Integration of lipidomics and transcriptomics data towards a systems biology model of sphingolipid metabolism

<p>Abstract</p> <p>Background</p> <p>Sphingolipids play important roles in cell structure and function as well as in the pathophysiology of many diseases. Many of the intermediates of sphingolipid biosynthesis are highly bioactive and sometimes have antagonistic activities, for example, ceramide promotes apoptosis whereas sphingosine-1-phosphate can inhibit apoptosis and induce cell growth; therefore, quantification of the metabolites and modeling of the sphingolipid network is imperative for an understanding of sphingolipid biology.</p> <p>Results</p> <p>In this direction, the LIPID MAPS Consortium is developing methods to quantitate the sphingolipid metabolites in mammalian cells and is investigating their application to studies of the activation of the RAW264.7 macrophage cell by a chemically defined endotoxin, Kdo₂-Lipid A. Herein, we describe a model for the C₁₆-branch of sphingolipid metabolism (i.e., for ceramides with palmitate as the N-acyl-linked fatty acid, which is selected because it is a major subspecies for all categories of complex sphingolipids in RAW264.7 cells) integrating lipidomics and transcriptomics data and using a two-step matrix-based approach to estimate the rate constants from experimental data. The rate constants obtained from the first step are further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data for all species. The robustness of the model is validated through parametric sensitivity analysis.</p> <p>Conclusions</p> <p>A quantitative model of the sphigolipid pathway is developed by integrating metabolomics and transcriptomics data with legacy knowledge. The model could be used to design experimental studies of how genetic and pharmacological perturbations alter the flux through this important lipid biosynthetic pathway.</p

Regulation of Lipid Biosynthesis in Saccharomyces cerevisiae by Fumonisin B\u3csub\u3e1\u3c/sub\u3e

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingo-lipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases

Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids

Native and polyubiquitinated forms of dihydroceramide desaturase are differentially linked to human embryonic kidney cell survival

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival with studies showing that it can both promote and protect against apoptosis. We have therefore, investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) or fenretinide, but not the Degs1 inhibitor, GT11 (((N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide)) induced the polyubiquitination of Degs1 (Mr=40-140 kDa) via a mechanism involving oxidative stress, p38 MAPK and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit ‘gain of function’ and activate pro-survival pathways, p38 MAPK, JNK and X-box protein-1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) at concentrations of 25-50 uM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast with SKi, ABC294640 (25 uM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from pro-apoptotic to pro-survival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme on cell survival/apoptosis

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

Fumonisins-Occurrence, Toxicology and Mechanism of Action

[Synopsis] Fumonisins are fungal toxins commonly found on corn worldwide.They are produced by species of Fusarium, most notably Fusarium moniliforme. Typically, the fumonisin B_1 (FB_1) level in processed corn products averages 5 ppm sometimes occur in corn-products and home-grown corn. High levels of fumonisins in feed are correlated with outbreaks of equine leucoencephalomalacia and porcine pulmonary oedema syndrome. The liver is a target in most species and kidney is a target in some species. FB_1 is considered non-genotoxic but produced liver tumors in male BDIX rats and female B6C3F1 mice, and renal tumors in male F344N rats. Reproductive effects are secondary to maternal toxicity. FB_1 induces apoptosis and alters cell growth, can modify immune response, inhibit the biosynthesis of receptors for pathogens and toxins, can sensitize macrophages to endotoxins, and alters cytokine expression in vivo. FB_1 is an inhibitor of ceramide synthase, a key enzyme in sphingolipid biosynthesis. Inhibition results in an elevation in sphingoid bases and alters the amounts of the 1- phosphates and N-acetyl-derivatives of sphinganine. Disruption of sphingolipid metabolism is correlated with the animal toxicity and rodent carcinogenicity of FB_1. FB_1 also inhibits processes mediated by ceramide generated de novo. All of these changes must be considered when evaluating the effects of fumonisins

Fumonisins-Occurrence, Toxicology and Mechanism of Action

none[Synopsis] Fumonisins are fungal toxins commonly found on corn worldwide.They are produced by species of Fusarium, most notably Fusarium moniliforme. Typically, the fumonisin B_1 (FB_1) level in processed corn products averages < 1 ppm. Nonetheless, levels of FB_1 > 5 ppm sometimes occur in corn-products and home-grown corn. High levels of fumonisins in feed are correlated with outbreaks of equine leucoencephalomalacia and porcine pulmonary oedema syndrome. The liver is a target in most species and kidney is a target in some species. FB_1 is considered non-genotoxic but produced liver tumors in male BDIX rats and female B6C3F1 mice, and renal tumors in male F344N rats. Reproductive effects are secondary to maternal toxicity. FB_1 induces apoptosis and alters cell growth, can modify immune response, inhibit the biosynthesis of receptors for pathogens and toxins, can sensitize macrophages to endotoxins, and alters cytokine expression in vivo. FB_1 is an inhibitor of ceramide synthase, a key enzyme in sphingolipid biosynthesis. Inhibition results in an elevation in sphingoid bases and alters the amounts of the 1- phosphates and N-acetyl-derivatives of sphinganine. Disruption of sphingolipid metabolism is correlated with the animal toxicity and rodent carcinogenicity of FB_1. FB_1 also inhibits processes mediated by ceramide generated de novo. All of these changes must be considered when evaluating the effects of fumonisins

The regulation of p53, p38 MAPK, JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knock down of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knock down of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knock down of SK2 and increased by knock down of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells

Ceramide transfer protein function is essential for normal oxidative stress response and lifespan

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein. We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display \u3e70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal