The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B 1 , an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3 H]sphinganine or L-3-[ 3 H]serine demonstrated that uog1 conferred fumonisin B 1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB 1 , and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B 1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.Interest in determining the regulatory mechanisms of ceramide metabolism has been stimulated over the past decade by the realization that ceramides formed by turnover of complex sphingolipids, and by de novo synthesis, influence key aspects of cell growth, regulation, differentiation, and death (1-6). Ceramides are formed de novo by N-acylation of sphinganine to dihydroceramide, which is subsequently desaturated by dihydroceramide desaturase (7-9). The N-acyltransferase(s), which are referred to herein as (dihydro)ceramide synthase(s), acylate various long chain bases, including sphinganine, sphingosine, and 4-hydroxysphinganine, utilize a wide spectrum of fatty acyl-CoAs, and are inhibited by the mycotoxin, fumonisin B 1 (FB 1 ) 1 (10 -12). Kinetic evidence has been obtained for multiple (dihydro)ceramide synthases, but no biochemical or molecular evidence has been obtained to prove their existence. One reason for suggesting that multiple (dihydro)ceramide synthases exist is that FB ...

Low-grade inflammation observed in obesity is a risk factor for cardiovascular disease. Recent studies revealed that this would be linked to gut-derived endotoxemia during fat digestion in high-fat diets, but nothing is known about the effect of lipid composition. The study was designed to test the impact of oil composition of high-fat diets on endotoxin metabolism and inflammation in mice. C57/Bl6 mice were fed for 8 wk with chow or isocaloric isolipidic diets enriched with oils differing in fatty acid composition: milk fat, palm oil, rapeseed oil, or sunflower oil. In vitro, adipocytes (3T3-L1) were stimulated or not with lipopolysaccharide (LPS; endotoxin) and incubated with different fatty acids. In mice, the palm group presented the highest level of IL-6 in plasma ( P < 0.01) together with the highest expression in adipose tissue of IL-1β and of LPS-sensing TLR4 and CD14 ( P < 0.05). The higher inflammation in the palm group was correlated with a greater ratio of LPS-binding protein (LBP)/sCD14 in plasma ( P < 0.05). The rapeseed group resulted in higher sCD14 than the palm group, which was associated with lower inflammation in both plasma and adipose tissue despite higher plasma endotoxemia. Taken together, our results reveal that the palm oil-based diet resulted in the most active transport of LPS toward tissues via high LBP and low sCD14 and the greatest inflammatory outcomes. In contrast, a rapeseed oil-based diet seemed to result in an endotoxin metabolism driven toward less inflammatory pathways. This shows that dietary fat composition can contribute to modulate the onset of low-grade inflammation through the quality of endotoxin receptors.

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca 2؉ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca 2؉ release from rat brain microsomes. GlcCer had no direct effect on Ca 2؉ release, but rather augmented agonist-stimulated Ca 2؉ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca 2؉ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca 2؉ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca 2؉ release, although only galactosylsphingosine mediated Ca 2؉ release via the RyaR. Finally, we demonstrated that GlcCer levels were ϳ10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca 2؉ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.

Consumption of EPA+DHA esterified to different carriers had different effects on the incorporation of these FAs in blood fractions and on the visual sustained attention performance in children. This trial was registered at clinicaltrials.gov as NCT00382616.