Background: Skeletal muscles, metabolically one of the most active tissues, and an important site of insulin-dependent postprandial glucose uptake, are involved in maintaining normoglycaemia. For maintaining energy homeostasis is important AMP activated protein kinase (AMPK). AMPK is also a promising target for the development of new therapies for insulin resistance and type 2 diabetes, as activated enhances glucose homeostasis Aim: AMPK is activated allosterically and by phosphorylation of Thr172 on the catalytic α subunit, while phosphorylation of the Ser485 decreases its activity. Our aim was to determine how insulin and adrenaline affect the function of AMPK or AMPK pharmacological activators in skeletal muscle cells. Hypotheses: 1) Insulin impairs AMPK activity in skeletal muscle cells by suppressing AMPKα Thr172 phosphorylation and promoting AMPKα Ser485 phosphorylation. 2) Adrenaline increases AMPK activity in skeletal muscle cells, as it stimulates AMPKα Thr172 phosphorylation and inhibits AMPKα Ser485 phosphorylation. 3) Insulin decreases while adrenaline increases the effects of AMPK activator A-769662 on phosphorylation of AMPKα Thr172 and Ser485. 4) Direct and indirect AMPK pharmacological activators increase the phosphorylation of AMPKα Thr172 and decrease the phosphorylation of AMPKα Ser485 in skeletal muscle cells. 5) Adrenaline, despite AMPK activation, reduces basal and insulin-induced glucose uptake by L6 skeletal muscle cells. Methods: We investigated the regulation of AMPK in the rat skeletal muscle cell line L6. The phosphorylation sites of AMPKα Thr172 and Ser485 were analyzed with the Western blot method. Activation of AMPK was estimated by measuring phosphorylation of acetyl-CoA carboxylase (Ser79), a direct downstream target of AMPK. Glucose uptake in cells was assessed with the [3H]-2-deoxyglucose uptake assay. Results: We confirmed the first hypothesis, as insulin stimulated AMPKα Ser485 phosphorylation, inhibited AMPKα Thr172 phosphorylation and decreased AMPK activity. In contrast to the second hypothesis, adrenaline had similar effects on phosphorylation and AMPK activity as insulin. The third hypothesis was only partially supported, as prior stimulation with insulin and adrenaline reduced the effects of A-769662 on AMPKα Thr172 phosphorylation but increased the effects of A-769662 on AMPKα Ser485 phosphorylation. Most of the tested pharmacological activators stimulated the phosphorylation of AMPKα Thr172, but they did not affect or even incrased the AMPKα phosphorylation, which is contrary to the fourth hypothesis. We rejected the fifth hypothesis because adrenaline increased basal and insulin-induced glucose uptake in L6 skeletal muscle cells. Conclusion: Our results highlight that adrenaline and insulin act similarly on regulation of AMPK and glucose uptake in L6 skeletal muscle cells and that some pharmacological activators simultaneously increase
phosphorylation of AMPKα Thr172 and AMPKα Ser485, indicating existence of multiple regulatory signalling pathways that may present new opportunities for the development of new strategies for treatment of type 2 diabetes.
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