Long-chain Ceramides Research Articles

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.

Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins

Increased mitochondrial ceramide levels are associated with the initiation of apoptosis. There is evidence that ceramide is causal. Thus, the conversion of the precursor, dihydroceramide, to ceramide by the enzyme dihydroceramide desaturase may be important in preparing the cell for apoptosis. Ceramide can initiate apoptosis by permeabilizing the mitochondrial outer membrane to apoptosis-inducing proteins. However, the mitochondrion's ability to produce ceramide may be limited by its proteome. Here, we show that ceramide synthesized in isolated mammalian endoplasmic reticulum (ER) vesicles from either C8-dihydroceramide or sphingosine to produce long-chain ceramide can transfer to isolated mitochondria. The rate of transfer is consistent with a simple collision model. The transfer of the long-chain ceramide is faster than expected for an uncatalyzed process. Sufficient ceramide is transferred to permeabilize the outer membrane to cytochrome c and adenylate kinase. The mitochondria-associated membranes, ER-like membranes that are tightly associated with isolated mitochondria, can produce enough ceramide to permeabilize the outer membrane transiently. Thus, this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria to increase ceramide levels to the critical value required for functional changes, such as ceramide channel self-assembly followed by protein release.

Upregulation of the Human Alkaline Ceramidase 1 and Acid Ceramidase Mediates Calcium-Induced Differentiation of Epidermal Keratinocytes

Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs. haCER1 is the human ortholog of mouse alkaline ceramidase 1 that we previously identified. haCER1 catalyzed the hydrolysis of very long-chain ceramides to generate sphingosine (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER1 in HEKs decreased the levels of D-e-C(24:1)-ceramide and D-e-C(24:0)-ceramide but elevated the levels of both SPH and its phosphate (S1P), whereas RNA interference-mediated knockdown of haCER1 caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER1 overexpression, Ca2+(o) increased the levels of SPH and S1P, and this was attenuated by haCER1 knockdown. haCER1 knockdown also inhibited the Ca2+(o)-induced growth arrest of HEKs and the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Ca2+(o); and its knockdown attenuated the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. These results strongly suggest that upregulation of haCER1 and AC mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs by generating SPH and S1P.

Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.

Recycling of Sphingosine Is Regulated by the Concerted Actions of Sphingosine-1-phosphate Phosphohydrolase 1 and Sphingosine Kinase 2

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(1), an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.

Glucosylceramide synthase decrease in frontal cortex of Alzheimer brain correlates with abnormal increase in endogenous ceramides: Consequences to morphology and viability on enzyme suppression in cultured primary neurons

Abnormal increase in native long-chain ceramides ( lcCer) in AD implicates roles in neuronal atrophy and cognitive dysfunction especially in view of divergent roles this second messenger plays in cell function. Since clearance is mediated by glucosylceramide synthase (GCS, EC 2.4.1.80) levels of the enzyme were compared for 18 samples of AD Brodmann area 9/10 frontal cortex with 11 age-matched controls. Western analysis for ir GCS showed a significant decrease in AD brain ( p < 0.01) consistent with the hypothesis that enzyme dysfunction contributes to neuronal decay. To examine kinetics and consequences to morphology, cerebellar granule cells were treated in vitro with d- threo-P4 (P4). This potent inhibitor of GCS induced a time- and concentration-dependent increase in lcCer parallel to loss of viability and dramatic changes in neuron/neurite morphology via caspase-independent pathways distinct from those of apoptosis or necrosis. Fluorescent labeling with NBD-sphingolipids or immunostaining with anti-synaptic or cytoskeletal markers showed unusual formation of globular swellings along neurites rich in synaptophysin that may resemble formation of dystrophic neurites in AD. Effects of the inhibitor were verified by changes in lcCer mass and turnover of 14[C]-acetate and -galactose or NBD-labeled anabolic products. Addition of a panel of inhibitors of other pathways confirms GCS as the major route for clearance in the present model. Pretreatment with GM 1 whose turnover is compromised was protective and pointed to useful therapeutic applications by supplementing existing membrane stores prior to GSC dysfunction.

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides

Epidermal differentiation results in the formation of the extracellular lipid barrier in the stratum corneum, which mainly consists of ceramides, free fatty acids, and cholesterol. Differentiating keratinocytes of the stratum granulosum synthesize a series of complex long-chain ceramides and glucosylceramides with different chain lengths and hydroxylation patterns at intracellular membranes of the secretory pathway. Formation of complex extracellular ceramides parallels the transition of keratinocytes from the stratum granulosum to the stratum corneum, where their precursors, complex glucosylceramides and sphingomyelin, are secreted and exposed to extracellular lysosomal lipid hydrolases. Submerged cultures used so far showed a reduced ceramide content compared to the native epidermis or the air-exposed, organotypic culture system. In order to investigate the sphingolipid metabolism during keratinocyte differentiation, we optimized a simple cell culture system to generate the major barrier sphingolipids. This optimized model is based on the chemically well-defined serum-free MCDB medium. At low calcium ion concentrations (0.1 mM), keratinocytes proliferate and synthesize mainly Cer(NS) and a small amount of Cer(NP). Supplementation of the MCDB cell culture medium with calcium ions (1.1 mM) and 10 μM linoleic acid triggered differentiation of keratinocytes and synthesis of a complex pattern of free and covalently bound ceramides as found in native epidermis or air-exposed organotypic cultures, though at a reduced level. The mRNA levels of the differentiation markers keratin 10 and profilaggrin increased, as well as those of ceramide glucosyltransferase and glucosylceramide- β-glucosidase. The described culture system was thus suitable for biochemical studies of the sphingolipid metabolism during keratinocyte differentiation. The addition of serum or vitamin A to the medium resulted in a decrease in ceramide and glucosylceramide content. Lowering the medium pH to 6, while maintained cell viability, led to an increase in the processing of probarrier lipids glucosylceramide and sphingomyelin to free ceramides and protein-bound ceramide Cer(OS).

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin.

Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT-GM1 complexes with sphingomyelin (SM)- and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.

Adipose Tissue Inflammation and Increased Ceramide Content Characterize Subjects With High Liver Fat Content Independent of Obesity

We sought to determine whether adipose tissue is inflamed in individuals with increased liver fat (LFAT) independently of obesity. A total of 20 nondiabetic, healthy, obese women were divided into normal and high LFAT groups based on their median LFAT level (2.3 +/- 0.3 vs. 14.4 +/- 2.9%). Surgical subcutaneous adipose tissue biopsies were studied using quantitative PCR, immunohistochemistry, and a lipidomics approach to search for putative mediators of insulin resistance and inflammation. The groups were matched for age and BMI. The high LFAT group had increased insulin (P = 0.0025) and lower HDL cholesterol (P = 0.02) concentrations. Expression levels of the macrophage marker CD68, the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha, and plasminogen activator inhibitor-1 were significantly increased, and those of peroxisome proliferator-activated receptor-gamma and adiponectin decreased in the high LFAT group. CD68 expression correlated with the number of macrophages and crown-like structures (multiple macrophages fused around dead adipocytes). Concentrations of 154 lipid species in adipose tissue revealed several differences between the groups, with the most striking being increased concentrations of triacylglycerols, particularly long chain, and ceramides, specifically Cer(d18:1/24:1) (P = 0.01), in the high LFAT group. Expression of sphingomyelinases SMPD1 and SMPD3 were also significantly increased in the high compared with normal LFAT group. Adipose tissue is infiltrated with macrophages, and its content of long-chain triacylglycerols and ceramides is increased in subjects with increased LFAT compared with equally obese subjects with normal LFAT content. Ceramides or their metabolites could contribute to adverse effects of long-chain fatty acids on insulin resistance and inflammation.

Raft Domain Reorganization Driven by Short- and Long-Chain Ceramide: A Combined AFM and FCS Study

Naturally occurring long-chain ceramides (Cer) are known to alter the lateral organization of biological membranes. In particular, they produce alterations of microdomains that are involved in several cellular processes, ranging from apoptosis to immune response. In order to induce similar biological effects, short-chain Cer are extensively used in in vivo experiments to replace their long-chain analogues. In this work, we used the combined approach of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS) to investigate the effect of Cer chain length in lipid bilayers composed of sphingomyelin, dioleoyl-phosphatidylcholine, and cholesterol. Our results show that only long-chain Cer, like C18 and C16, are able to segregate from the liquid-ordered phase, forming separate Cer-enriched domains. Conversely, short-chain Cer do not form a separate phase but alter the physical properties of the liquid-ordered domains, decreasing their stability and viscosity and perturbing the lipid packing. These differences may contribute to the explanation of the different physiological effects that are often observed for the long- and short-chain Cer.

Obesity resistance of the stearoyl-CoA desaturase-deficient (scd1 -/-) mouse results from disruption of the epidermal lipid barrier and adaptive thermoregulation

Targeted deletion of the stearoyl-CoA desaturase 1 gene (scd1) in mouse causes obesity resistance and a severe skin phenotype. Here, we demonstrate that SCD1 deficiency disrupts the epidermal lipid barrier and leads to uncontrolled transepidermal water loss, breakdown of adaptive thermoregulation and cold resistance, as well as a metabolic wasting syndrome. The loss of omega-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with omega-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1-/- mutants. Artificial occlusion of the skin by topical lipid application largely reconstituted the epidermal barrier and also reversed dysregulation of thermogenesis and cold resistance, as well as the metabolic disturbances. Interestingly, SCD1 deficiency abolished expression of the key transcription factor Lef1, which is essential for interfollicular epidermis, sebaceous glands, and hair follicle development. Finally, the occurrence of SCD1 and a newly described hSCD5 (ACOD4) gene in humans suggests that the scd1-/- mouse mutant might be a valuable animal model for the study of human skin diseases associated with epidermal barrier defects.

On the presence of C2-ceramide in mammalian tissues: possible relationship to etherphospholipids and phosphorylation by ceramide kinase

C(2)-ceramide (N-acetyl-sphingenine) is often used as an analog to study ceramide-mediated cellular processes. According to Lee et al. [J. Biol. Chem. 271 (1996), 209-217], C(2)-ceramide is formed by an acetyl transfer from platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to sphingenine. To substantiate these unconfirmed findings, we (i) developed a method to quantify C(2)-ceramide and (ii) analyzed C(2)-ceramide levels in Pex5(-/-) mice, a model for Zellweger syndrome, in which the synthesis of ether lipids such as PAF is impaired. The presence of C(2)-ceramide could be established in brain (+/-10 pmol/g) and liver (+/-25 pmol/g) from control mice, and was approximately 5000-fold less than the main long-chain ceramide species. In Pex5(-/-) mice, C(2)-ceramide levels did not differ significantly compared to control tissues. Given the presence of a ceramide kinase in mammals, phosphorylation of C(2)-ceramide by human ceramide kinase (HsCERK) was tested. C(2)-ceramide appears to be a good substrate when albumin is used as carrier. In CHO cells overexpressing HsCERK, phosphorylation of exogenously added C(2)-ceramide could also be demonstrated. Our data indicate that C(2)-ceramide is present in mammalian tissues and can be converted to C(2)-ceramide-1-phosphate, in addition to other documented metabolic alterations, but does not seem to be linked to ether lipid metabolism.

Sphingosine, a Product of Ceramide Hydrolysis, Influences the Formation of Ceramide Channels

Ceramides are known to have a regulatory function in apoptosis, including the release of cytochrome c and other proapoptotic factors from the mitochondrial intermembrane space. Ceramides can form large, stable channels in the outer mitochondrial membrane, leading to the proposal that ceramide channels are the pathway through which these proteins are released. Here, we report that sphingosine, a product of ceramide hydrolysis by ceramidase, is capable of destabilizing ceramide channels, leading to their disassembly. Sphingosine is directly responsible for the disassembly of ceramide channels in planar membrane experiments and markedly reduces the ability of ceramide to induce the release of intermembrane space proteins from mitochondria in vitro. Low concentrations of both L and D sphingosine potentiate the release of intermembrane space proteins by long-chain ceramide and channel formation in liposomes. These results provide evidence for a mechanism by which the disassembly of ceramide channels, as initiated by ceramidase, could be accelerated by the direct interaction of the hydrolysis product with the ceramide channels themselves. This mechanism therefore could form a positive feedback loop for rapid shut-down of ceramide channels. However, potentiation of ceramide channel formation is also possible and thus both effects could influence the propensity for mitochondria-mediated apoptosis.

Isolation, Structure Elucidation, Total Synthesis, and Evaluation of New Natural and Synthetic Ceramides on Human SK-MEL-1 Melanoma Cells

Two new long-chain ceramides, trametenamides A (1) and B (2), were isolated from the methanolic extract of the fruiting body of the fungus Trametes menziesii. The structures were elucidated by spectroscopic analyses and chemical transformations, and the absolute stereochemistry of trametenamide B (2) was determined by stereoselective total synthesis of four possible diastereomers. The acetyl derivative of the natural ceramide (1a) and synthetic ceramides (24-27) showed cytotoxicity on the human melanoma cell line SK-MEL-1, which was caused by induction of apoptosis as determined by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and procaspase-9 and -8 processing.

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4 degrees C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.

Cationic long-chain ceramide LCL-30 induces cell death by mitochondrial targeting in SW403 cells

Ceramides are sphingolipid second messengers that are involved in the mediation of cell death. There is accumulating evidence that mitochondria play a central role in ceramide-derived toxicity. We designed a novel cationic long-chain ceramide [omega-pyridinium bromide D-erythro-C16-ceramide (LCL-30)] targeting negatively charged mitochondria. Our results show that LCL-30 is highly cytotoxic to SW403 cells (and other cancer cell lines) and preferentially accumulates in mitochondria, resulting in a decrease of the mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 and caspase-9. Ultrastructural analyses support the concept of mitochondrial selectivity. Interestingly, levels of endogenous mitochondrial C16-ceramide decreased by more than half, whereas levels of sphingosine-1-phosphate increased dramatically and selectively in mitochondria after administration of LCL-30, suggesting the presence of a mitochondrial sphingosine kinase. Of note, intracellular long-chain ceramide levels and sphingosine-1-phosphate remained unaffected in the cytosolic and extramitochondrial (nuclei/cellular membranes) cellular fractions. Furthermore, a synergistic effect of cotreatment of LCL-30 and doxorubicin was observed, which was not related to alterations in endogenous ceramide levels. Cationic long-chain pyridinium ceramides might be promising new drugs for cancer therapy through their mitochondrial preference.

Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg2+ ions, much less on Ca2+ ions. A clear preference for the d-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but N-hexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and N-tetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg2+ dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain. No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.