Ceramide induction of cell cycle arrest in muscle cell cultures

High fat diets induce insulin resistance and the accumulation of intramyocellular lipids and ceramide in skeletal muscle, but their effect on senescence and proliferative potential of skeletal muscle is less clear. Typically metformin, a common anti-diabetic drug, is prescribed to lower glucose levels and promote lipid mobilization. However, metformin induces apoptosis and cell cycle arrest in cancerous, but less is known in non-cancerous cells. The aim of this study was to determine how ceramide and metformin alter cell cycle in skeletal muscle cells. C2C12 myoblasts were cultured and plated in DMEM containing 10% FBS and 1% antibiotics for experiments. Western blot analysis examined protein expression, BrDu cell proliferation assay kit and FACS flow cytometry were used for cell cycle analysis, and beta-galactosidase staining for senescent cells. Myoblast cultures showed a decrease in cell proliferation and were halted in the G2 phase of the cell cycle after a 8h treatment with 50&mgr;M C2-ceramide. They also showed positive staining for beta-galactosidase at 8h treatment with C2-ceramide treatment. Akt activation was lower in insulin-stimulated cultures receiving C2-treatment at 50 &mgr;M versus control. There was no increase in p21 and p53 protein expression on treatment with C2-ceramide. C2-ceramide induced cell cycle arrest in the G2 phase was ameliorated by metformin. Metformin thus ameliorates the deleterious effects of ceramide-induced cell cycle arrest, supporting the role that AMPK agonists may play in decreasing obesity-associated skeletal muscle mass loss.