Sphingolipids are comprised of a backbone sphingoid base that may be phosphorylated, acylated, glycosylated, bridged to various headgroups through phosphodiester linkages, or otherwise modified. Organisms usually contain large numbers of sphingolipid subspecies and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, "sphingolipidomic" analysis is becoming feasible (at least for important subsets such as all of the backbone "signaling" subspecies: ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry, and these profiles are revealing many surprises, such as that under certain conditions cells contain significant amounts of "unusual" species: N-mono-, di-, and tri-methyl-sphingoid bases (including N,N-dimethylsphingosine); 3-ketodihydroceramides; N-acetyl-sphingoid bases (C2-ceramides); and dihydroceramides, in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4-hydroxyphenylretinamide). The elevation of DHceramides by fenretinide is befuddling because the 4,5-trans-double bond of ceramide has been thought to be required for biological activity; however, DHceramides induce autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.
Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [ 3 H]palmitic acid showed that 4-HPR elicited a doseresponsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/ electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor D-erythro-N,N -dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR -induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther 2008;7(9):2967 -76]
Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo2-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 109 molecules per cell in 24 h, based on the sum of subspecies analyzed by “lipidomic” mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-13C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.
Dysregulated sphingolipid metabolism causes neuronal cell death and is associated with insulin resistance and diseases. Thus, we hypothesized that diabetes-induced changes in retinal sphingolipid metabolism may contribute to neuronal pathologies in diabetic retinopathy. ESI-MS/MS was used to measure ceramide content and ceramide metabolites in whole retinas after 2, 4, and 8 weeks of streptozotocin-induced diabetes. After 4 and 8 weeks of diabetes, a ϳ30% decrease in total ceramide content was observed, concomitant with a significant ϳ30% increase in glucosylceramide levels in fed diabetic rats compared with their age-matched controls. Acute insulin therapy as well as a short-term lowering of glucose via fasting did not affect the increase in glucosylceramide composition. To assess the putative biological consequences of the increase in glucosylceramide composition, R28 retinal neurons were treated with glucosylceramide synthase inhibitors. Inhibiting glycosphingolipid metabolism increased insulin sensitivity in retinal neurons. Glycosphingolipid inhibitors augmented insulin-stimulated p70 S6kinase activity in the presence of inhibitory concentrations of high glucose or glucosamine. Inhibition of glycosphingolipid synthesis also suppressed glucosamine-and interleukin-1-induced death. Consistent with these inhibitor studies, pharmacological accumulation of glycosphingolipids increased activation of the endoplasmic reticulum stress response, a putative modulator of insulin resistance and neuronal apoptosis. It is speculated that an increase in glucosylceramide, and possibly higher-order glycosphingolipids, could contribute to the pathogenesis of diabetic retinopathy by contributing to local insulin resistance, resulting in neuronal cell death. Thus, dysfunctional glycosphingolipid metabolism may contribute to metabolic stress in diabetes, and therapeutic strategies to restore normal sphingolipid metabolism may be a viable approach for treatment of diabetic retinopathy. Diabetes 55: [3573][3574][3575][3576][3577][3578][3579][3580] 2006
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