·糖尿病基础研究·

　　高春林 赵亚萍 陈小慧 季晨博 张春梅 朱春 朱金改 郭锡熔

　　作者单位：210002 南京，南京军区南京总医院儿科(高春林);南京医科大学附属南京妇幼保健院(赵亚萍、陈小慧、季晨博、张春梅、朱春、朱金改、郭锡熔)

　　通讯作者：郭锡熔，E-mail：xrguo@njmu.edu.cn

　　基金项目：国家自然科学基金资助项目(30973231)

　　【摘要】 目的 研究不同浓度的葡萄糖和游离脂肪酸(FFA)对骨骼肌L6细胞胰岛素敏感性及细胞内活性氧(ROS)的影响。 方法 L6 细胞诱导分化成熟后，分为6组，以5 mmol/L葡萄糖为低糖对照组，其中加0.3或1 mmol/L的混合FFA为低糖低FFA或低糖高FFA组，以25 mmol/L葡萄糖为高糖对照组，其中加0.3和1 mmol/L的FFA的培养基为高糖低FFA或高糖高FFA组。干预48 h， 3H-D-葡萄糖摄取实验(加与不加100 nmol/L胰岛素37℃ 30 min )验证L6细胞胰岛素敏感性，经荧光探针H2DCFDA观察细胞内ROS含量的变化。 结果 在5 mmol/L葡萄糖组，低糖对照组基础摄糖值为22777.6±6608.7，低糖低FFA组为26027.5±4085.7，低糖高FFA组为146805.1±21099.4。胰岛素刺激后，低糖对照组为76586.5±18450.1，低糖低FFA组为43738±9203.6，低糖高FFA组为32050.6±3362.3，较低糖对照组显著降低(P<0.01)。在25 mmol/L葡萄糖组，基础葡萄糖摄取量为11793.8±551.4，高糖低FFA组11887.3±2082.3高糖高FFA组为13886.1±3872.9，差别无统计学意义(P>0.05)，但较低糖对照组显著降低(P<0.01);加入胰岛素刺激后，高糖对照组为42798.8±9441.5，高糖低FFA组为23946.9±3839.6，高糖高FFA组为13886.1±3872.9，各组之间差别显著(P<0.01)。对细胞内ROS的研究发现，低糖低FFA组为2234.2±477.2，低糖高FFA组为1969.0±480.9，高糖对照组为1969.0±229.4，高糖低FFA组为2184.5±734.2，高糖高FFA组为2571.3±96.7，可以显著增加的L6细胞内ROS，与低糖对照组(615.3±244.3)相比差别显著(P<0.01)。除对照组之外的各组之间差别无统计学意义(P>0.05)。 结论 高糖和高FFA 可以诱导L6细胞出现氧化应激，同时高糖和高FFA是导致骨骼肌细胞胰岛素抵抗的重要原因之一。

　　【关键词】骨骼肌细胞;胰岛素抵抗;活性氧;游离脂肪酸;葡萄糖

　　doi:10.3969/j.issn.1006-6187.2011.03.018

　　The effect of different concentrations of glucose and free fatty acid on insulin sensitivity and intracellular ROS in L6 myocytes

　　GAO Chun-lin, ZHAO Ya-ping, CHEN Xiao-hui, et al. Department of Pediatrics, Nanjing General Hospital of Nanjing Command, Nanjing 210002, China

　　Corresponding author： GUO Xi-rong, E-mail: xrguo@njmu.edu.cn

　　【Abstract】 Objective To explore the the effect of high levels of glucose and free fatty acid (FFA) on insulin sensitivity and intracellular reactive oxygen species (ROS) in L6 myocytes. Methods L6 myocytes were cultured and differentiated into mature myocytes, then were cultured for 48 hours in DMEM medium containing 5 mmol/L and 25 mmol/L glucose, or 0.3 mmol/L or 1 mmol/L FFA.Insulin sensitivity of L6 myocytes were assessed by 3H-2-D-glucose uptake stimulated by insulin, and the intracellular ROS was determined according to fluorescence intensity of H2DCFDA. Results After treatment with glucose and /or FFA,the basal glucose uptake of L6 in control was 22777.6±6608.7. And the 0.3 mmol/L of FFA did not put any further effect on the basal glucose uptake(26027.5±4085.7). The 1 mmol/L FFA greatly decreased the basal glucose uptake, and the treatment with 25 mmol/L glucose or 25 mmol/L glucose plus 0.3 mmol/L or 1 mmol/L FFA significantly decreased the basal glucose uptake. The insulin stimulated glucose uptake was drastically elevated in control and 25 mmol/L glucose group, but the absolute glucose uptake was lowered in 25 mmol/L glucose group. The insluin sensitivity of L6 myocytes reached the lowest in 25 mmol/L glucose plus 1mmol/L FFA group. On the other hand , all the treatments obviously induced oxidation stress. Conclusions Both high glucose and high FFA induce obvious oxidation stress in L6 myocytes, and are the important causes of insulin resistance of L6 myocytes.

　　【Key words】Skeletal muscle; Insulin resistance; Reactive oxygen species; Free fatty acid; Glucose