Ceramide, a sphingolipid, has a causal role in cardiometabolic disease. Ceramides containing saturated acyl tails of 16 and 18 carbons are particularly deleterious, due to frequent association with metabolic diseases. Ceramide synthase (CerS), the enzyme catalysing ceramide production, has six isoforms, each demonstrating specific fatty-acid substrate preference and tissue localisation. Selective pharmacological inhibition of CerS isoforms producing deleterious ceramides, could prevent cardiometabolic disease. Our study aimed to characterise the anti-obesogenic effects of ET2.39, a novel CerS inhibitor.
HEK293 cells were treated with ET2.39 and effects on ceramide levels and cell viability were evaluated. A 4 week high-fat diet (HFD) study was undertaken with male C57BL/6 mice provided Chow, HFD, or HFD + ET2.39 (~10 mg/kg/day) and physiological measurements (weight gain, fat and lean mass, glucose tolerance) taken throughout. Subcellular fractions were extracted from muscle and liver using ultracentrifugation and an iodixanol gradient. Enrichment was verified via western blotting. Lipids extracted from cells, whole tissue lysates and subcellular fractions, were analysed via targeted liquid chromatography-mass spectrometry. ET2.39 significantly inhibited C16:0 and C18:0 ceramide production in HEK293 cells, without impacting cell viability. Dietary administration of ET2.39 prevented HFD-induced weight gain, but not glucose intolerance. ET2.39 slowed fat accretion, significantly decreasing epididymal and inguinal fat pad mass in mice.
Subcellular fractions demonstrated unique sphingolipid composition, with greatest sphingolipid content in the membrane and nucleus. HFD significantly increased deleterious ceramides across all fractions. ET2.39 treatment significantly reduced C18 ceramides in quadriceps (50% reduction), with similar trends across cellular fractions. There was no observed impact of ET2.39 on C16:0 ceramides in tissues from mice. Different sphingolipid distributions exist across cellular fractions, and can be altered by diet and pharmacological inhibition. Our novel CerS inhibitor, ET2.39 demonstrates significant anti-obesogenic effects in HFD-fed mice and could limit the accumulation of deleterious C18:0 ceramides in key metabolic tissues.