METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting.
RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C.
CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKβ/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.
方法:WEPE对葡萄糖摄取的影响,GLUT4易位,在C2C12肌管和棕榈酸酯处理的肌管中研究了AMPK和AKT的磷酸化。使用AMPK抑制剂和siRNA来探索WEPE的机制。葡萄糖摄取使用2-(N-(7-硝基苯基-2-氧杂-1,3-二唑-4-基)氨基)-2-脱氧葡萄糖(2-NBDG)摄取测定,通过蛋白质印迹法评估蛋白质表达和GLUT4易位。
结果:在正常肌管中,WEPE在125和250µg/mL的浓度下显着刺激葡萄糖摄取和GLUT4转运到质膜。这伴随着AMPK及其下游靶标的磷酸化增加。然而,化合物C和AMPKsiRNA均阻断WEPE诱导的GLUT4易位和葡萄糖摄取。此外,用钙/钙调蛋白依赖性蛋白激酶激酶β(CaMKKβ)抑制剂STO-609预处理,抑制WEPE诱导的AMPK磷酸化并减弱WEPE刺激的葡萄糖摄取和GLUT4易位。在用棕榈酸酯处理的肌管中,WEPE通过增强胰岛素介导的葡萄糖摄取和AKT磷酸化来预防棕榈酸诱导的胰岛素抵抗。它还恢复了胰岛素介导的GLUT4从细胞质到膜的易位。然而,用化合物C预处理可阻断WEPE对葡萄糖摄取和GLUT4易位的这些影响。
结论:WEPE通过CaMKβ/AMPK途径显着刺激基础葡萄糖摄取,并通过激活C2C12肌管的AMPK途径显着改善棕榈酸诱导的胰岛素抵抗。