Background: Type 2 diabetes is the most common form of diabetes. Type 2 diabetes is characterized by insulin resistance in skeletal muscles, which represent a key site of postprandial glucose uptake. Pharmacological activation of AMP-activated protein kinase (AMPK) is a promising strategy to reduce insulin resistance in skeletal muscle. Drugs that act as AMPK activators, may be useful in treatment of type 2 diabetes.
Aim: Sulfasalazine, like antirheumatic drug methotrexate, inhibits 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase
(ATIC) and reduces clearance of ZMP. ZMP (AICAR-monophosphate) is a pharmacologically active form of the most widely used experimental AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR), indicating sulfasalazine might also act as an AMPK activator. The aim of our study was to determine whether sulfasalazine and/or its metabolites sulfapyridine and 5-aminosalicylic acid promote AMPK activation in skeletal muscle.
Hypotheses: (1) Sulfasalazine promotes AMPK activation in skeletal muscle cells independently of 5-aminosalicylic acid and sulfapyridine; (2) Sulfasalazine enhances AICAR-induced AMPK activation in skeletal muscle cells independently of 5-aminosalicylic acid and sulfapyridine; (3) Derivatives of salicylic acid, balsalazide, olsalazine and diflunisal, activate AMPK in skeletal muscle cells.
Methods: As an experimental model we used primary human skeletal muscle cells and rat L6 skeletal muscle cells. AMPK activation was estimated with western blot, namely with measuring AMPK phosphorylation (Thr172) and phosphorylation of his substrate acetyl-CoA carboxylase (ACC, Ser79). We also measured phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2, Thr202/Tyr204).
Results: Our results of 8-hour treatment of cultured myotubes with sulfasalazine indicate, that sulfasalazine promotes AMPK activation in rat L6 myotubes. While it does not act as an AMPK activator in primary human myotubes. Sulfapyridine and 5-aminosalicylic acid did not stimulate AMPK activation in rat or human skeletal muscle cells. So our results partially confirm our first hypothesis. Our results also show that sulfasalazine or its metabolites do not increase AICAR-induced AMPK activation in rat L6 or human myotubes, which does not support our second hypothesis. We partially confirmed our third hypothesis, because after an 1 hour and an 8 hour treatment of rat L6 myotubes, diflunisal increased ACC phosphorylation with statistical significance, while balsalazide and olsalazine did not give us clear results regarding AMPK activation.
Conclusions: Sulfasalazine promotes AMPK activation in rat L6 skeletal muscle cells and does not act as an AMPK activator in primary human skeletal muscle cells, while its two metabolites, sulfapyridine and 5-aminosalicylic acid, do not act as AMPK activators in these experimental models. Sulfasalazine and its metabolites do not act as enhancers of AICAR-induced AMPK activation. We also found that diflunisal is the most effective AMPK activator from the studied derivatives of salicylic acid in rat L6 skeletal muscle cells.