Identification of adenine modulating AMPK activation in NIH/3T3 cells by proteomic approach.

AMP-activated protein kinase (AMPK) is a metabolic master switch maintaining the energy homeostasis in cells and thought to modulate cellular response to stresses. Adenine as well as a pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), induced the phosphorylation of AMPK and acetyl-CoA carboxylase in NIH/3T3 cells. Administration of adenine or AICAR increased the expression and translocation of glucose transporter 4, enhanced the cellular glucose uptake, and elevated the intracellular ATP level. To better understand the proteomic changes in response to exogenous adenine treatment, we performed two-dimensional difference gel electrophoresis (2DE-DIGE) and grouped protein spots with similar intensities prior to MS analysis. These process allowed us to exclude these constant expressed proteins, reduce the coverage from abundant signals and increase the identification of middle/lower expressed proteins. Bioinformatics analysis on the proteomic alterations suggested that both of adenine and AICAR could induce up-regulation of a panel of proteins associated with glucose metabolism. We also found that adenine upregulated expression of the glycolytic enzyme, hexokinase 2, indicating a link between adenine and AMPK-mediated glycolysis. Taken together, by demonstrating the adenine-mediated proteome changes in NIH/3T3 cells, our study provides useful information for the characteristics of adenine-induced AMPK activation and development of efficient AMPK activator. BIOLOGICAL SIGNIFICANCE: AMPK is a fuel sensing enzyme that responds to a central role of energy homeostasis and contributes to the acceleration of insulin signaling. Recently, we have shown that exogenous adenine exerted anti-inflammatory effects through activation of AMPK, suggesting the treatment is a potent therapeutic strategy against hyperglycemia. Adenine had similar effects with 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) in modulating glucose uptake via AMPK-mediated signaling. In this study, we performed a 2DE-DIGE/MS-based approach to investigate the mechanism of exogenous adenine in NIH/3T3 cells. Our results provide evidence of a novel role for adenine in AMPK-mediated signaling and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.