Neutral ceramidase regulates breast cancer progression by metabolic programming of TREM2-associated macrophages.

The tumor microenvironment is reprogrammed by cancer cells and participates in all stages of tumor progression. Neutral ceramidase is a key regulator of ceramide, the central intermediate in sphingolipid metabolism. The contribution of neutral ceramidase to the reprogramming of the tumor microenvironment is not well understood. Here, we find that deletion of neutral ceramidase in multiple breast cancer models in female mice accelerates tumor growth. Our result show that Ly6C+CD39+ tumor-infiltrating CD8 T cells are enriched in the tumor microenvironment and display an exhausted phenotype. Deletion of myeloid neutral ceramidase in vivo and in vitro induces exhaustion in tumor-infiltrating Ly6C+CD39+CD8+ T cells. Mechanistically, myeloid neutral ceramidase is required for the generation of lipid droplets and for the induction of lipolysis, which generate fatty acids for fatty-acid oxidation and orchestrate macrophage metabolism. Metabolite ceramide leads to reprogramming of macrophages toward immune suppressive TREM2+ tumor associated macrophages, which promote CD8 T cells exhaustion.

Recognition of pathogen-derived sphingolipids in Arabidopsis.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apoplastic ceramidase, NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by a plasma membrane lectin receptor-like kinase, RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2). 9-Methyl-branched sphingoid base is specific to microbes and induces plant immune responses by physically interacting with RDA2. Loss of RDA2 or NCER2 function compromised Arabidopsis resistance against an oomycete pathogen. Thus, we elucidated the recognition mechanisms of pathogen-derived lipid molecules in plants.

Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection.

It is not clear how the complex interactions between diet and intestinal immune cells protect the gut from infection. Neutral ceramidase (NcDase) plays a critical role in digesting dietary sphingolipids. We find that NcDase is an essential factor that controls intestinal immune cell dynamics. Mice lacking NcDase have reduced cluster of differentiation (CD) 8αß+ T cells and interferon (IFN)-γ+ T cells and increased macrophages in the intestine and fail to clear bacteria after Citrobacter rodentium infection. Mechanistically, cellular NcDase or extracellular vesicle (EV)-related NcDase generates sphingosine, which promotes macrophage-driven Th1 immunity. Loss of NcDase influences sphingosine-controlled glycolytic metabolism in macrophages, which regulates the bactericidal activity of macrophages. Importantly, administration of dietary sphingomyelin and genetic deletion or pharmacological inhibition of SphK1 can protect against C. rodentium infection. Our findings demonstrate that sphingosine profoundly alters macrophage glycolytic metabolism, leading to intestinal macrophage activation and T cell polarization, which prevent pathogen colonization of the gut.

Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury.

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.

Measurement of neutral ceramidase activity in vitro and in vivo.

Neutral ceramidase is a hydrolase of ceramide that has been implicated in multiple biologic processes, including inflammation and oncogenesis. Ceramides and other sphingolipids, belong to a family of N-acyl linked lipids that are biologically active in signaling, despite their limited structural functions. Ceramides are generally pro-apoptotic, while sphingosine and sphingosine-1-phosphate (S1P) exert proliferative and pro-oncogenic effects. Ceramidases are important regulators of ceramide levels that hydrolyze ceramide to sphingosine. Thus, ceramidase inhibition significantly increases the quantities of ceramide and its associated signaling. To better understand the function of ceramide, biochemical and cellular assays for enzymatic activity were developed and validated to identify inhibitors of human neutral ceramidase (nCDase). Here we review the measurement of nCDase activity both in vitro and in vivo.

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced ß-cell damage via inhibition of neutral ceramidase.

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to ß-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on ß-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic ß-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced ß-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced ß-cell injury by promoting ceramide synthesis.

New candidate blood biomarkers potentially associated with white matter hyperintensities progression.

We aimed to discover blood biomarkers associated with longitudinal changes in white matter hyperintensities (WMH). This study was divided into a discovery phase and a replication phase. Subjects in both studies were patients with hypertension, aged 50-70, who underwent two magnetic resonance imaging (MRI) sessions and blood extractions over a 4-year follow-up period. In the discovery phase, we screened 1305 proteins in 12 subjects with WMH progression and in 12 matched control subjects. We found that 41 proteins were differentially expressed: 13 were upregulated and 28 were downregulated. We subsequently selected three biomarkers for replication in baseline and follow-up samples in 80 subjects with WMH progression and in 80 control subjects. The selected protein candidates for the replication were MMP9 (matrix metalloproteinase-9), which was higher in cases, MET (hepatocyte growth factor receptor) and ASAH2 (neutral ceramidase), which were both lower in cases of WMH progression. Baseline biomarker concentrations did not predict WMH progression. In contrast, patients with WMH progression presented a steeper decline in MET over time. Furthermore, cases showed higher MMP9 and lower ASAH2 levels than controls at the follow-up. These results indicate that MMP9, MET, and ASAH2 are potentially associated with the progression of WMH, and could therefore be interesting candidates to validate in future studies.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

Neutral Ceramidase Mediates Nonalcoholic Steatohepatitis by Regulating Monounsaturated Fatty Acids and Gut IgA+ B Cells.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is associated with obesity and an increased risk for liver cirrhosis and cancer. Neutral ceramidase (NcDase), which is highly expressed in the intestinal brush border of the small intestine, plays a critical role in digesting dietary sphingolipids (ceramide) to regulate the balance of sphingosine and free fatty acids. It remains unresolved whether obesity-associated alteration of NcDase contributes to the manifestation of NASH. Here, we revealed that NcDase deficiency in murine models of NASH prevents hepatic inflammation and fibrosis but not steatosis. APPROACH AND RESULTS: NcDase-/- mice display reduced stearoyl-CoA desaturase (SCD) 1 expression with a compositional decrease of monounsaturated fatty acids (MUFAs) under the different dietary conditions. We further found that NcDase is a functional regulator of intestinal B cells and influences the abundance and quality of the secretory IgA response toward commensal bacteria. Analysis of composition of the gut microbiota found that Clostridiales colonization was increased in NcDase-/- mice. The colonization of germ-free mice with gut microbiota from NcDase-/- mice resulted in a greater decrease in the expression of SCD1 and the level of MUFAs in the liver relative to gut microbiota from wild-type littermates, which are associated with the alternation of IgA-bound bacteria, including increase of Ruminococcaceae and reduction of Desulfovibrio. Mechanistically, NcDase is a crucial link that controls the expression of SCD1 and MUFA-mediated activation of the Wnt/ß-catenin. Very importantly, our experiments further demonstrated that Wnt3a stimulation can enhance the activity of NcDase in hepatocytes. CONCLUSIONS: Thus, the NcDase-SCD1-Wnt feedback loop promotes the diet-induced steatohepatitis and fibrosis through the regulation of intestinal IgA+ immune cells.

Colon Cancer and Perturbations of the Sphingolipid Metabolism.

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

Manipulation of the Sphingolipid Rheostat Influences the Mediator of Flow-Induced Dilation in the Human Microvasculature.

Background Elevated levels of ceramide, a sphingolipid known to cause a transition from nitric oxide (NO)- to hydrogen peroxide-dependent flow-induced dilation (FID) in human arterioles, correlate with adverse cardiac events. However, elevations of ceramide are associated with changed concentrations of other sphingolipid metabolites. The effects of sphingolipid metabolites generated through manipulation of this lipid pathway on microvascular function are unknown. We examined the hypothesis that inhibition or activation of the ceramide pathway would determine the mediator of FID. Methods and Results Using videomicroscopy, internal diameter changes were measured in human arterioles collected from discarded adipose tissue during surgery. Inhibition of neutral ceramidase, an enzyme responsible for the hydrolysis of ceramide, favored hydrogen peroxide-dependent FID in arterioles from healthy patients. Using adenoviral technology, overexpression of neutral ceramidase in microvessels from diseased patients resulted in restoration of NO-dependent FID. Exogenous sphingosine-1-phosphate, a sphingolipid with opposing effects of ceramide, also restored NO as the mediator of FID in diseased arterioles. Likewise, exogenous adiponectin, a known activator of neutral ceramidase, or, activation of adiponectin receptors, favored NO-dependent dilation in arterioles collected from patients with coronary artery disease. Conclusions Sphingolipid metabolites play a critical role in determining the mediator of FID in human resistance arterioles. Manipulating the sphingolipid balance towards ceramide versus sphingosine-1-phosphate favors microvascular dysfunction versus restoration of NO-mediated FID, respectively. Multiple targets exist within this biolipid pathway to treat microvascular dysfunction and potentially improve patient outcomes.

Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma.

Purpose: To determine major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Methods: Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extracted using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), respectively. Lipids were extracted after weighing the tissue; the protein amounts in the corresponding aqueous phase of organic solvent extraction were recorded. High-resolution mass spectrometry was performed using a Q-exactive mass spectrometer coupled with an EASY-nLC 1000 liquid chromatograph instrument. Bioinformatics and statistical analysis were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amounts were determined using Bradford's method. Western blot, ELISA, and immunohistochemistry utilized established protocols and were performed for protein quantification and localization, respectively. Additional donor tissues were utilized for Western blot, ELISA, and immunohistochemistry. Results: Principal component analysis (PCA) placed control and glaucomatous ONs in two distinct groups based on analysis of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. Conclusions: We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymatic pathways in lysosomal and nonlysosomal cellular compartments.

Neutral ceramidase: Advances in mechanisms, cell regulation, and roles in cancer.

Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of ceramidases, neutral ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.

Functions of neutral ceramidase in the Golgi apparatus.

Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/ß-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.

AKT as a key target for growth promoting functions of neutral ceramidase in colon cancer cells.

Despite advances in the field, colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Research into bioactive sphingolipids over the past two decades has played an important role in increasing our understanding of the pathogenesis and therapeutics of CRC. In the complex metabolic network of sphingolipids, ceramidases (CDases) have a key function. These enzymes hydrolyze ceramides into sphingosine (SPH) which in turn is phosphorylated by sphingosine kinases (SK) 1 and 2 to generate sphingosine-1 phosphate (S1P). Importantly, we have recently shown that inhibition of neutral CDase (nCDase) induces an increase of ceramide in colon cancer cells which decreases cellular growth, increases apoptosis and modulates the WNT/ß-catenin pathway. We have also shown that the deletion of nCDase protected mice from the onset and progression of colorectal cancer in the AOM carcinogen model. Here, we demonstrate that AKT is a key target for the growth suppressing functions of ceramide. The results show that inhibition of nCDase activates GSK3ß through dephosphorylation, and thus is required for the subsequent phosphorylation and degradation of ß-catenin. Our findings show that inhibition of nCDase also inhibits the basal activation status of AKT, and we further establish that a constitutively active AKT (AKT T308D, S473D; AKTDD) reverses the effect of nCDase on ß-catenin degradation. Functionally, the AKTDD mutant is able to overcome the growth suppressive effects of nCDase inhibition in CRC cells. Moreover, nCDase inhibition induces a growth delay of xenograft tumors from control cells, whereas xenograft tumors from constitutively active AKT cells become resistant to nCDase inhibition. Taken together, these results provide important mechanistic insight into how nCDase regulates cell proliferation. These findings demonstrate a heretofore unappreciated, but critical, role for nCDase in enabling/maintaining basal activation of AKT and also suggest that nCDase is a suitable novel target for colon cancer therapy.

Gene cloning of a neutral ceramidase from the sphingolipid metabolic pathway based on transcriptome analysis of Amorphophallus muelleri.

Amorphophallus is a perennial herbaceous plant species mainly distributed in the tropics or subtropics of Asia and Africa. It has been used as a traditional medicine for a long time and now is utilized for the pharmaceutical, chemical and agriculture industries as a valued economic crop. Recently, Amorphophallus has attracted tremendous interest because of its high ceramide content. However, the breeding and genome studies are severely limited by the arduous whole genome sequencing of Amorphophallus. In this study, the transcriptome data of A. muelleri was obtained by utilizing the high-throughput Illumina sequencing platform. Based on this information, the majority of the significant genes involved in the proposed sphingolipid metabolic pathway were identified. Then, the full-length neutral ceramidase cDNA was obtained with the help of its candidate transcripts, which were acquired from the transcriptome data. Furthermore, we demonstrated that this neutral ceramidase was a real ceramidase by eukaryotic expression in the yeast double knockout mutant Δypc1 Δydc1, which lacks the ceramidases-dihydroCDase (YDC1p), phytoCDase (YPC1p). In addition, the biochemical characterization of purified A. muelleri ceramidase (AmCDase) exhibited classical Michaelis-Menten kinetics with an optimal activity ranging from pH 6.5 to 8.0. Based on our knowledge, this study is the first to report the related information of the neutral ceramidase in Amorphophallus. All datasets can provide significant information for related studies, such as gene expression, genetic improvement and application on breeding in Amorphophallus.

A neutral ceramidase, NlnCDase, is involved in the stress responses of brown planthopper, Nilaparvata lugens (Stål).

Ceramidases (CDases) are vital enzymes involved in the biosynthesis of sphingolipids, which are essential components of eukaryotic membranes. The function of these enzymes in insects, however, is poorly understood. We identified a neutral ceramidase (NlnCDase) from the brown planthopper, Nilaparvata lugens, one of the most destructive hemipteran pests of rice. The C12-ceramide was the most preferred substrate for the NlnCDase enzyme. The activity of the NlnCDase enzyme was highest in the neutral-pH range (pH 6.0). It was inhibited by EGTA, Cs+ and Fe2+, while stimulated by EDTA and Ca2+. Moreover, the NlnCDase has higher transcript level and activity in adults than in eggs and nymphs, and in the reproductive organs (ovaries and spermaries) than in other tissues (i.e. heads, thorax, legs, midguts), which suggested that the NlnCDase might be elevated to mediate developmental process. In addition, transcripts and activity of the NlnCDase were up-regulated under abiotic stresses including starvation, abnormal temperature, and insecticides, and biotic stress of resistant rice varieties. Knocking down NlnCDase by RNA interference increased female survival under starvation and temperature stresses, suggesting that NlnCDase might be involved in the stress response in N. lugens.

Neutral ceramidase activity inhibition is involved in palmitate-induced apoptosis in INS-1 cells.

Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic ß cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.

Ceramidases, roles in sphingolipid metabolism and in health and disease.

Over the past three decades, extensive research has been able to determine the biologic functions for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (Hannun, 1996; Hannun et al., 1986; Okazaki et al., 1989). These studies have managed to define the metabolism, regulation, and function of these bioactive sphingolipids. This emerging body of literature has also implicated bioactive sphingolipids, particularly S1P and ceramide, as key regulators of cellular homeostasis. Ceramidases have the important role of cleaving fatty acid from ceramide and producing sphingosine, thereby controlling the interconversion of these two lipids. Thus far, five human ceramidases encoded by five different genes have been identified: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). These ceramidases are classified according to their optimal pH for catalytic activity. AC, which is localized to the lysosomal compartment, has been associated with Farber's disease and is involved in the regulation of cell viability. Neutral ceramidase, which is localized to the plasma membrane and primarily expressed in the small intestine and colon, is involved in digestion, and has been implicated in colon carcinogenesis. ACER1 which can be found in the endoplasmic reticulum and is highly expressed in the skin, plays an important role in keratinocyte differentiation. ACER2, localized to the Golgi complex and highly expressed in the placenta, is involved in programed cell death in response to DNA damage. ACER3, also localized to the endoplasmic reticulum and the Golgi complex, is ubiquitously expressed, and is involved in motor coordination-associated Purkinje cell degeneration. This review seeks to consolidate the current knowledge regarding these key cellular players.

Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells.

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from ß-cells on free fatty acid (FFA)-induced ß-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway. Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination. Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio. Conclusions: ß-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected ß-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for ß-cell lipotoxicity and type 2 diabetes.