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

Nutrition & Metabolism

Nathania Soekamto¹  Jorge Bolinaga1¹  Harsh Patel¹  Srinidi Mohan¹ 

[1] Department of Pharmaceutical Sciences, College of Pharmacy, University of New England, 716 Stevens Avenue, Portland, ME 04103, USA

关键词: Endothelial nitric oxide synthase;    Peroxynitrite;    Superoxide;    Nitric oxide;    AMPK;    L-Arginine;   

Others  :  804441
DOI  :  10.1186/1743-7075-10-40

 received in 2013-02-11, accepted in 2013-05-25,  发布年份 2013

【 摘 要 】

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 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.

Results

Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-N^G-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.

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.

【 授权许可】

   
2013 Mohan et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

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