Background: AMP-activated protein kinase (AMPK) has a significant role in regulating metabolic processes and maintaining energy homeostasis in skeletal muscle. AMPK activation in skeletal muscle stimulates glucose uptake independently of insulin action thereby bypassing defects in insulin signaling. Insulin resistance in skeletal muscle considerably contributes to development of type 2 diabetes, therefore pharmacological activators of AMPK in skeletal muscle present a promising strategy for its treatment. Methotrexate and various other compounds that target purine metabolic pathways were shown to promote AMPK activation.
Aim: AICAR, a pharmacological activator of AMPK, is metabolized to uric acid, which is an independent risk factor of development insulin resistance, type 2 diabetes and numerous other diseases. AICAR application combined with alopurinol, inhibitor of xanthine oxidoreductase, reduces the plasma levels of uric acid. The aim of the master thesis was to investigate the effect of alopurinol on the action of AMPK and insulin in L6 skeletal muscle cells.
Hypotheses: 1) Alopurinol does not activate AMPK, but enhances the AICAR-induced AMPK activation in L6 skeletal muscle cells. 2) In the presence of methotrexate alopurinol does not affect the AICAR-induced AMPK activation in L6 skeletal muscle cells. 3) Alopurinol does not affect the insulin-induced Akt and ERK1/2 phosphorylation in L6 skeletal muscle cells. 4) Alopurinol enhances the AICAR-induced glucose uptake, but does not affect the insulin-induced glucose uptake in L6 skeletal muscle cells.
Methods: To investigate our hypotheses we used cultured L6 skeletal muscle cells. AMPK activation was monitored with western blot by measuring the phosphorylation of AMPK (Thr172) and acetyl-CoA carboxylase (ACC, Ser79). We also determined the phosphorylation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204). Metabolic effects were evaluated by estimating the upake of 3H-2-deoxyglucose. The mRNA expression of xanthine oxidoreductase was estimated with quantitative real-time PCR.
Results: Alopurinol itself (100 µM) or in combination with AICAR did not promote the activation of AMPK in skeletal muscle cells. Alopurinol also did not affect the AICAR-induced AMPK phosphorylation in the presence of methotrexate. In addition, alopurinol reduced the insulin-induced phosphorylation of Akt, but did not affect the phosphorylation of ERK1/2. Finally, alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells.
Conclusion: 1) Alopurinol did not activate AMPK or enhanced AICAR-induced AMPK activation. 2) In the presence of methotrexate alopurinol did not affect the AICAR-induced AMPK activation. 3) Alopurinol reduced the insulin-induced Akt activation, but did not affect the ERK1/2 activation. 4) Alopurinol did not affect the AICAR or insulin-induced glucose uptake in skeletal muscle cells. Our results therefore, support the second hypothesis. The first, third and fourth hypothesis are only partially consistent with the obtained results.