The link between lipid oversupply, ceramide production and insulin resistance is well established, although the mechanisms of ceramide accumulation have not been fully explored. We previously showed that insulin resistant mice fed polyunsaturated or saturated high-fat diets have increased ceramide content in skeletal muscle, associated with altered expression of various isoforms of ceramide synthase (CerS). These enzymes generate ceramide via acylation of sphinganine or sphingosine, incorporating different fatty acid (FA) side-chains onto the sphingolipid backbone.

We hypothesised that distinct CerS activities govern inhibitory ceramide species accretion in skeletal muscle, promoting insulin resistance in response to lipid oversupply. Our aim was to modify individual CerS isoforms via adenovirally-mediated overexpression or siRNA knock-down in L6 myotubes, to determine the impact of each isoform on insulin action. Cells were treated in the absence or presence of the FA palmitate, and analysis of glucose disposal and insulin signalling was performed.

Infection of myotubes with recombinant adenoviruses induced a dose-dependent increase in CerS isoforms as determined by immunoblotting. Surprisingly, this was associated with an enhancement of insulin-stimulated glycogen synthesis in myotubes overexpressing CerS1 (p<0.05), and myotubes overexpressing CerS6 exhibited a similar tendency. Both isoforms also promoted insulin-stimulated Ser473-Akt phosphorylation in the absence and presence of palmitate (p<0.05). siRNA-mediated knock-down reduced CerS mRNA expression by 40-60 % for all isoforms (p<0.01 vs. scrambled control). CerS5 and CerS6 knock-down was inhibitory on insulin-stimulated glycogen synthesis (p<0.05), and CerS6 siRNA also impaired insulin signalling at Tyr612-IRS1 and Ser473-Akt (p<0.05), in both the absence and presence of palmitate.

Contrary to our original hypothesis, these data indicate that specific CerS isoforms, in particular CerS1 and CerS6, may play positive roles in the regulation of glucose metabolism. Establishing the intracellular location of these isoforms and their effects on the myotube sphingolipid profile are expected to help decipher this unexpected paradox.