Characterisation of the first specific inhibitor of ceramide synthase 1

The signalling lipid ceramide has been implicated in the pathogenesis of obesity-related insulin resistance. Ceramide is synthesized by a family of six ceramide synthases in mammals (CerS1-6), each of which preferentially utilises fatty acids of particular acyl chain length (C14-C28). In skeletal muscle (SkM), C18 ceramide, which is synthesized almost exclusively by ceramide synthase 1 (CerS1), has been suggested to mediate systemic insulin resistance in obese humans and rodent models. Therefore, targeted reduction of SkM C18 ceramide may improve detrimental metabolic outcomes induced by a high fat diet (HFD). This thesis describes the characterisation of a new, selective inhibitor of CerS1, P053. P053 inhibits CerS1 with an IC50 of 0.5 micromolar and selectively reduced C18 ceramide in cultured cells. Lipidomic profiling revealed that P053 administration specifically reduced C18 ceramide levels in SkM of mice fed with standard chow or HFD for 4-6 weeks. The reduction of SkM C18 ceramide was coupled to a compensatory increase in C24 ceramides, which are synthesized by CerS2, and loss of triacylglycerols. No other complex lipid species were affected by P053 treatment. At the physiological level, P053 impeded fat deposition induced by a HFD. Despite its effect on adiposity, P053 treatment had no effect on glucose tolerance and insulin sensitivity, assessed by glucose tolerance test and hyperinsulinemic-euglycemic clamps. Unexpectedly, prolonged CerS1 inhibition with P053 improved mitochondrial oxidative capacity in SkM. This effect was associated with significant increases in mitochondrial respiratory chain complex levels, increased activity of TCA cycle and β-oxidation enzymes, and increased mitochondrial respiratory capacity in SkM. Enhanced mitochondrial metabolism may therefore underlie the reduced lipid accretion in mice treated with P053. A significant positive correlation between SkM C18 ceramide and body adiposity was identified. In contrast, SkM C24 ceramides were inversely correlated with adiposity. Thus, P053 may reduce adiposity both directly by reducing SkM C18 ceramide and indirectly through the associated increase in C24 ceramides. In conclusion, we have generated the first selective CerS1 inhibitor. Results from this thesis revealed a potential new role for CerS1 as an endogenous inhibitor of mitochondrial oxidative function in SkM and regulator of whole-body adiposity