Background/Purpose:

Methotrexate (MTX) is a first-line medication effective in multiple forms of inflammatory arthritis. The anti-inflammatory effects of MTX are more prolonged than its plasma half-life might suggest and most likely are the result of accumulation of polyglutamate metabolites in tissues. Inhibition of  5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase by methotrexate metabolites result in elevation of intracellular levels of AICAR, which is a potent inhibitor of AMP deaminase and adenosine deaminase. The consequent increase in intracellular and released adenosine has led to the hypothesis that adenosine mediates the anti-inflammatory effects of MTX.  In addition to regulating nucleotide metabolism AICAR is also is also an endogenous activator of   the intracellular energy sensor AMPK, a highly conserved protein kinase that exists in all eukaryotic cells. AMPK is activated by an increasing cellular AMP/ATP ratio secondary to metabolic stresses or accelerated ATP consumption, and promotes ATP production by promoting catabolism and autophagy and suppressing energy-consuming biosynthetic pathways. Parallel studies of immune cell metabolism have shown that activating catabolic pathways can have anti-inflammatory effects. We hypothesize that AMPK activation through AICAR may mediates a major portion of the anti-inflammatory effects of MTX, and that this may account for some of the efficacy of MTX in rheumatic diseases.

Methods:

Human monocytes derived macrophages (MDM) and murine bone marrow-derived macrophages were treated with MTX, AICAR, folic acid and A769662, a small-molecule that activates AMPK independently of AMP.  AMPK phosphorylation and total AMPK was measured by Western blotting. We have also used compound C, a selective ATP-competitive inhibitor of AMPK in order to determine whether MTX exhibits anti-inflammatory through AMPK. Cells were then stimulated with LPS and production of pro-inflammatory cytokines were measured in the supernatant. 

Results:

MTX induced AMPK phosphorylation in a time and dose-dependent manner, with effects comparable to the synthetic AMPK activator A769662 and AICAR. Mitochondrial oxygen consumption and the ATP/ADP ratio were not altered by MTX, suggesting that AMPK activation by MTX was not due to suppression of ATP generation. MTX-induced AMPK activation was associated with a reduction in production of pro-inflammatory cytokines (IL-6 and TNF) in response to LPS. Compound C is able to partially reverse the effects of MTX on LPS-induced cytokine production, suggesting that AMPK activation is responsible for these anti-inflammatory effects. 

Conclusion: Methotrexate is able to induces AMPK activation in both human and mouse macrophages, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results are being confirmed genetically in macrophages deficient in AMPK subunits. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the action of current ‘antimetabolite’ anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/amp-activated-protein-kinase-as-an-anti-inflammatory-target-for-methotrexate/