Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.     

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C_24:0 CerS activity, and while CerS5 and CerS6 each confer ～ 50% of the C_16:0 CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.     

Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis

Mitochondria mediate both cell survival and death. The intrinsic apoptotic pathway is initiated by the permeabilization of the mitochondrial outer membrane to pro-apoptotic inter-membrane space (IMS) proteins. Many pathways cause the egress of IMS proteins. Of particular interest is the ability of ceramide to self-assemble into dynamic water-filled channels. The formation of ceramide channels is regulated extensively by Bcl-2 family proteins and dihydroceramide. Here, we show that the chain length of biologically active ceramides serves as an important regulatory factor. Ceramides are synthesized by a family of six mammalian ceramide synthases (CerS) each of which produces a subset of ceramides that differ in their fatty acyl chain length. Various ceramides permeabilize mitochondria differentially. Interestingly, the presence of very long chain ceramides reduces the potency of C₁₆-mediated mitochondrial permeabilization indicating that the intercalation of the lipids in the dynamic channel has a destabilizing effect, reminiscent of dihydroceramide inhibition of ceramide channel formation (Stiban et al., 2006). Moreover, mitochondria isolated from cells overexpressing the ceramide synthase responsible for the production of C₁₆-ceramide (CerS5) are permeabilized faster upon the exogenous addition of C₁₆-ceramide whereas they are resistant to permeabilization with added C₂₄-ceramide. On the other hand mitochondria isolated from CerS2-overexpressing cells show the opposite pattern, indicating that the product of CerS2 inhibits C₁₆-channel formation ex vivo and vice versa. This interplay between different ceramide metabolic enzymes and their products adds a new dimension to the complexity of mitochondrial-mediated apoptosis, and emphasizes its role as a key regulatory step that commits cells to life or death.     

A novel C-terminal DxRSDxE motif in ceramide synthases involved in dimer formation

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-Lag-CLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6^Δ338–344 indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis.     

Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C_12:0), N-myristoyl- (C_14:0) and N-palmitoyl- (C_16:0) sphingosine (C_18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C_18:0) substrate. The activity towards N-linolenoylsphingosine (C_18:3/C_18:1), N-linoleoylsphingosine (C_18:2/C_18:1) and N-oleoylsphingosine (C_18:1/C_18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C_18:0/C_18:1). The activity towards N-stearoyldihydrosphingosine (C_18:0/C_18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C_6:0/C_18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher K_m values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.     

Inhibitors of specific ceramide synthases

Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C_14:0-Cer – C_26:0-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure–activity relationships and the potential biological impact of these compounds are discussed.     

Oxidized Phospholipids Induce Ceramide Accumulation in RAW 264.7 Macrophages: Role of Ceramide Synthases

Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC) are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs). These OxPLs are factors contributing to pro-atherogenic effects of oxidized LDLs (OxLDLs), including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer) which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C₁₆-, C₂₂-, and C_24:0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.     

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II³SO₃LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C₆ to C₈ fatty acids, C₆-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C₁₆ and C₁₈ fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.     

Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.     

Ceramide biosynthesis in keratinocyte and its role in skin function

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1–CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.     

Stinging Nettle (Urtica dioica L.) Attenuates FFA Induced Ceramide Accumulation in 3T3-L1 Adipocytes in an Adiponectin Dependent Manner

Objective

Excess dietary lipids result in the accumulation of lipid metabolites including ceramides that can attenuate insulin signaling. There is evidence that a botanical extract of Urtica dioica L. (stinging nettle) improves insulin action, yet the precise mechanism(s) are not known. Hence, we examined the effects of Urtica dioica L. (UT) on adipocytes.

Research Design

We investigated the effects of an ethanolic extract of UT on free fatty acid (palmitic acid) induced inhibition of insulin-stimulated Akt serine phosphorylation and modulation of ceramidase expression in 3T3-L1 adipocytes. Adipocytes were exposed to excess FFAs in the presence or absence of UT. Effects on adiponectin expression, ceramidase expression, ceramidase activity, ceramide accumulation and insulin signaling were determined.

Results

As expected, FFAs reduced adiponectin expression and increased the expression of ceramidase enzymes but not their activity. FFA also induced the accumulation of ceramides and reduced insulin-stimulated phosphorylation of Akt in adipocytes. The effects of FFA were partially reversed by UT. UT enhanced adiponectin expression and ceramidase activity in the presence of excess FFAs. UT abated ceramide accumulation and increased insulin sensitivity via enhanced Akt phosphorylation. A siRNA knockdown of adiponectin expression prevented UT from exerting positive effects on ceramidase activity but not Akt phosphorylation.

Conclusions

In adipocytes, the ability of UT to antagonize the negative effects of FFA by modulating ceramidase activity and ceramide accumulation is dependent on the presence of adiponectin. However, the ability of UT to enhance Akt phosphorylation is independent of adiponectin expression. These studies demonstrate direct effects of UT on adipocytes and suggest this botanical extract is metabolically beneficial.     

Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via KCa1.1 upregulation

K_Ca1.1 regulates smooth muscle contractility by modulating membrane potential, and age‐associated changes in K_Ca1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K_Ca1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2‐null mice. The levels of C16‐ and C18‐ceramides, sphinganine, sphingosine, and sphingosine 1‐phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild‐type and young CerS2‐null mice. The altered SL composition upregulated K_Ca1.1 and increased K_Ca1.1 currents, while no change was observed in K_Ca1.1 channel activity. The upregulation of K_Ca1.1 impaired intracellular Ca²⁺ mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3‐kinase, protein kinase C_ζ, c‐Jun N‐terminal kinases, and nuclear factor kappa‐B were found to be involved in K_Ca1.1 upregulation. Our findings suggest that age‐associated changes in SL composition or CerS2 ablation upregulate K_Ca1.1 via the phosphoinositide 3‐kinase/protein kinase C_ζ/c‐Jun N‐terminal kinases/nuclear factor kappa‐B‐mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2‐null mice exhibited similar effects to aged wild‐type mice; therefore, CerS2‐null mouse models may be utilized for investigating the pathogenesis of aging‐associated motility disorders.     

Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C₆, C₁₂, C₁₆, C_18:1, and C_24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe²⁺. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.     

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.     

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism

Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.     

Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C_18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C_18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C_18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C_18:1-ceramide, but not C_18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C_18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C_18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C_18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.¹ In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,² allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.³ demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C₁₆-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.⁴ found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.⁵ revealed that LPS and palmitic acid synergistically increase C₁₆-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C₁₆-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.⁶ These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.⁷ Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.³ demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.⁸ showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C_18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3^−/−) and their wild-type (Acer3^+/+) littermates, we further discovered that the inverse regulation of Acer3 and C_18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C_18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases.     

Ceramide levels in blood plasma correlate with major depressive disorder severity and its neutralization abrogates depressive behavior in mice

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis that will affect ∼10% of people during their lifetime. Therapy for MDD requires prolonged treatment and often fails, predicating a need for novel treatment strategies. Here, we report increased ceramide levels in the blood plasma of MDD patients and in murine stress-induced models of MDD. These blood plasma ceramide levels correlated with the severity of MDD in human patients and were independent of age, sex, or body mass index. In addition, intravenous injection of anti-ceramide antibodies or neutral ceramidase rapidly abrogated stress-induced MDD, and intravenous injection of blood plasma from mice with MDD induced depression-like behavior in untreated mice, which was abrogated by ex vivo preincubation of the plasma with anti-ceramide antibodies or ceramidase. Mechanistically, we demonstrate that ceramide accumulated in endothelial cells of the hippocampus of stressed mice, evidenced by the quantitative measurement of ceramide in purified hippocampus endothelial cells. We found ceramide inhibited the activity of phospholipase D (PLD) in endothelial cells in vitro and in the hippocampus in vivo and thereby decreased phosphatidic acid in the hippocampus. Finally, we show intravenous injection of PLD or phosphatidic acid abrogated MDD, indicating the significance of this pathway in MDD pathogenesis. Our data indicate that ceramide controls PLD activity and phosphatidic acid formation in hippocampal endothelial cells and thereby mediates MDD. We propose that neutralization of plasma ceramide could represent a rapid-acting targeted treatment for MDD.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.