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PubMed1990

Time-course of utilization of [stearic or lignoceric acid]sphingomyelin from high-density lipoprotein by rat tissues.

Utilization of stearic and lignoceric acids supplied by high-density lipoprotein (HDL) sphingomyelin to different tissues was followed for 24 h after rats were injected with HDL containing [[1-14C]stearic (18:0) or [1-14C]lignoceric (24:0) acid [Me-3H]choline]sphingomyelin. Both isotopes reached a maximum in tissue lipids 3-12 h after injection and were recovered mainly in the liver (30%) and small intestine (3%), whereas the other tissues contained approx. 1% or less of the injected dose. All the tissues were able to take up some intact sphingomyelin from HDL and hydrolyze it. In the lung and erythrocytes, the 3H:14C ratio of sphingomyelin remained unchanged throughout the studied period, while an increase in the isotopic ratio was observed in the kidney due to the 3H choline moiety re-used for synthesis of new sphingomyelin. Conversely, the isotopic ratio of sphingomyelin decreased in the liver, indicating a saving of the 14C-labelled fatty acids, especially 24:0. Furthermore, [24:0]ceramide in the liver remained at a high level (6% of the injected dose), whereas [18:0]ceramide decreased to 1%. When the tissues were examined 24 h after injection, the proportion of the 14C linked to sphingomyelin in the total 14C was always higher for both kinds of sphingomyelin than the molar proportion of sphingomyelin in the whole of lipid classes. However, in the majority of the extra-hepatic tissues, more [14C]18:0 than [14C]24:0 was recovered in sphingomyelin, and more 14C radioactivity from 18:0 than from 24:0 was redistributed in the other lipids. The choline moiety from both kinds of sphingomyelin was re-used to synthesize phosphatidylcholine, especially in the liver (up to 20% of the injected dose). All these results show that utilization of sphingomyelin from HDL by tissues normally occurs in vivo and that this phenomenon should be taken into account in the study of the phospholipid turnover of cell membranes. They also show that metabolism of sphingomyelin from HDL in the liver and other tissues is dependent on the sphingomyelin acyl moiety.

Animals↗
PubMed1991

Sphingomyelin synthesis is involved in adherence during macrophage differentiation of HL-60 cells.

Prior studies demonstrated that sphingomyelin degradation via a sphingomyelinase antagonized phorbol ester-mediated differentiation of HL-60 cells into macrophages (Kolesnick, R.N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies show that phorbol esters induce early sphingomyelin synthesis in HL-60 cells and that this event may play a direct role in development of an adherent macrophage population. A maximally effective concentration of the potent phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 x 10(-7) M) stimulated an elevation in the sphingomyelin level at 24 h from 560 to 700 pmol/10(6) cells; a peak level of 1400 pmol/10(6) cells was achieved at 48 h. Phosphatidylcholine levels did not change significantly, indicating sphingomyelin synthesis was selective. The phosphatidylcholine:sphingomyelin ratio decreased from 10.3 to 7.9 at 24 h and to 5.3 at 48 h. Phorbol ester-induced sphingomyelin synthesis was biphasic. A burst of synthesis, detectable within 1 h and linear for 4 h, was followed by a prolonged phase at a slower rate. Ceramide synthesis was also biphasic. Ceramide levels decreased initially consistent with activation of the enzyme, phosphatidylcholine:ceramide cholinephosphotransferase and increased during the prolonged phase of sphingomyelin synthesis. During phorbol ester-induced differentiation, an adherent macrophage population was demonstrable by 14 h. This population contained the entire elevation of sphingomyelin levels. This demonstrates that early sphingomyelin synthesis defines a population of cells destined to become adherent macrophages. Studies were performed to directly manipulate sphingomyelin levels. Small unilamellar vesicles containing sphingomyelin did not directly induce macrophage differentiation but rather potentiated the effect of submaximal concentrations of phorbol ester. Sphingomyelin vesicles (2 x 10-6 M) enhanced TPA (5 x 10-10 M)-induced adherence 2-fold from 12 to 24% of the total population. Sphingosylphosphorylcholine (5 x 10-6 M), which may be acylated to sphingomyelin, was similarly effective. Further, exogenous sphingomyelinase, but not various phospholipases A2 and C, induced detachment of adherent macrophages. In sum, these studies demonstrate that phorbol esters induce early, selective synthesis of sphingomyelin in HL-60 cells. This event defines a population of cells destined to become adherent macrophages and may play a direct role in the adherence process.

Cell Adhesion↗
PubMed1996

Comparative study of the metabolic pools of sphingomyelin and phosphatidylcholine sensitive to tumor necrosis factor.

The metabolism and localization of the pools of sphingomyelin and phosphatidylcholine (PtdCho) which are hydrolyzed upon activation of the sphingomyelin signal transduction pathway were studied in human skin fibroblasts treated with tumor necrosis factor alpha (TNF-alpha). In a first series of experiments, cellular phospholipids were labeled with [3H]choline under conditions that inhibit the vesicular traffic to the plasma membrane. Thus, in human fibroblasts metabolically labeled in the presence of brefeldin A, monensin or at 20 degree C, the arrival of newly synthesized sphingomyelin to the cell surface was prevented, supporting previous conclusions for a vesicular mechanism of sphingomyelin transport to the plasma membrane. Under these conditions, TNF-alpha induced the hydrolysis of PtdCho but did not promote the hydrolysis of 3H-labeled sphingomyelin, suggesting that the sphingomyelin signaling pool resides in a compartment distal to the Golgi apparatus, and possibly in the plasma membrane. TNF was also unable to trigger the breakdown of a radioactive sphingomyelin, [ceramide-3H]sphingomyelin, exogenously added to the cells to label the exoplasmic side of the cell surface. However, TNF caused PtdCho and sphingomyelin degradation in fibroblasts that had been treated with bacterial sphingomyelinase to degrade the sphingomyelin pool of the external leaflet of the plasma membrane. A similar result was obtained at 4 degree C, i.e. under conditions which inhibit endocytosis, thereby excluding the endosomes as a potential site for TNF-induced sphingomyelin hydrolysis. Altogether, these results strongly argue for a localization of the sphingomyelin signaling pool at the inner leaflet of the plasma membrane, but neither in the endolyso-somal nor the Golgi compartments. In addition, when [3H]choline-labeled fibroblasts were treated under non-lytic conditions with bacterial phospholipase C to degrade the external pool of PtdCho, TNF was still able to stimulate the hydrolysis of PtdCho. This demonstrates that the pool of PtdCho involved in TNF-alpha signaling (and which is hydrolyzed concurrently with sphingomyelin to generate diacylglycerol), is not located in the outer leaflet of the plasma membrane.

Brefeldin A↗
PubMed2001

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase, and the resultant accumulation of sphingomyelin in cells and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.

Animals↗
PubMed1988

Sphingomyelin liposomes with defined fatty acids: metabolism and effects on reverse cholesterol transport.

Small unilamellar liposomes prepared from sphingomyelins with defined 14C-labeled fatty acids were studied after injection into rats. The liposomes contained trace amounts of [3H]cholesteryl linoleyl ether (CLE), which served as a nonexchangeable and nonhydrolyzable marker. The liposomes were cleared from the circulation with an initial t1/2 of about 90 min. [14C]18:0- and [14C]18:1-containing sphingomyelins were cleared at a similar rate, but [14C]18:2-sphingomyelin disappeared much faster. The liver accounted for up to 70% of [3H]cholesteryl ether injected with 18:0-sphingomyelin liposomes, and for up to 50% with liposomes prepared from 18:1 or 18:2-sphingomyelin. The initial uptake of the liver appeared to be of the entire particle, and the loss of 14C label with time indicated metabolism of the sphingomyelins. With [14C]18:0-sphingomyelin liposomes, up to 8% of liver radioactivity was recovered in neutral lipids 6 h after injection, and this value was 17 and 22% with [14C]18:2- and [14C]18:1-sphingomyelins, respectively. The recovery in 'carcass' of [3H]cholesteryl ether 3 h after injection of [14C]18:2-sphingomyelin liposomes was 33% and of 14C label, 21%. Injection of 18:1- or 18:2-sphingomyelin liposomes (5.4 mumol/100 g body weight) resulted in a 2-fold increase of plasma unesterified cholesterol; a 30% increase was seen with 18:0 liposomes (2.63 mumol/100 g body weight). In experiments with cultured cells, the unsaturated sphingomyelin liposomes alone enhanced cholesterol efflux more extensively than the saturated ones, but their efficacies became similar when mixed with apoprotein (apo) A-I. At equimolar concentration, apo C-III1 or C-III2 had a smaller effect than apo A-I. It is concluded that 18:1- or 18:2-sphingomyelin tends to form small unilamellar liposomes which may reach also extrahepatic tissues. The liposomes able to enhance cholesterol release in vitro and in vivo. Since they are not a substrate for lecithin-cholesterol acyltransferase, they should be able to deliver the free cholesterol to the liver, where they are also rapidly metabolized.

Adrenal Glands↗
PubMed1991

Metabolism of 3-[3H]sphingosine sphingomyelin labeled with [14C]palmitic or [14C]linoleic acid by Hep G2 cells and rat liver in vivo.

The metabolism of sphingomyelin labeled with 3-[3H]sphingosine and [14C]16:0 or [14C]18:2 fatty acid was studied in cultured Hep G2 cells or macrophages and after injection into rats. In pulse-chase experiments, the loss of 3H and 14C-label was more rapid when the cells had been pulsed with 18:2 than with 16:0 sphingomyelin. At the end of 24 h chase, the labeled ceramide contained more [14C]18:2 fatty acid than [14C]16:0. In addition, the 3H-label derived from 3-[3H]sphingomyelin was recovered also as free sphingosine. After injection in vivo, more [3H]sphingosine-labeled sphingomyelin was present in the liver 3 and 24 h after injection of 16:0 than after injection of 18:2 sphingomyelin. The ratio of [3H]ceramide derived from 16:0 sphingomyelin to that derived from 18:2 sphingomyelin as percent of injected dose was 1.84 3 h after injection and 1.31 after 24 h. The ratio of 3H/14C in liver ceramide was 6.4 3 h after injection of 18:2 sphingomyelin and 3.4 after 16:0 sphingomyelin. The present results show that 3-[3H]sphingomyelin is metabolized quite extensively and that the fate of the sphingosine moiety is related to the type of fatty acid present in the phospholipid. These findings indicate that there is little or no reutilization of 18:2 ceramide for sphingomyelin formation and suggest that sphingosine derived from 18:2 sphingomyelin is channeled primarily for catabolism.

Animals↗
PubMed1975

Permeability and integrity properties of lecithin-sphingomyelin liposomes.

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acid's composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C24: 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1:1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1:1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1:1 molar ratio. (8) When the temperature is abruptly lowered to about 2 degrees C, lipsomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.

Animals↗
PubMed1995

Neurotrophins induce sphingomyelin hydrolysis. Modulation by co-expression of p75NTR with Trk receptors.

We examined neurotrophin-induced sphingomyelin hydrolysis in cells expressing solely the low affinity neurotrophin receptor, p75NTR, and in PC12 cells that co-express p75NTR and Trk receptors. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-5 stimulated sphinomyelin hydrolysis with similar kinetics in p75NTR-NIH-3T3 cells. Although brain-derived neurotrophic factor (10 ng/ml) was slightly more potent than NGF at inducing sphingomyelin hydrolysis, NT-3 and NT-5 induced significant hydrolysis (30-35%) at 0.1 to 1 ng/ml in p75NTR-NIH-3T3 cells. NT-3 did not induce sphingomyelin hydrolysis in Trk C-NIH-3T3 cells nor in cells expressing a mutated p75NTR containing a 57-amino acid cytoplasmic deletion, thus demonstrating the role of p75NTR in this signal transduction pathway. In p75NTR-NIH-3T3 cells, neurotrophin-induced sphingomyelin hydrolysis 1) localized to an internal pool of sphingomyelin, 2) was not a consequence of receptor internalization, and 3) showed no specificity with respect to the molecular species of sphingomyelin hydrolyzed. In contrast to cells expressing solely p75NTR, NGF (100 ng/ml) did not induce sphingomyelin hydrolysis in PC12 cells. Interestingly, NT-3 (10 ng/ml) induced the same extent of sphingomyelin hydrolysis in PC12 cells as was apparent in p75NTR-NIH-3T3 cells. However, in the presence of NGF, NT-3 was unable to induce sphingomyelin hydrolysis, raising the possibility that Trk was modulating p75NTR-dependent sphingomyelin hydrolysis. Inhibition of Trk tyrosine kinase activity with 200 nM K252a enabled both NGF and NT-3 in the presence of NGF to induce sphingomyelin hydrolysis. These data support that p75NTR serves as a common signaling receptor for neurotrophins through induction of sphingomyelin hydrolysis and that crosstalk pathways exist between Trk and p75NTR-dependent signaling pathways.

3T3 Cells↗
PubMed2004

Non-raft forming sphingomyelin-cholesterol mixtures.

Sphingomyelin from biological membranes forms segregated domains with cholesterol in fluid bilayers. However, a synthetic form of sphingomyelin with an oleoyl chain linked to sphingosine is not incorporated into cholesterol-rich domains. We have studied the properties of mixtures of oleoyl-sphingomyelin and cholesterol as well as mixtures of oleoyl-sphingomyelin with 1-stearoyl-2-oleoyl-phosphatidylcholine by DSC and NMR. Cholesterol has a high miscibility with oleoyl-sphingomyelin and it does not separate in crystalline form until the mol fraction of cholesterol reaches a value above 0.6. A large fraction of the cholesterol crystals that are formed are in the monohydrate form. Furthermore, these crystals rehydrate relatively rapidly compared with pure cholesterol crystals in the absence of phospholipid. The environment of the carbonyl group of the phospholipid indicates that it is similar to other forms of sphingomyelin with saturated acyl chains. Also similar to other forms of sphingomyelin, the quaternary ammonium group of oleoyl-sphingomyelin is more rigid than that of phosphatidylcholines, as indicated by the strong resonance observed with cross-polarization/magic angle spinning. Additionally, oleoyl-sphingomyelin produces a larger alteration than egg sphingomyelin of the phase transition of 1-stearoyl-2-oleoyl-phosphatidylcholine. These studies indicate that oleoyl-sphingomyelin, unlike saturated forms of sphingomyelin, does not form segregated domains with cholesterol because of its greater miscibility with phosphatidylcholine.

Cholesterol↗
PubMed2003

Chromatin-associated sphingomyelin: metabolism in relation to cell function.

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.

Animals↗
PubMed1987

A sphingomyelinase-resistant pool of sphingomyelin in the nuclear membrane of hen erythrocytes.

Experiments in which hen erythrocytes were exposed to the action of exogenous sphingomyelinase (Staphylococcus aureus) or to their endogenous plasma membrane sphingomyelinase showed that about 15% of the total sphingomyelin was resistant to breakdown either in intact or lysed cells. This resistant pool of sphingomyelin seems likely to reside in the nuclear membranes of the cells, so that essentially all the plasma membrane sphingomyelin can be broken down by exogenous sphingomyelinase acting on intact cells, suggesting that plasma membrane sphingomyelin is exclusively localised in the outer lipid leaflet. Paradoxically, introduction of Ca2+ into the intact cells using A23187 causes the breakdown of up to 30% of total cell sphingomyelin inside the cells but without apparently affecting the putative nuclear pool of sphingomyelin and this suggests that Ca2+ may alter the original disposition of sphingomyelin in the membrane so that originally outer leaflet sphingomyelin becomes accessible to the endogenous sphingomyelinase inside the cells. No differences were seen in the fatty acid compositions of sphingomyelin degradable by exogenous sphingomyelinase, sphingomyelin degradable in the presence of A23187/Ca2+ or the enzyme-resistant pool of sphingomyelin.

Animals↗
PubMed1999

Comparison of the biophysical properties of racemic and d-erythro-N-acyl sphingomyelins.

In this study stereochemically pure d-erythro-sphingomyelins (SMs) with either 16:0 or 18:1(cisDelta9) as the N-linked acyl-chain were synthesized. Our purpose was to examine the properties of these sphingomyelins and acyl-chain matched racemic (d-erythro/l-threo) sphingomyelins in model membranes. Liquid-expanded d-erythro-N-16:0-SM in monolayers was observed to pack more densely than the corresponding racemic sphingomyelin. Cholesterol desorption to beta-cyclodextrin was significantly slower from d-erythro-N-16:0-SM monolayers than from racemic N-16:0-SM monolayers. Significantly more condensed domains were seen in cholesterol/d-erythro-N-16:0-SM monolayers than in the corresponding racemic mixed monolayers, when [7-nitrobenz-2-oxa-1, 3-diazol-4-yl]phosphatidylcholine was used as a probe in monolayer fluorescence microscopy. With monolayers of N-18:1-SMs, both the lateral packing densities (sphingomyelin monolayers) and the rates of cholesterol desorption (mixed cholesterol/sphingomyelin monolayers) was found to be similar for d-erythro and racemic sphingomyelins. The phase transition temperature and enthalpy of d-erythro-N-16:0-SM in bilayer membranes were slightly higher compared with the corresponding racemic sphingomyelin (41.1 degrees C and 8.4 +/- 0.4 kJ/mol, and 39.9 degrees C and 7.2 +/- 0.2 kJ/mol, respectively). Finally, d-erythro-sphingomyelins in monolayers (both N-16:0 and N-18:1 species) were not as easily degraded at 37 degrees C by sphingomyelinase (Staphylococcus aureus) as the corresponding racemic sphingomyelins. We conclude that racemic sphingomyelins differ significantly in their biophysical properties from the physiologically relevant d-erythro sphingomyelins.

Biophysics↗
PubMed1994

Regulation of the threshold for lipoprotein-induced acyl-CoA:cholesterol O-acyltransferase stimulation in macrophages by cellular sphingomyelin content.

Macrophage acyl-CoA:cholesterol O-acyltransferase (ACAT), a key enzyme in atheroma foam cell formation, is stimulated by lipoproteins only after a "threshold" amount of cholesterol has accumulated in the cell. The present study explores the hypothesis that cellular sphingomyelin, by increasing the capacity of the cell to accommodate excess cholesterol, can influence the threshold of ACAT stimulation by lipoproteins. When the sphingomyelin content of macrophages was increased by either incubation with exogenous sphingomyelin or ceramide (a stimulator of endogenous sphingomyelin synthesis), the ability of acetyl-LDL to stimulate whole-cell ACAT activity was substantially reduced despite similar lipoprotein uptake and total cholesterol accumulation as in control cells. When the sphingomyelin content of macrophages was decreased by sphingomyelinase treatment, the ability of acetyl-LDL to stimulate whole-cell ACAT activity was enhanced despite no change in lipoprotein uptake. Importantly, microsomes isolated from control, sphingomyelin-, or sphingomyelinase-treated macrophages showed no difference in ACAT activity when assayed in vitro in the presence of exogenous cholesterol, suggesting that these treatments affected cholesterol trafficking. Lastly, a corollary of the hypothesis, that cells might adapt to a large increase in free cholesterol by increasing their sphingomyelin content, was supported by showing that the sphingomyelin content of macrophages increased 2.6-fold when the cells were induced to accumulate free cholesterol by incubation with acetyl-LDL plus an ACAT inhibitor. Thus, the sphingomyelin content of macrophages can influence the threshold at which ACAT is stimulated by lipoprotein delivery of cholesterol, and the cholesterol content of macrophages can influence the sphingomyelin content of the cell. These findings are consistent with a model in which cellular sphingomyelin plays an important role in accommodating pools of cellular cholesterol that result from the uptake of atherogenic lipoproteins by macrophages.

Animals↗
PubMed1999

Consumption of casein instead of soybean protein produces a transient rise in the concentration of sphingomyelin in VLDL in rats.

In rats fed cholesterol-rich diets, dietary casein vs. soybean protein raises VLDL cholesterol concentrations. Because sphingomyelin may be an essential, structural component of VLDL, we tested whether casein feeding would raise VLDL-sphingomyelin. Rats were fed cholesterol-rich semipurified diets containing either soybean protein (35 g/100 g) or casein for up to 21 d. Consistent with previous work, casein consumption increased hepatic and VLDL cholesterol concentrations. Dietary casein also significantly raised the amount of sphingomyelin in the VLDL fraction, but this effect was transient. Casein feeding transiently lowered LDL- and HDL-2-sphingomyelin concentrations. We suggest that an increase in hepatic VLDL secretion after casein consumption imposed an increased demand for sphingomyelin in the liver. The activity of key enzymes of sphingomyelin synthesis, i.e., serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholinetransferase and phosphatidylethanolamine:ceramide phosphoethanolaminetransferase and sphingomyelin degradation, i.e., acid sphingomyelinase, were enhanced and depressed, respectively, by casein consumption. Again these effects were transient. Thus, these data indicate that the extra sphingomyelin needed after short-term casein feeding came about through enhanced rates of biosynthesis and reduced rates of degradation in the liver. In addition, plasma transfer of sphingomyelin from HDL-2 to VLDL might have contributed to the increase in VLDL sphingomyelin in the casein-fed rats. This study shows that dietary casein vs. soybean protein transiently influences sphingomyelin metabolism in rats.

Animals↗
PubMed1983

HDL sphingomyelin determinations in normal individuals and patients with type IV-hyperlipoproteinaemia.

In 138 men and 79 women sphingomyelin values in the supernatant of sera obtained by precipitation of apolipoprotein B-containing lipoproteins ( = high density lipoprotein-sphingomyelin, HDL sphingomyelin) were determined using an enzymatic method. In men, lower HDL sphingomyelin values were obtained (0.22 +/- 0.06 mmol/l) than for women (0.24 +/- 0.05 mmol/l) (p less than 0.05). In both sexes HDL sphingomyelin values were independent of age, with the exception that in men greater than or equal to 55 years, HDL sphingomyelin concentrations were lower. The correlation coefficients calculated for HDL sphingomyelin and hypertriglyceridaemia and relative body weight were lower than the correlation coefficients calculated for the corresponding HDL cholesterol values and both risk factors. In women with hypertriglyceridaemia (greater than or equal to 2.28 mmol/l) HDL sphingomyelin values were lower than those for normotriglyceridaemic women within the same age group, while in male hypertriglyceridaemic patients within the same age group there were no differences in HDL sphingomyelin values in comparison to normotriglyceridaemic male subjects. In both sexes the differences measured between hypertriglyceridaemic and normotriglyceridaemic subjects for HDL sphingomyelin were lower than for the corresponding HDL cholesterol values. The results obtained show that HDL sphingomyelin values are not simply a reflection of HDL cholesterol values.

Adolescent↗
PubMed1990

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase.

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.

Adenylyl Cyclases↗
PubMed1999

Dietary pectin lowers sphingomyelin concentration in VLDL and raises hepatic sphingomyelinase activity in rats.

There is evidence that cholesterol and sphingomyelin metabolism are interrelated, and thus the hypothesis tested was that dietary pectin, because it can alter hepatic cholesterol metabolism, would also alter hepatic sphingomyelin metabolism. For that purpose, 4-wk-old female Wistar rats were fed a diet without or with pectin (20 g/100 g) up to 21 d. In accordance with previous work, pectin consumption caused a significant (P < 0.001) reduction in hepatic (65%), whole plasma (37%), and VLDL (80%) cholesterol levels. Pectin also significantly reduced VLDL sphingomyelin concentrations (57%), but raised the amount of sphingomyelin in the high density lipoproteins (HDL)-2 fractions (58%), so that the level of sphingomyelin in whole plasma remained unaffected. Pectin did not affect the sphingomyelin concentration in the liver. Pectin consumption did not affect the hepatic sphingomyelin synthesizing enzymes, serine palmitoyltransferase, phosphatidylcholine:ceramide phosphocholine transferase, or phosphatidylethanolamine:ceramide phosphoethanolamine transferase. In contrast, dietary pectin activated both lysosomal (28%) and plasma membrane (26%) sphingomyelinase and thus may have enhanced sphingomyelin degradation. An attempt was made to describe the effects of dietary pectin on sphingomyelin metabolism in terms of altered fluxes through liver and plasma, with whole liver and whole plasma concentrations of sphingomyelin remaining unaffected.

Animals↗
PubMed1990

Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin.

Cell Line, Transformed↗