PDF(Pubmed)
Abstract：
BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes.
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.
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.
摘要：
背景：余甘子的果实，中国和印度的传统医学，用于治疗糖尿病。其水提取物（WEPE）已证明对糖尿病大鼠的降血糖作用，但其对骨骼肌葡萄糖利用和胰岛素抵抗的作用机制尚不清楚。因此,本研究旨在探讨使用C2C12肌管的WEPE对葡萄糖利用和胰岛素抵抗的影响和潜在机制。
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。
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。
