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Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. We sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely to be a result of increased Drp1 (dynamin-related protein 1) action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O₂ consumption (i.e. respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fibre bundles. Ceramide treatment also increased H₂O₂ levels and reduced Akt/PKB (protein kinase B) phosphorylation in …