Mossakowski Medical Research Centre Polish Academy of Sciences, Gdańsk, Poland.
Biochem Biophys Res Commun. 2010 Mar 5;393(2):268-73. doi: 10.1016/j.bbrc.2010.01.119. Epub 2010 Feb 1.
Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms. The experiments were performed in normal (NG, 5.6mM) and high (HG, 30mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-(3)H]-glucose. ROS production increased by about 27% (187+/-8 vs. 238+/-9 arbitrary units AU, P<0.01) in HG. Metformin (2mM, 2h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58+/-0.07 to. 0.99+/-0.06, and from 0.53+/-0.03 to 0.64+/-0.03; P<0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C. We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.
高血糖会增加活性氧（ROS）的产生。NAD(P)H 氧化酶产生超氧阴离子，是糖尿病足细胞中 ROS 的主要来源，其产生导致糖尿病肾病的发展。我们研究了一种抗糖尿病药物二甲双胍对培养的足细胞中超氧阴离子产生的影响，并试图阐明其潜在机制。实验在正常（NG，5.6mM）和高（HG，30mM）葡萄糖浓度下进行。通过 DCF 探针的荧光来测量总体 ROS 产生。通过化学发光法测量 NAD(P)H 氧化酶的活性。通过免疫印迹测定 AMP 依赖的激酶（AMPK）的活性，测量磷酸化 AMPK 与总 AMPK 的比值。使用 2-脱氧-[1,2-(3)H]-葡萄糖测量葡萄糖积累。HG 中 ROS 产生增加约 27%（187+/-8 与 238+/-9 个任意单位 AU，P<0.01）。二甲双胍（2mM，2 小时）在 NG 中显著降低 45%的 ROS 产生，在 HG 中降低 60%。二甲双胍降低 NG（36%）和 HG（86%）中的 NAD(P)H 氧化酶活性。二甲双胍在 NG 和 HG 中增加 AMPK 活性（从 0.58+/-0.07 到 0.99+/-0.06，从 0.53+/-0.03 到 0.64+/-0.03；P<0.05）。在存在 AMPK 抑制剂化合物 C 的情况下，二甲双胍对 NAD(P)H 氧化酶和 AMPK 活性的影响被消除。我们表明，二甲双胍通过降低 NAD(P)H 氧化酶活性来减少 ROS 的产生。我们还证明了 NAD(P)H 氧化酶和 AMPK 活性之间的关系。
