Acid ceramidase and human disease

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago, and is deficient in the inherited lipid storage disorder, Farber Lipogranulomatosis (Farber disease). Importantly, AC not only hydrolyzes ceramide into sphingosine, but also can synthesize ceramide from sphingosine and free fatty acids in vitro and in situ. This "reverse" enzymatic activity occurs at a distinct pH from the hydrolysis ("forward") reaction (6.0 vs. 4.5, respectively), suggesting that the enzyme may have diverse functions within cells dependent on its subcellular location and the local pH. Most information concerning the role of AC in human disease stems from work on Farber disease. This lipid storage disease is caused by mutations in the gene encoding AC, leading to a profound reduction in enzymatic activity. Recent studies have also shown that AC activity is aberrantly expressed in several human cancers, and that the enzyme may be a useful cancer drug target. For example, AC inhibitors have been used to slow the growth of cancer cells, alone or in combination with other established, anti-oncogenic treatments. Aberrant AC activity also has been described in Alzheimer's disease, and overexpression of AC may prevent insulin resistant (Type II) diabetes induced by free fatty acids. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.     

Insertional mutagenesis of the mouse acid ceramidase gene leads to early embryonic lethality in homozygotes and progressive lipid storage disease in heterozygotes

Ceramide is an important cellular lipid involved in signal transduction and the biosynthesis of complex sphingolipids. It can be hydrolyzed into sphingosine, another important signaling lipid, by the activity of ceramidases. Point mutations in the gene (Asah1) encoding one ceramidase, acid ceramidase (AC), lead to the lysosomal storage disorder Farber disease (FD). To investigate the role of AC in mammalian development, we disrupted the mouse gene Asah1 in embryonic stem cells by homologous recombination mediated insertion of an AC targeting vector into the wild-type sequence. Genotype analysis of over 150 offspring or embryos from heterozygous intercrosses revealed an absence of Asah1(-/-) individuals at embryonic day (E) 8.5 or later, although the ratio of wild-type to Asah1(+/-) individuals from these intercrosses was 1:2. Northern blot analysis showed that AC expression was turned on early in development, by E7.0, and continued through at least E17. In contrast, expression of the related lipid hydrolase, acid sphingomyelinase, was shut down by E11. Asah1(+/-) mice survived and lived a normal lifespan, but developed a progressive lipid storage disease in several of their organs, particularly the liver. These histopathological findings in Asah1(+/-) animals correlated with an up to twofold increase in the ceramide content of these tissues and a reduction n AC activity, confirming that the gene insertion event disrupted AC activity and ceramide metabolism. These results provide direct in vivo evidence that normal ceramide metabolism, and AC activity in particular, is essential for mammalian development. The animals and embryos described here should be a valuable resource for investigators studying the role of ceramide in cell growth and development, as well as those interested in the pathogenesis of FD and other sphingolipid storage disorders.     

Overexpression and mass spectrometry analysis of mature human acid ceramidase

Human acid ceramidase catalyzes the last step of lysosomal sphingolipid degradation, the hydrolysis of ceramide to sphingosine and free fatty acid. Inherited deficiency of acid ceramidase activity leads to Farber disease (Farber lipogranulomatosis). In this study, we describe the overexpression and processing of recombinant human acid ceramidase in Sf21 insect cells, its purification and characterization. Infection of Sf21 cells with a recombinant baculovirus encoding acid ceramidase precursor led to a mixture of human acid ceramidase precursor and mature enzyme secreted into the medium. Acidification of the cell culture supernatant to pH 4.2-4.3 triggered the processing of the precursor and resulted in a homogeneous sample of mature human acid ceramidase. The enzyme was purified by chromatography on Concanavalin A Sepharose and Octyl Sepharose yielding 1 mg purified protein per liter of supernatant. The recombinant enzyme was deglycosylated with peptide N-glycosidase F and the main component of the released oligosaccharides was identified as GlcNAc(2)(Fuc)Man(3) by electrospray mass spectrometry. Apparently, five of the six potential N-glycosylation sites were used. Tryptic digestion of the functional recombinant enzyme and matrix-assisted laser desorption/ionization time-of-flight- and electrospray ionization-mass spectrometry analysis of the resulting peptides indicated disulfide bridges between C10-C319, C122-C271 and C367-C371.     

Acid ceramidase overexpression prevents the inhibitory effects of saturated fatty acids on insulin signaling

Recent studies indicate that insulin resistance and type 2 diabetes result from the accumulation of lipids in tissues not suited for fat storage, such as skeletal muscle and the liver. To elucidate the mechanisms linking exogenous fats to the inhibition of insulin action, we evaluated the effects of free fatty acids (FFAs) on insulin signal transduction in cultured C2C12 myotubes. As we described previously (Chavez, J. A., and Summers, S. A. (2003) Arch. Biochem. Biophys. 419, 101-109), long-chain saturated FFAs inhibited insulin stimulation of Akt/protein kinase B, a central regulator of glucose uptake and anabolic metabolism. Moreover, these FFAs stimulated the de novo synthesis of ceramide and sphingosine, two sphingolipids shown previously to inhibit insulin action. To determine the contribution of either sphingolipid in FFA-dependent inhibition of insulin action, we generated C2C12 myotubes that constitutively overexpress acid ceramidase (AC), an enzyme that catalyzes the lysosomal conversion of ceramide to sphingosine. AC overexpression negated the inhibitory effects of saturated FFAs on insulin signaling while blocking their stimulation of ceramide accumulation. By contrast, AC overexpression stimulated the accrual of sphingosine. These results support a role for aberrant accumulation of ceramide, but not sphingosine, in the inhibition of muscle insulin sensitivity by exogenous FFAs.     

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.     

Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling

This research paper provides direct evidence concerning the localisation of free fatty acids in stratum corneum lipid model membranes. We employed partially deuterated free fatty acids to gain further information about the assembly of a stratum corneum lipid model membrane based on a ceramide of the phytosphingosine-type (ceramide [AP]) with particular respect to the position of the deuterated groups of the free fatty acids. The application of behenic-22,22,22-d₃-acid and cerotic-12,12,13,13-d₄-acid confirmed that the short-chain ceramide [AP] forces the longer-chained free fatty acids to incorporate into the bilayer created by ceramide [AP]. The ceramide [AP] molecules determine the structural assembly of this model membrane and obligate the long-chain free fatty acids to either arrange inside this formation or to separate as a fatty acid rich phase.     

The reverse activity of human acid ceramidase

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.     

A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.     

Effect of cyclodextrins on the solubilization of lignoceric acid, ceramide, and cerebroside, and on the enzymatic reactions involving these compounds

alpha-Cyclodextrin at concentrations of 1-8 mM helps dissolve, in aqueous solution, fatty acids such as lignoceric, stearic, and palmitic, and complex lipids such as ceramide and cerebroside that contain these acids. Formation of an inclusion complex was indicated on examination of the solution by gel filtration. alpha-Cyclodextrin strikingly increased synthesis of ceramide from sphingosine and either free lignoceric or stearic acid by rat brain preparations. These results suggest the further use of alpha-cyclodextrin in lipid enzymology, especially in relation to sphingolipid metabolism.     

n-3 PUFA and lipotoxicity

Excess lipid accumulation in nonadipose tissues may occur in the setting of high levels of plasma free fatty acids or triglycerides (TGs) in a process called “lipotoxicity”. Evidence from human studies and animal models suggests that lipid accumulation in the heart, skeletal muscle, pancreas, and liver play an important role in the pathogenesis of heart failure, obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM). During the past few years, several studies have shown that n-3 polyunsaturated fatty acids (PUFA) have potentially cardioprotective effects, especially in high-risk patients with dyslipidemia, and might therefore be expected to be of benefit in T2DM. Moreover, new information has demonstrated the beneficial effects of consuming n-3 PUFA in preventing the complications of lipotoxicity. n-3 PUFA dietary intake thus had positive effects on fatty liver in patients with non-alcoholic fatty liver disease (NAFLD), with an improvement in liver echotexture and a significant regression of hepatic brightness, associated with improved liver hemodynamics. The n-3 PUFA also had beneficial effects on ectopic fat accumulation inside the heart, with stabilization of cardiac myocytes and antiarrhythmic effects. On the other hand, recent data from animal models suggest that oral dosing of eicosapentaenoic acid (EPA) could contribute to protect against β-cell lipotoxicity. This review discusses the latest hypotheses regarding lipotoxicity, concentrating on the impact of the n-3 PUFA that contribute to ectopic lipid storage, affecting organ function. Further human studies are needed to test the evidence and elucidate the mechanisms involved in this process.     

Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization

Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.     

Short chain ceramides as substrates for glucocerebroside synthetase. Differences between liver and brain enzymes.

In order to increase the sensitivity of the assay for ceramide: UDPGlc glucosyltransferase, the enzyme that makes glucocerebroside, we synthesized a variety of ceramide homologues that might be better substrates than the naturally occurring ceramides. N-Octanoyl sphingosine proved to be the best lipid tested in liver and brain. It could be added to the tissue homogenate in the dry form, as a thin layer coated on Celite, or in liposomes, prepared from lecithin and cerebroside sulfate. The liposomal form produced better replication of assay values. It is suggested that the addition of cerebroside sulfate to liposomal preparations might be a good, and more physiological, replacement for the commonly used dicetyl phosphate. A new homologue of DL-sphinganine, decasphinganine, was synthesized by an efficient series of steps and acylated with different fatty acids to form ceramide homologues. The best substrate in this series was the lauroyl amide and it is suggested that this lipid be used in cerebroside synthetase assays because of the convenience of preparing it, even though it is not as good as octanoyl sphingosine. Both compounds are distinctly better than natural ceramide or DL-sphinganine amides. From comparisons of enzyme activity under various conditions, the tentative conclusion is drawn that the enzymes in liver and brain have different properties, and that liver has two different synthetases.     

FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix

IR spectroscopic studies are reported for N-stearyl-d-erythro-phytosphingosine (Cer NP) and N-stearyl-2-hydroxy-d-erythro-phytosphingosine (Cer AP) in a hydrated model of the skin lipid barrier comprised of equimolar mixtures of each ceramide with cholesterol and d₃₅-stearic acid. Examination of the methylene stretching, rocking and bending modes reveal some rotational freedom and hexagonal packing in both the ceramide and stearic acid chains. Analysis of the acid carbonyl stretch and the ceramide Amide I modes show both shift to higher frequencies, indicating weaker hydrogen bonding, in the mixed systems compared to the pure materials. For both systems, the fatty acid chain disordering temperatures are significantly increased from those of the pure acids. The observed behaviors of these phytosphingosine ceramide systems are fundamentally different from the previously reported analogous sphingosine ceramide systems. The implications of these observations for lipid organization in the stratum corneum are briefly discussed.     

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.     

Sulfatide and Sphingomyelin Loading of Living Cells as Tools for the Study of Ceramide Turnover by Lysosomal Ceramidase - Implications for the Diagnosis of Farber-Disease

The ceramide turnover by lysosomal ceramidase in intact, living cells was investigated by loading radiolabeled sulfatide or sphingomyelin in situ on skin fibroblasts and lymphoid cells. The cells originated from normal individuals and from patients with acid ceramidase deficiency (Farber disease). While fibroblasts from individuals with Farber disease exhibited some impairment in the degradation of the ceramide produced by sulfatide hydrolysis, lymphoid cells from individuals with Farber disease metabolized the ceramide as readily as did normal cells, suggesting the existence in lymphoid cells of a nonlysosomal degradation pathway for the sulfatide-derived ceramide, In contrast, sphingomyelin loading in the presence of serum showed a considerably decreased turnover of ceramide in both fibroblasts and lymphoid cells from individuals with Farber disease. Further methodologic variation led to the use of LDL-associated radioactive sphingomyelin; LDL-association promoted the targeting of exogenous sphingomyelin to lysosomes. As a result, an almost complete deficiency of ceramide degradation was found in cells from severely affected patients with Farber disease. Our data with this novel method show that sphingomyelin loading of intact living cells is a simple, alternative means for determining ceramide degradation by lysosomal ceramidase and for diagnosing Farber disease.     

Effect of cholesterol on miscibility and phase behavior in binary mixtures with synthetic ceramide 2 and octadecanoic acid. Infrared studies

The three main lipid components of the stratum corneum, namely ceramides, free fatty acids and cholesterol, play a fundamental role in the maintenance of the skin barrier. The current investigation is aimed toward understanding the miscibility and intermolecular interactions of these lipids. Toward this end, Fourier transform infrared spectroscopic studies of the three possible equimolar binary mixtures of cholesterol, a synthetic non-hydroxylated fatty acid N-acyl sphingosine with a C18 chain length (N-stearoylsphingosine, approximating human ceramide 2), and stearic acid were undertaken. The thermotropic responses of the methylene stretching and scissoring vibrations were used to evaluate chain conformation and packing respectively. Selective perdeuteration, of either the stearic acid or the ceramide acid chains, permitted separate and simultaneous evaluation of the conformational order and packing properties of the sphingosine chain, the amide linked fatty acid chains and/or the stearic acid chain. Whereas cholesterol mixed well with ceramide at physiological temperatures, the stearic acid was miscible with the cholesterol only at relatively high temperatures where the fatty acid is disordered. A complex interaction between stearic acid and ceramide was detected. A separate fatty acid-rich phase persisted until at least 50 degrees C, whereas at higher temperatures the components appear to be quite miscible. However, a preferential association of the fatty acid with the ceramide base chain is indicated. None of the binary systems studied exhibit miscibility and interactions resembling those in the ternary mixtures of these substances, which is widely used to model stratum corneum. The role of cholesterol in controlling the miscibility characteristics in the ternary system is evident.     

The effects of N-hexyl-O-glucosyl sphingosine on normal cultured human fibroblasts: a chemical model for Gaucher's disease.

Normal human skin fibroblasts were grown in the presence of N-hexyl-O-glucosyl sphingosine (HGS), an inhibitor of aryl glucosidase and glucocerebrosidase. Tests of the cells with aryl glycosides showed that beta-glucosidase activity in the cells was drastically reduced while other enzyme activities (alpha-glucosidase, beta-galactosidase, and N-acetyl-beta-hexosaminidase) were normal or elevated. Exposure of cells to HGS for 28 days resulted in increased values for cell weight per plate, glucocerebroside concentration, and galactosyl-galactosylglucosyl ceramide concentration. The concentrations of total lipid, cholesterol, and protein were unchanged, as was the fatty acid distribution within the glycolipids. Chemically, the inhibitor-treated cells exhibited a model form of Gaucher's disease. Although many membranous cytoplasmic inclusions were induced by HGS, they were unlike the characteristic inclusions seen in individuals with the genetic disorder. Skin fibroblasts from a Gaucher patient showed no abnormalities in composition or appearance.