Background: Type 2 diabetes is characterized by insulin resistance. Skeletal muscle is the main site for glucose uptake postprandially, thus representing an important target tissue to prevent or treat insulin resistance and hyperglicemia. AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis and represents a new pharmacological target in treatment of type 2 diabetes. In skeletal muscle AMPK activation promotes glucose uptake into the tissue, fatty acid oxidation and mitochondrial biogenesis, which helps to ameliorate metabolic dysregulation such as hyperglicemia and insulin resistance in type 2 diabetes. Metabolic dysregulation, such as type 2 diabetes is a common condition, also developed after an organ transplantation and is at least partially caused by immunosupressive therapy. Mycophenolate mofetil is a part of commonly used triple transplantation immunosupressive regimen. It inhibits inosine monophosphate dehydrogenase (IMP-dehydrogenase), preventing the coversion of IMP to xanthosine monophosphate.
Aim: Inhibition of IMP dehydrogenase results in accumulation of IMP and its precursors in de novo synthesis of guanosine monophosphate. Therefore, mycophenolate mofetil, like antirheumatic drug methotrexate, could reduce clearance of ZMP (5-aminoimidazole-4-carboxamide-1-?-D-ribofuranosyl 5’-monophosphate), a precursor of IMP in the last two steps of the de novo purine synthesis. ZMP is an AMP analogue and an active form of AICAR, the most commonly used experimental AMPK activator. Aim of our research was to investigate, if mycophenolate mofetil and alanosine, an inhibitor of conversion of IMP to adenylosuccinate, could activate AMPK in skeletal muscle.
Hypotheses: 1) Mycophenolate mofetil promotes AMPK activation in cultured rat and human skeletal muscle cells; 2) Mycophenolate mofetil and alanosine synergistically activate AMPK in rat skeletal muscle cells; 3) Mycophenolate mofetil modulates the Akt signalling pathway in rat skeletal muscle cells.
Methods: To investigate our hypotheses we used cultured rat skeletal muscle cells (L6) and primary human skeletal muscle cells. We evaluated the effect of mycophenolate mofetil and other agents on AMPK activation with western blot, by measuring the phosphorylation of AMPK (Thr172) and its substrate acetyl-CoA carboxylase (ACC, Ser79). We assessed activation of Akt signalling pathway by measuring the phosphorylation of Akt (Ser473), its substrate AS160 (Ser588) and glycogen synthase kinase-3?/? (GSK-3?/?, Ser21/9).
Results: Mycophenolate mofetil (5?M) increased AICAR-stimulated phosphorylation of AMPK and ACC in rat skeletal muscle cells, but did not activate AMPK when used alone. In human skeletal muscle cells, mycophenolate mofetil did not increase AICAR stimulated AMPK activation. These results only partially support our first hypothesis. In rat skeletal muscle cells mycophenolate mofetil and alanosine increased phosphorylation of ACC, indicating that they activate AMPK sinergistically, which supports our second hypothesis. Finally, we investigated the effect of mycophenolate mofetil on the Akt signalling pathway. Insulin increased Akt phosphorylation in the presence of mycophenolate mofetil. Further, mycophenolate mofetil alone increased Akt phosphorylation, which supports our third hypothesis.
Conclusions: We can conclude that: 1) Mycophenolate mofetil alone does not activate AMPK, but enhances AICAR-stimulated AMPK activation in rat skeletal muscle cells, demonstrating it promotes AMPK activation. In human skeletal muscle cells mycophenolate mofetil did not promote AMPK activation. 2) Mycophenolate mofetil and alanosine synergistically activate AMPK in rat skeletal muscle cells. 3) Mycophenolate mofetil promotes activation of the Akt signalling pathway in rat skeletal muscle cells.
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