Increased glucosylceramide production leads to decreased cell energy metabolism and lowered tumor marker expression in non-cancerous liver cells.

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.

Update on Glycosphingolipids Abundance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation.

UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells.

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.

The UDP-glucose ceramide glycosyltransferase (UGCG) and the link to multidrug resistance protein 1 (MDR1).

The UDP-glucose ceramide glycosyltransferase (UGCG) is a key enzyme in the sphingolipid metabolism by generating glucosylceramide (GlcCer), the precursor for all glycosphingolipids (GSL), which are essential for proper cell function. Interestingly, the UGCG is also overexpressed in several cancer types and correlates with multidrug resistance protein 1 (MDR1) gene expression. This membrane protein is responsible for efflux of toxic substances and protects cancer cells from cell damage through chemotherapeutic agents. Studies showed a connection between UGCG and MDR1 overexpression and multidrug resistance development, but the precise underlying mechanisms are unknown. Here, we give an overview about the UGCG and its connection to MDR1 in multidrug resistant cells. Furthermore, we focus on UGCG transcriptional regulation, the impact of UGCG on cellular signaling pathways and the effect of UGCG and MDR1 on the lipid composition of membranes and how this could influence multidrug resistance development. To our knowledge, this is the first review presenting an overview about UGCG with focus on the relationship to MDR1 in the process of multidrug resistance development.

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity.

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2+/+ and CerS2-/- mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2-/- mice developed more severe disease than CerS2+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2-/- mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2-/- mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2-/- mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4+ and Th17+ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.

R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4.

R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

Influence of glycosphingolipids on cancer cell energy metabolism.

A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.

Ether lipid and sphingolipid expression patterns are estrogen receptor-dependently altered in breast cancer cells.

Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.

UGCG overexpression leads to increased glycolysis and increased oxidative phosphorylation of breast cancer cells.

The only enzyme in the glycosphingolipid (GSL) metabolic pathway, which produces glucosylceramide (GlcCer) de novo is UDP-glucose ceramide glucosyltransferase (UGCG). UGCG is linked to pro-cancerous processes such as multidrug resistance development and increased proliferation in several cancer types. Previously, we showed an UGCG-dependent glutamine metabolism adaption to nutrient-poor environment of breast cancer cells. This adaption includes reinforced oxidative stress response and fueling the tricarboxylic acid (TCA) cycle by increased glutamine oxidation. In the current study, we investigated glycolytic and oxidative metabolic phenotypes following UGCG overexpression (OE). UGCG overexpressing MCF-7 cells underwent a metabolic shift from quiescent/aerobic to energetic metabolism by increasing both glycolysis and oxidative glucose metabolism. The energetic metabolic phenotype was not associated with increased mitochondrial mass, however, markers of mitochondrial turnover were increased. UGCG OE altered sphingolipid composition of the endoplasmic reticulum (ER)/mitochondria fractions that may contribute to increased mitochondrial turnover and increased cell metabolism. Our data indicate that GSL are closely connected to cell energy metabolism and this finding might contribute to development of novel therapeutic strategies for cancer treatment.

GPER1 influences cellular homeostasis and cytostatic drug resistance via influencing long chain ceramide synthesis in breast cancer cells.

The G protein-coupled estrogen receptor 1 (GPER1) is involved in the regulation of physiological processes such as cellular growth and proliferation, but also in pathophysiological processes such as tumor development. The role of GPER1 in breast cancer is contradictory. Therefore, we investigated the influence of GPER1 overexpression on cellular processes in MCF-7 breast cancer cells. GPER1 overexpression leads to a cell cycle arrest in the G1 phase, induction of autophagy and reduced proliferation. Reduced proliferation was accompanied by a reduced basal respiration and reduced glycolysis rate in GPER1 overexpressing cells. This is presumably ascribable to mitophagy induction following GPER1 overexpression. However, GPER1 overexpressing cells were less sensitive against doxorubicin as compared to control cells. In previous work we showed the effect of transient GPER1 overexpression on the synthesis of several ceramide synthases (CerS) thereby influencing the sphingolipid pathway. Therefore, we investigated CerS expression and sphingolipid level in stable GPER1 overexpressing and control cells. Stable GPER1 overexpression strongly reduced CerS4, CerS5 and CerS6 promoter activity and CerS5 and CerS6 mRNA expression, whereas CerS2 mRNA expression was upregulated. The GPER1 effect on CerS5 promoter is mediated by GSK-3ß signaling. In addition, other enzymes of the sphingolipid pathway were upregulated. Our study provides new insights into the role of GPER1 and the activated sphingolipid pathways and how GPER1 may influence cellular processes such as cancer cell survival following chemotherapy. Further studies are needed to investigate the molecular mechanisms leading to these cellular effects. Finding new therapeutic targets for modulating specifically GPER1 in breast tumors may improve endocrine breast cancer therapy.

UGCG influences glutamine metabolism of breast cancer cells.

UDP-glucose ceramide glucosyltransferase (UGCG) is the key enzyme in glycosphingolipid (GSL) metabolism by being the only enzyme that generates glucosylceramide (GlcCer) de novo. Increased UGCG synthesis is associated with pro-cancerous processes such as increased proliferation and multidrug resistance in several cancer types. We investigated the influence of UGCG overexpression on glutamine metabolism in breast cancer cells. We observed adapted glucose and glutamine uptake in a limited energy supply environment following UGCG overexpression. Glutamine is used for reinforced oxidative stress response shown by increased mRNA expression of glutamine metabolizing proteins such as glutathione-disulfide reductase (GSR) resulting in increased reduced glutathione (GSH) level. Augmented glutamine uptake is also used for fueling the tricarboxylic acid (TCA) cycle to maintain the proliferative advantage of UGCG overexpressing cells. Our data reveal a link between GSL and glutamine metabolism in breast cancer cells, which is to our knowledge a novel correlation in the field of sphingolipid research.

Role of ceramide synthase 2 in G-CSF signaling and G-CSF-R translocation into detergent-resistant membranes.

Ceramides are sphingolipids with defined acyl chain lengths, which are produced by corresponding ceramide synthases (CerS1-6). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), the ablation of CerS2 suppresses EAE-pathology by reducing neutrophil migration into the central nervous system. This migration is induced by granulocyte-colony stimulating factor (G-CSF) signaling. G-CSF signaling leads to a signal cascade including the phosphorylation of Lyn kinase and STAT3. This in turn regulates expression of the neutrophil surface receptor chemokine receptor 2 (CXCR2) and causes translocation of the receptor into detergent-resistant membranes (DRMs). In this study we investigated the role of ceramides in G-CSF signaling. We found, that G-CSF treatment of wild type bone marrow cells (BMCs) leads to translocation of G-CSF-receptor (G-CSF-R) into DRMs. G-CSF also induces downregulation of ceramides in WT and CerS2 null BMCs, as well as upregulation of very long chain lactosylceramides. However, in CerS2 null BMCs, G-CSF failed to induce translocation of G-CSF-R into DRMs, leading to reduced phosphorylation of Lyn and reduced CXCR2 expression. Interestingly, G-CSF signaling in CerS6 null BMCs was not affected. In conclusion, very long chain ceramides are important for G-CSF signaling and translocation of G-CSF-R into DRMs.

Chemosensitivity of human colon cancer cells is influenced by a p53-dependent enhancement of ceramide synthase 5 and induction of autophagy.

Resistance against chemotherapy is a life-threatening complication in colon cancer therapy. To increase response rate, new additional targets that contribute to chemoresistance are still needed to be explored. Ceramides, which belong to the group of sphingolipids, are well-known regulators of cell death and survival, respectively. Here, we show that in human wild-type (wt) p53 HCT-116 colon cancer cells treatment with oxaliplatin or 5-fluorouracil (5-FU) leads to a strong increase in ceramide synthase 5 (CerS5) expression and C16:0-ceramide levels, which was not shown in HCT-116 lacking p53 expression (HCT-116 p53-/-). The increase in CerS5 expression occurs by stabilizing CerS5 mRNA at the 3'-UTR. By contrast, in the p53-deficient cells CerS2 expression and CerS2-related C24:0- and C24:1-ceramide levels were elevated which is possibly related to enhanced polyadenylation of the CerS2 transcript in these cells. Stable knockdown of CerS5 expression using CerS5-targeting shRNA led to an increased sensitivity of HCT-116 p53wt cells, but not of p53-/- cells, to oxaliplatin and 5-FU. Enhanced sensitivity was accompanied by an inhibition of autophagy and inhibition of mitochondrial respiration in these cells. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines. In conclusion, in p53wt colon cancer cells chemosensitivity against oxaliplatin or 5-FU could be enhanced by downregulation of CerS5 expression leading to reduced autophagy and mitochondrial respiration.

The enigma of ceramide synthase regulation in mammalian cells.

Ceramide synthases (CerS) are key enzymes in the lipid metabolism of eukaryotic cells. Their products, ceramides (Cer), are components of cellular membranes but also mediate signaling functions in physiological processes such as proliferation, skin barrier function and cerebellar development. In pathophysiological processes such as multiple sclerosis and tumor progression, ceramide levels are altered, which can be ascribed, partly, to dysregulation of CerS gene transcription. Most publications deal with the effects of altered ceramide levels on physiological and pathophysiological processes, but the regulation of the appropriate CerS is frequently not investigated. This is insufficient for the clarification of the role of ceramides, because most ceramide species are generated by at least two CerS. The mechanisms of CerS regulation are manifold and it seems that each CerS isoform is regulated individually. For this reason, we discuss the different CerS separately in this review. From transcriptional regulation to alteration of protein activity, the possibilities to influence CerS are diverse. Furthermore, CerS are influenced by a variety of molecules including hormones and lipids. Without claiming completeness, we provide a résumé of the regulatory mechanisms for each CerS in mammalian cells and how dysregulation of these mechanisms during physiological processes may lead to pathophysiological processes.

Ceramide synthases CerS4 and CerS5 are upregulated by 17ß-estradiol and GPER1 via AP-1 in human breast cancer cells.

Ceramide synthases (CerS) are important enzymes of the sphingolipid pathway, responsible for the production of ceramides with distinct chain lengths. In human breast cancer tissue, we detected a significant increase in CerS4 and CerS6 mRNA in estrogen receptor positive (ER+) cancer tissue. To clarify the molecular mechanism of this upregulation, we cloned CerS2, -4, -5 and CerS6 promoter and 3'-UTR fragments into luciferase reporter gene plasmids and determined luciferase activity in MCF-7 (ERα/ß) and MDA-MB-231 (ERß) cells after 17ß-estradiol treatment. Only the activities of CerS4 and CerS5 promoter Luc constructs, as well as CerS2- and CerS5-3'-UTR Luc constructs increased after estradiol treatment in MCF-7 cells, and this could be inhibited by the anti-estrogen fulvestrant. Co-transfection with the G protein-coupled estrogen receptor 1 (GPER1) also enhanced CerS2, CerS4 and CerS6 promoter activity whereas CerS5 promoter activity was inhibited in both cell lines. Promoter deletion and mutation constructs from CerS4 and CerS5 promoters revealed that estradiol and GPER1 mediate their effects on both promoters by activating AP-1, most likely through dimerization of c-Jun and c-Fos. At least we could show, that cell proliferation induced by estradiol could be blocked by co-treatment with Fumonisin B1, indicating that upregulation of CerS in breast cancer cells by estrogen is important for cell proliferation and possibly tumor development. In conclusion, our data highlight transcriptional and posttranscriptional mechanisms regulating CerS expression in human cells which provide the basis for further studies investigating the regulation of CerS expression and ceramide synthesis after diverse stimuli in physiological and pathophysiological processess.

Regulation of ceramide synthase 6 in a spontaneous experimental autoimmune encephalomyelitis model is sex dependent.

Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.

The equilibrium between long and very long chain ceramides is important for the fate of the cell and can be influenced by co-expression of CerS.

Ceramides are synthesized by six different ceramide synthases (CerS1-6), which differ in their specificity to produce ceramides of distinct chain length. We investigated the impact of CerS-co-transfection on ceramide production and apoptosis and proliferation in HCT-116 cells. Over-expression of CerS4 and CerS6 enhanced the level of C(16:0)-Cer twofold, that of C(18:0)- and C(20:0)-Cer up to sevenfold, in comparison to vector control transfected cells, whereas over-expression of CerS2 had no effect on the level of very long chain ceramide C(24:0)- and C(24:1)-Cer. Instead over-expression of CerS2 together with CerS4 or CerS6 increased the activity of CerS2 against very-long-chain ceramides about twofold. In contrast, co-expression of CerS4 with CerS6 inhibited slightly the production of C20:0-ceramide in comparison to cells over-expressing CerS4 alone, whereas the activity of CerS6 seemed not to be affected by other CerS. Interestingly, down-regulation of ELOVL1 had a comprehensive effect on the synthesis of very long chain ceramides which possibly point to a requirement for ELOVL1 expression for full CerS2-activity. Co-expression of CerS2 with CerS4/CerS6 reversed the inhibitory effect of long chain ceramides on cell proliferation and the induction of apoptosis. Even though we observed a twofold increase in total ceramide levels after co-expression of CerS2 with CerS4/CerS6, we detected no effect on cell proliferation. These data indicate that an increase in ceramide production per se is not critical for cell survival, but the equilibrium between long and very long chain ceramides and possibly protein/protein interactions determine the fate of the cell.

Ceramide metabolism in mouse tissue.

Ceramides with different N-acyl chains can act as second messengers in various signaling pathways. They are involved in cell processes such as apoptosis, differentiation and inflammation. Ceramide synthases (CerS) are key enzymes in the biosynthesis of ceramides and dihydroceramides. Six isoenzymes (CerS1-6) catalyze the N-acylation of the sphingoid bases, albeit with strictly acyl-Coenzyme A (CoA) chain length specificity. We analyzed the mRNA expression, the protein expression, the specific activity of the CerS, and acyl-CoA, dihydroceramide and ceramide levels in different tissues by LC-MS/MS. Our data indicate that each tissue express a distinct composition of CerS, whereby the CerS mRNA expression levels do not correlate with the respective protein expression levels in the tissues. Furthermore, we found a highly significant negative correlation between the protein expression level of CerS6 and the C16:0-acyl-CoA amounts as well as between the protein expression of CerS2 and C24:0-acyl-CoA amounts. These data indicate that in mouse tissues low substrate availability is compensated by higher CerS protein expression level and vice versa. Apart from the expression level and the specific activity of the CerS, other enzymes of the sphingolipid pathway also influence the composition of ceramides with distinct chain lengths in each cell. Acyl-CoA availability seems to be less important for ceramide composition and might be compensated for by CerS expression/activity.