulin receptor substrate 1 (IRS1) (Tyr632), which were purchased from Abcam; and AKT, ATP1A1, p38 MAPK and β-actin, which were obtained from Proteintech
Summary
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. It is pivotal to T2DM research that we find ways to promote glucose uptake in skeletal muscle and lower glucose production in the liver. Glucose uptake in skeletal muscle is regulated by two distinct pathways: 1) stimulation by insulin through IRS1 and phosphatidylinositol 3 (PI3)-kinase [5]; and 2) stimulation by muscle contraction and exercise through the activation of AMPK [6]. AMPK can phosphorylate multiple kinases and other downstream target proteins to exert its various functions. Studies have revealed that AMPK can phosphorylate and activate p38 MAPK [10], whose role in promoting cell glucose uptake has already been clarified [10, 11]. AMPK is regulated by various upstream kinases, including but not limited to CaMKKβ, transforming growth factor (TGF) β-activated kinase 1 (TAK1) and liver kinase B1 (LKB1) [12]
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