AMP-activated protein kinase regulates L-arginine mediated cellular responses

作者：Mohan Srinidi^*; Patel Harsh; Bolinaga Jorge; Soekamto Nathania

来源：Nutrition & Metabolism, 2013, 10(1): 40.

DOI：10.1186/1743-7075-10-40

• L-Arginine
• AMPK
• Nitric oxide
• Superoxide
• Peroxynitrite
• Endothelial nitric oxide synthase

Background: 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.
Method: Human umbilical vein endothelial cells were incubated with 100 mu M 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. C-13(6) 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 (O-2(.)) was identified by EPR-spin-trap, and peroxynitrite (ONOO-) was measured by flow-cytometer using aminophenyl fluorescein dye.
Results: Short-term incubation of cells with 100 mu M ARG in the presence or absence of 30 mu M L-NG-Nitroarginine methyl ester (L-NAME) or 30 mu M 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 mu M ARG exposure, was suppressed in cells co-incubated with 30 mu M 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, O-2(-) and ONOO-. Gluconeogenesis inhibition with 30 mu M 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, O-2(-) and ONOO- to control conditions.
Conclusion: 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.

• 出版日期2013-5-29