Achari Arunkumar E, Jain Sushil K
Department of Pediatrics, Louisiana State University Health Sciences Center, Shreveport, LA 71103, United States.
Department of Pediatrics, Louisiana State University Health Sciences Center, Shreveport, LA 71103, United States.
Arch Biochem Biophys. 2017 Sep 15;630:54-65. doi: 10.1016/j.abb.2017.07.016. Epub 2017 Jul 27.
Diabetic patients have lower blood levels of l-cysteine (LC) and glutathione (GSH). This study examined the hypothesis that LC supplementation positively up regulates the effects of insulin on GSH and glucose metabolism in 3T3-L1 adipocyte model. 3T3L1 adipocytes were treated with LC (250 μM, 2 h) and/or insulin (15 or 30 nM, 2 h), and high glucose (HG, 25 mM, 20 h). Results showed that HG caused significant increase (95%) in ROS and reduction in the protein levels of DsbA-L (43%), adiponectin (64%), GCLC (20%), GCLM (21%), GSH (50%), and GLUT-4 (23%) in adipocytes. Furthermore, HG caused a reduction in total (35%) and HMW adiponectin (30%) secretion. Treatment with insulin alone significantly (p < 0.05) reduced ROS levels as well as increased DsbA-L, adiponectin, GCLC, GCLM, GSH, and GLUT-4 protein levels, glucose utilization, and improved total and HMW adiponectin secretion in HG treated adipocytes compared to HG alone. Interestingly, LC supplementation along with insulin caused greater reduction in ROS levels and significantly (p < 0.05) boosted the DsbA-L (41% vs LC, 29% vs Insulin), adiponectin (92% Vs LC, 84% Vs insulin) protein levels and total (32% Vs LC, 22% Vs insulin) and HMW adiponectin (75% Vs LC, 39% Vs insulin) secretion compared with the either insulin or LC alone in HG-treated cells. In addition, LC supplementation along with insulin increased GCLC (21% Vs LC, 14% insulin), GCLM (28% Vs LC, 16% insulin) and GSH (25% Vs LC and insulin) levels compared with the either insulin or LC alone in HG-treated cells. Furthermore, LC and insulin increases GLUT-4 protein expression (65% Vs LC, 18% Vs Insulin), glucose utilization (57% Vs LC, 27% Vs insulin) compared with the either insulin or LC alone in HG-treated cells. Similarly, LC supplementation increased insulin action significantly in cells maintained in medium contained control glucose. To explore the beneficial effect of LC is mediated by the upregulation of GCLC, we knocked down GCLC using siRNA in adipoctyes. There was a significant decrease in DsbA-L and GLUT-4 mRNA levels and GSH levels in GCLC knockdown adipocytes and LC supplementation up regulates GCLC, DsbA-L and GLUT-4 mRNA expression and GSH levels in GCLC knockdown cells. These results demonstrated that LC along with insulin increases GSH levels thereby improving adiponectin secretion and glucose utilization in adipocytes. This suggests that LC supplementation can increase insulin sensitivity and can be used as an adjuvant therapy for diabetes.
糖尿病患者血液中的L-半胱氨酸（LC）和谷胱甘肽（GSH）水平较低。本研究检验了以下假设：在3T3-L1脂肪细胞模型中，补充LC可正向上调胰岛素对GSH和葡萄糖代谢的作用。用LC（250μM，2小时）和/或胰岛素（15或30 nM，2小时）以及高糖（HG，25 mM，20小时）处理3T3L1脂肪细胞。结果显示，HG导致脂肪细胞中活性氧显著增加（95%），DsbA-L（43%）、脂联素（64%）、谷氨酸半胱氨酸连接酶催化亚基（GCLC，20%）、谷氨酸半胱氨酸连接酶调节亚基（GCLM，21%）、GSH（50%）和葡萄糖转运蛋白4（GLUT-4，23%）的蛋白质水平降低。此外，HG导致总脂联素分泌减少（35%），高分子量脂联素分泌减少（30%）。与单独使用HG相比，单独用胰岛素处理可显著（p<0.05）降低活性氧水平，并增加HG处理的脂肪细胞中DsbA-L、脂联素、GCLC、GCLM、GSH和GLUT-4的蛋白质水平、葡萄糖利用率，并改善总脂联素和高分子量脂联素的分泌。有趣的是，在HG处理的细胞中，与单独使用胰岛素或LC相比，补充LC与胰岛素一起可更大程度地降低活性氧水平，并显著（p<0.05）提高DsbA-L（与LC相比增加41%，与胰岛素相比增加29%）、脂联素（与LC相比增加92%，与胰岛素相比增加84%）的蛋白质水平以及总脂联素（与LC相比增加32%，与胰岛素相比增加22%）和高分子量脂联素（与LC相比增加75%，与胰岛素相比增加39%）的分泌。此外，在HG处理的细胞中，与单独使用胰岛素或LC相比，补充LC与胰岛素一起可增加GCLC（与LC相比增加21%，与胰岛素相比增加14%）、GCLM（与LC相比增加28%，与胰岛素相比增加16%）和GSH（与LC和胰岛素相比均增加25%）水平。此外，与单独使用胰岛素或LC相比，在HG处理的细胞中，LC和胰岛素可增加GLUT-4蛋白表达（与LC相比增加65%，与胰岛素相比增加18%）、葡萄糖利用率（与LC相比增加57%，与胰岛素相比增加27%）。同样，在含有对照葡萄糖的培养基中培养的细胞中，补充LC可显著增强胰岛素作用。为了探究LC的有益作用是否由GCLC的上调介导，我们在脂肪细胞中使用小干扰RNA敲低GCLC。在GCLC敲低的脂肪细胞中，DsbA-L和GLUT-4的mRNA水平以及GSH水平显著降低，而补充LC可上调GCLC敲低细胞中GCLC、DsbA-L和GLUT-4的mRNA表达以及GSH水平。这些结果表明，LC与胰岛素一起可增加GSH水平，从而改善脂肪细胞中脂联素的分泌和葡萄糖利用率。这表明补充LC可增加胰岛素敏感性，并可作为糖尿病的辅助治疗方法。
