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目的以原代培养的大鼠骨骼肌细胞为研究对象,观察亮氨酸对骨骼肌细胞及渥曼青霉素(wortmannin)处理后葡萄糖摄取能力及胰岛素信号通路关键分子蛋白激酶B(AKT)表达的影响,从而为探讨亮氨酸与胰岛素抵抗的关系和相关机制提供依据。方法组织块法培养大鼠原代骨骼肌细胞,不同浓度的亮氨酸(0,0.4,2.0,4.0,8.0mmol/L)作用80min或45min,酶法测定基础状态下和胰岛素刺激下葡萄糖摄取能力,确定亮氨酸作用的最佳浓度和作用时间;在此基础上,应用PI-3K抑制剂wortmannin作用30min,测定葡萄糖摄取能力,Western blot检测AKT及磷酸化AKT表达水平。结果胰岛素刺激状态下,2mmol/L亮氨酸作用45min骨骼肌细胞葡萄糖摄取能力达最大;2mmol/L亮氨酸干预可改善wortmannin对胰岛素刺激下骨骼肌细胞葡萄糖摄取的抑制作用,显著增强胰岛素信号传导关键分子pSer473 AKT的表达。结论亮氨酸可改善骨骼肌细胞胰岛素抵抗。这种作用不完全依赖于PI-3K胰岛素信号传导通路,并且可能与其增强AKT磷酸化水平有关。
OBJECTIVE: To investigate the effects of leucine on glucose uptake and expression of key protein kinase B (AKT) in insulin signaling pathway in skeletal muscle cells and wortmannin treated rat primary cultured skeletal muscle cells , So as to explore the relationship between leucine and insulin resistance and related mechanisms. Methods Primary cultured rat skeletal muscle cells were cultured with different concentrations of leucine (0, 0.4, 2.0, 4.0, 8.0 mmol / L) for 80 min or 45 min. Enzyme assay was used to determine glucose uptake under basal condition and insulin stimulation Ability to determine the best leucine concentration and duration of action; on this basis, the application of PI-3K inhibitor wortmannin for 30min, the determination of glucose uptake capacity, Western blot detection of AKT and phosphorylated AKT expression levels. Results When stimulated with 2mmol / L leucine for 45min, glucose uptake capacity of skeletal muscle cells reached the maximum under insulin stimulated condition; 2mmol / L leucine treatment could improve the inhibitory effect of wortmannin on glucose uptake by skeletal muscle cells stimulated by insulin and significantly enhance the insulin signaling Conducting the expression of the key molecule pSer473 AKT. Conclusion Leucine can improve insulin resistance in skeletal muscle cells. This effect is not completely dependent on the PI-3K insulin signaling pathway, and may be related to its enhanced AKT phosphorylation level.