Widespread tissue distribution and synthetic pathway of polyunsaturated C24:2 sphingolipids in mammals

Abstract

Sphingolipids are multifunctional lipids and a major constituent of the cell membranes of eukaryotes. Although the fatty acid (FA) moiety of sphingolipids is usually a saturated or monounsaturated FA, polyunsaturated FA (PUFA)-containing species also exist in mammalian tissues. In the present study, we showed that C24:2 PUFA-containing ceramide is one of the seven major ceramide species in a wide range of tissues. C24:2 ceramide levels were especially high in spleen and small intestine; in the former, it was the fourth most abundant ceramide species. However, both the synthetic pathway and the physiological function of C24:2 ceramide had yet to be identified. Tracer analysis using deuterium-labeled linoleic acid (C18:2) revealed that C24:2 ceramide is produced via elongation of linoleic acid. We also found that the FA elongase ELOVL1 and the ceramide synthase CERS2 were involved in C24:2 ceramide production. Sphingolipids are known to form lipid microdomains in membranes; however, in a detergent-resistant membrane (DRM) assay, we observed a lower proportion of C24:2 sphingomyelin in the DRM fraction than of saturated sphingomyelins, suggesting that C24:2 sphingolipids may act to negatively regulate lipid microdomain formation. Our findings expand our knowledge of sphingolipid diversity, and provide insight into how different sphingolipid molecular species play different functions in biological membranes.

Biosynthesis of the anti‐lipid‐microdomain sphingoid base 4,14‐sphingadiene by the ceramide desaturase FADS3

“…Various ceramide species differing in FA chain length and degree of unsaturation exist in mammalian tissues and the composition of these ceramide species differs among tissues . The seven most abundant SPH ceramide species in many tissues contain a C16:0, C18:0, C20:0, C22:0, C24:0, C24:1, or C24:2 FA; however, some tissues have ceramide species with a unique FA moiety: C28‐C34 polyunsaturated FAs (PUFAs) in testis and C26‐C36 saturated or monounsaturated FAs in epidermis . Consistent with our previous report, the most abundant SPH ceramide species were a C18:0 ceramide in brain and skeletal muscle, C24 (C24:0, C24:1, and C24:2) ceramides in spleen and liver, and a C16:0 ceramide in kidney, colon, and small intestine (Figure B).…”

“…Similar results have been observed for sphingolipids with a cis double bond in the FA moiety. 36 We hypothesize that FADS3 negatively regulates lipid microdomain formation by increasing the levels of anti-lipid-microdomain sphingolipids (SPD sphingolipids) with concomitant decreases in those of pro-lipid-microdomain sphingolipids (sphingolipids containing other sphingoid bases).…”

“…The DRM fraction was prepared, and lipids were extracted as described previously . Extracted lipids were dried, dissolved in 225 µL of chloroform/methanol (1:1, v/v), and subjected to alkaline treatment by incubating them with 71 µL of 0.5 M NaOH for 1 hour at 37°C.…”

“…The DRM fraction was prepared, and lipids were extracted as described previously. 36 Extracted lipids were dried, dissolved in 225 µL of chloroform/methanol (1:1, v/v), and subjected to alkaline treatment by incubating them with 71 µL of 0.5 M NaOH for 1 hour at 37°C. The samples were neutralized by the addition of 35.5 µL of 1 M formic acid, mixed with 106.5 µL of chloroform and 219 µL of water, and centrifuged (20 400g, room temperature, 3 min) to separate the phases.…”

Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS

“…Each ceramide class includes a variety of molecular species which differ in FA chain-length and/or the degree of unsaturation (8,27,28). Therefore, more than several hundred ceramide species exist in the human SC (5-7).…”