AMP-activated protein kinase regulates L-arginine mediated cellular responses.
AMP-activated protein kinase regulates L-arginine mediated cellular responses.
复制标题
DOI：
10.1186/1743-7075-10-40
复制
发表时间：
2013-05-29
影响因子：
4.5
通讯作者：
Soekamto N
中科院分区：
文献类型：
--
作者：
Mohan S;Patel H;Bolinaga J;Soekamto N
我们先前的研究揭示了连续暴露后短期L-精氨酸（ARG）治疗效果的丧失;导致耐受性的发展，由内皮型一氧化氮合酶（eNOS）下调介导，继发于氧化应激和诱导的葡萄糖蓄积。然而，调节ARG细胞反应的潜在因素目前尚不清楚。在存在或不存在其他试剂的情况下，将人脐静脉内皮细胞与100 μM ARG在缓冲液中孵育2小时（短期或急性），或在培养基中孵育7天并在缓冲液中激发2小时（连续或慢性）。通过分析细胞亚硝酸盐/硝酸盐（NO2-/NO3-）来测定eNOS活性，并且使用SAMS肽来测定AMP活化蛋白激酶（AMPK）活性。将13 C6葡萄糖加入培养基中以测量细胞处理期间的葡萄糖摄取，通过LC-MS/MS测定。用EPR自旋捕集器检测超氧阴离子（O2·-），用氨基苯基荧光素染料流式细胞仪检测过氧亚硝酸根（ONOO-）。在存在或不存在30 μM L-NG-硝基精氨酸甲酯（L-NAME）或30 μM AMPK抑制剂（化合物C，CMP-C）的情况下，将细胞与100 μM ARG短期孵育增加了细胞氧化应激和总体葡萄糖蓄积，而葡萄糖转运蛋白-1（GLUT-1）或AMPK活性与对照组无变化。在与30 μM CMP-C或L-NAME共孵育的细胞中，100 μM ARG暴露2 h后总NO2-/NO3-的增加被抑制。长期暴露于ARG（含或不含CMP-C或L-NAME）可抑制NO2-/NO3-、葡萄糖摄取、GLUT-1、AMPK表达和活性低于对照组，并增加总细胞葡萄糖、O2·-和ONOO-。在ARG暴露2 h期间，用30 μM 5-氯-2-N-2，5-二氯苯磺酰氨基-苯并恶唑（CDB）抑制葡萄糖异生，使总体细胞葡萄糖保持在对照水平，但增加了细胞葡萄糖摄取。连续与CDB和ARG共孵育增加NO2-/NO3-、葡萄糖摄取、GLUT-1、AMPK表达和活性，并将总体细胞葡萄糖、O2·-和ONOO-维持至对照条件。本研究为AMPK作为ARG细胞反应的主要调节剂以及在短期和连续ARG治疗期间调节葡萄糖积累模式提供了基本证据。
Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye. Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased cellular glucose uptake. Continuous co-incubation with CDB and ARG increased NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity, and maintained overall cellular glucose, O2•– and ONOO– to control conditions. The present study provides the fundamental evidence for AMPK as the primary modulator of ARG cellular responses and for regulating the mode of glucose accumulation during short-term and continuous ARG treatments.
登录
查看更多内容
影响因子：
5.6
作者：
Mohan S;Fung HL
通讯作者：
Fung HL
影响因子：
3.6
作者：
GITZELMANN, R
通讯作者：
GITZELMANN, R
DOI：
10.1152/ajprenal.00417.2005
发表时间：
2006-08-01
影响因子：
4.2
作者：
Kakoki, Masao;Kim, Hyung-Suk;Mattson, David L.
通讯作者：
Mattson, David L.
影响因子：
3.5
作者：
Oka, RK;Szuba, A;Cooke, JP
通讯作者：
Cooke, JP
影响因子：
37.8
作者：
Förstermann, U;Münzel, T
通讯作者：
Münzel, T