Exogenous long chain C14 ceramide prevents insulin‐stimulated glucose uptake in C2C12 muscle cells independent of Akt signaling

Abstract

Plasma ceramides have been linked to peripheral insulin resistance, however the cellular mechanism is unclear. We investigated whether incubation with the long chain C14 ceramide would reduce insulin‐stimulated glucose uptake in muscle cells and if so, whether it acts via insulin signaling through Akt. C2C12 muscle cells were exposed to either C6 or C14 ceramide (50 uM), or vehicle, for 1 hour. Glucose uptake was measured using 3H‐2‐deoxyglucose. Parallel studies using non‐labeled glucose were used to determine Akt (Ser473) phosphorylation via ELISA, and cellular content of ceramide via electrospray ionization tandem mass spectrometry after separation with high‐performance liquid chromatography. Insulin‐stimulated glucose uptake was significantly reduced following treatment with both C6 and C14 compared to untreated cells (P=0.001). Akt phosphorylation was reduced in C6 treated cells; but was unaffected by C14 treatment. Further, there was an increase in C6 content in the cells following treatment with C6; however there was no change in C14 content following C14 treatment, suggesting that this long chain ceramide is not transported into the cell. These preliminary data suggest that C14 ceramide down‐regulates insulin‐stimulated glucose uptake via a different molecular mechanism than C6 ceramide, possibly a second messenger system, and is independent of insulin signaling through Akt.