Lipidome Analysis in Brain and Peripheral Plasma Following Milk Fat Globule Membrane Supplementation in Rodents.

SCOPE: Milk fat globule membrane (MFGM) is an essential component of milk. Bovine MFGM (bMFGM) has been shown to support cognitive development and increase relative concentrations of serum phospholipids. This study investigates bioavailability of bMFGM components after oral administration in two preclinical models to explore whether dietary bMFGM induces parallel changes to plasma and brain lipidomes. METHODS AND RESULTS: Transgenic APOE*3.Leiden mice (n = 18 per group) and Sprague-Dawley rats (n = 12 per group) are fed bMFGM-enriched (MFGM+) or Control diet, followed by phospholipid profile-determination in plasma, hippocampus, and prefrontal cortex tissue by targeted mass spectrometry. Multivariate analysis of lipidomic profiles demonstrates a separation between MFGM+ and Control plasma across rodents. In plasma, sphingomyelins contributed the most to the separation of lipid patterns among both models, where three sphingomyelins (d18:1/14:0, d18:1/23:0, d18:1/23:1[9Z]) are consistently higher in the circulation of MFGM+ groups. A similar trend is observed in rat prefrontal cortex, although no significant separation of the brain lipidome is demonstrated. CONCLUSION: bMFGM-enriched diet alters plasma phospholipid composition in rodents, predominantly increasing sphingomyelin levels in the systemic circulation with similar, but non-significant, trends in central brain regions. These changes may contribute to the beneficial effects of bMFGM on neurodevelopment during early life.

Effects of dietary phosphatidylcholine and sphingomyelin on DSS-induced colitis by regulating metabolism and gut microbiota in mice.

Patients with inflammatory bowel diseases tend to show alteration of lipid profiles. It remains unknown whether dietary intake with specific lipids, such as phosphatidylcholine (PC) and sphingomyelin (SM), have distinguishable effects against IBD. Here, a preclinical study using dextran sulphate sodium (DSS)-induced colitis mice model was applied to explore/compare the effects by PC, and SM. Results showed that PC treatment (p.o., 30 mg/kg b.w., 15 d) exerted higher inhibitory activity than the same dosage of SM supplementation on colonic tissue lesions and pro-inflammatory cytokines expressions induced by DSS. Integrative analysis of the metabolome and microbiome indicated that PC and SM supplementation could modulate endogenous tryptophan metabolism, arginine and proline metabolism, purine metabolism, bile secretion, as well as vitamin digestion and absorption, closely correlated with their regulation on the abundance of Lactobacillus, Faecalibacterium, Dubosiella, Turicibacter, and Parasutterella communities in the gut. Based on these data, PC is a more promising candidate for preventing colitis than SM. Our findings provided a scientific foundation for further clinical research to screen more efficient dietary intervention strategy for colitis prevention.

Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids.

SCOPE: Enhanced adiposity and metabolic inflammation are major features of obesity associated with altered gut microbiota and intestinal barrier. How these metabolic outcomes can be impacted by milk polar lipids (MPL), naturally containing 25% of sphingomyelin, is investigated in mice fed a mixed high-fat (HF) diet . METHODS AND RESULTS: Male C57Bl/6 mice receive a HF-diet devoid of MPL (21% fat, mainly palm oil, in chow), or supplemented with 1.1% or 1.6% of MPL (HF-MPL1; HF-MPL2) via a total-lipid extract from butterserum concentrate for 8 weeks. HF-MPL2 mice gain less weight versus HF (p < 0.01). Diets do not impact plasma markers of inflammation but in the liver, HF-MPL2 tends to decrease hepatic gene expression of macrophage marker F4/80 versus HF-MPL1 (p = 0.06). Colonic crypt depth is the maximum in HF-MPL2 (p < 0.05). In cecal microbiota, HF-MPL1 increases Bifidobacterium animalis versus HF (p < 0.05). HF-MPL2 decreases Lactobacillus reuteri (p < 0.05), which correlates negatively with the fecal loss of milk sphingomyelin-specific fatty acids (p < 0.05). CONCLUSION: In mice fed a mixed HF diet, MPL can limit HF-induced body weight gain and modulate gut physiology and the abundance in microbiota of bacteria of metabolic interest. This supports further exploration of how residual unabsorbed lipids reaching the colon can impact HF-induced metabolic disorders.

Dietary sphingomyelin attenuates hepatic steatosis and adipose tissue inflammation in high-fat-diet-induced obese mice.

Western-type diets can induce obesity and related conditions such as dyslipidemia, insulin resistance and hepatic steatosis. We evaluated the effects of milk sphingomyelin (SM) and egg SM on diet-induced obesity, the development of hepatic steatosis and adipose inflammation in C57BL/6J mice fed a high-fat, cholesterol-enriched diet for 10 weeks. Mice were fed a low-fat diet (10% kcal from fat) (n=10), a high-fat diet (60% kcal from fat) (HFD, n=14) or a high-fat diet modified to contain either 0.1% (w/w) milk SM (n=14) or 0.1% (w/w) egg SM (n=14). After 10 weeks, egg SM ameliorated weight gain, hypercholesterolemia and hyperglycemia induced by HFD. Both egg SM and milk SM attenuated hepatic steatosis development, with significantly lower hepatic triglycerides (TGs) and cholesterol relative to HFD. This reduction in hepatic steatosis was stronger with egg SM supplementation relative to milk SM. Reductions in hepatic TGs observed with dietary SM were associated with lower hepatic mRNA expression of PPARγ-related genes: Scd1 and Pparg2 in both SM groups, and Cd36 and Fabp4 with egg SM. Egg SM and, to a lesser extent, milk SM reduced inflammation and markers of macrophage infiltration in adipose tissue. Egg SM also reduced skeletal muscle TG content compared to HFD. Overall, the current study provides evidence of dietary SM improving metabolic complications associated with diet-induced obesity in mice. Further research is warranted to understand the differences in bioactivity observed between egg and milk SM.

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.

Improvement of skin condition by oral supplementation with sphingomyelin-containing milk phospholipids in a double-blind, placebo-controlled, randomized trial.

Sphingomyelin (SM), an essential phospholipid for the skin, is contained largely in the milk fat globule membrane surrounding milk fat, concentrated fractions of which are also generated concurrently during the manufacture of dairy products. Such an SM-containing milk phospholipid concentrate (SM-MPC) is useful for investigating the benefits of dietary SM. Here, we examined the effect of consuming SM-MPC on the condition of skin in a double-blind, placebo-controlled, randomized trial. Ninety-six healthy subjects aged 20 to 39 yr with low skin hydration were randomly assigned to 3 groups: a high-SM group supplemented with SM-MPC at a dose equivalent to 10 mg/d of SM, a low-SM group supplemented with SM-MPC equivalent to 5 mg/d of SM, and a placebo group fed a vehicle composed of olive oil and beeswax. During daily supplementation for 12 wk, parameters related to the condition of skin were evaluated at baseline and every 3 wk. Skin hydration at the heel was significantly increased at wk 9 and 12 in the low-SM group compared with the placebo group. Skin elasticity in the region below the eye was significantly increased at wk 9 in the high-SM group versus placebo. Questionnaire-based subjective perceptions of skin conditions were significantly improved for facial skin moisture at wk 3 and 12, and in the wrinkle around the eyes at wk 9 and 12 in the high-SM group versus placebo. Our results indicate that constant and long-term supplementation with SM-MPC is capable of improving the general condition of skin.

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

Effect of dietary sphingomyelin on absorption and fractional synthetic rate of cholesterol and serum lipid profile in humans.

BACKGROUND: Diets enriched with sphingolipids may improve blood lipid profiles. Studies in animals have shown reductions in cholesterol absorption and alterations in blood lipids after treatment with sphingomyelin (SM). However, minimal information exists on effect of SM on cholesterol absorption and metabolism in humans. The objective was to assess the effect of SM consumption on serum lipid concentrations and cholesterol metabolism in healthy humans. METHODS: Ten healthy adult males and females completed a randomized crossover study. Subjects consumed controlled diets with or without 1 g/day SM for 14 days separated by at least 4 week washout period. Serum lipid profile and markers of cholesterol metabolism including cholesterol absorption and synthesis were analyzed. RESULTS: Serum triglycerides, total, LDL- and VLDL- cholesterol were not affected while HDL cholesterol concentrations were increased (p = 0.043) by SM diet consumption. No change in cholesterol absorption and cholesterol fractional synthesis rate was observed with supplementation of SM compared to control. Intraluminal cholesterol solubilization was also not affected by consumption of SM enriched diet. CONCLUSIONS: In humans, 1 g/day of dietary SM does not alter the blood lipid profile except for an increased HDL-cholesterol concentration and has no effect on cholesterol absorption, synthesis and intraluminal solubilization compared to control. TRIAL REGISTRATION: Clinicaltrials.gov # NCT00328211.

Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice.

Controlling intestinal lipid absorption is an important strategy for maintaining lipid homeostasis. Accumulation of lipids in the liver is a major risk factor for metabolic syndrome and nonalcoholic fatty liver disease. It is well-known that sphingomyelin (SM) can inhibit intestinal cholesterol absorption. It is, however, unclear if dietary SM also lowers liver lipid levels. In the present study (i) the effect of pure dietary egg SM on hepatic lipid metabolism and intestinal cholesterol absorption was measured with [(14)C]cholesterol and [(3)H]sitostanol in male C57BL/6 mice fed a high-fat (HF) diet with or without 0.6% wt/wt SM for 18 days; and (ii) hepatic lipid levels and gene expression were determined in mice given a HF diet with or without egg SM (0.3, 0.6 or 1.2% wt/wt) for 4 weeks. Mice supplemented with SM (0.6% wt/wt) had significantly increased fecal lipid and cholesterol output and reduced hepatic [(14)C]cholesterol levels after 18 days. Relative to HF-fed mice, SM-supplemented HF-fed mice had significantly lower intestinal cholesterol absorption (-30%). Liver weight was significantly lower in the 1.2% wt/wt SM-supplemented mice (-18%). Total liver lipid (mg/organ) was significantly reduced in the SM-supplemented mice (-33% and -40% in 0.6% wt/wt and 1.2% wt/wt SM, respectively), as were triglyceride and cholesterol levels. The reduction in liver triglycerides was due to inactivation of the LXR-SREBP-1c pathway. In conclusion, dietary egg SM has pronounced hepatic lipid-lowering properties in mice maintained on an obesogenic diet.

[Effects of some membrane lipids on the hemolysis induced by hemolytic toxin from Karenia mikimotoi].

OBJECTIVE: To explore the effects of some membrane lipids on the hemolysis induced by hemolytic toxin from Karenia mikimotoi. METHODS: Effects of exogenous membrane lipids such as lecithin, sphingomyelin, L-alpha-phosphatidic acid,cholesterol and gangliosides on the hemolysis induced by the hemolytic toxin were observed. The sensitivities of some erythrocytes from different animals such as rabbit, rat and fish to the hemolytic toxin were evaluated. The total gangliosides in different erythrocytes membrane were detected by colorimetry. RESULTS: Only gangliosides significantly inhibited the hemolysis of the hemolytic toxin from K. mikimotoi (P <0.05). Hemolytic percentages decreased to 16.05% after 10 min addition of ganglioside, while those of control were 35.65%. The rabbit red blood cell was the most sensitive to the hemolytic toxin. The hemolytic percentages of rabbit erythrocyte were higher than those of rat (P < 0.05) and fish (P < 0.01). The amounts of lipid-bind sialic acid (LBSA) on frozen dried membrane of rabbit were 672.08 microg/g,and were higher than those of rat (585.97 microg/g) (P < 0.05) and that of fish (431.52 microg/g) (P < 0.01). CONCLUSION: Exogenous gangliosides could have a potent inhibition on the hemolysis induced by hemolytic toxin from K. mikimotoi. There was a significant correlation between the sensitivities of different erythrocytes to the hemolytic toxin and the amount of ganglioside on different erythrocytes membrane.

Phospholipids and a phospholipid-rich diet alter the in vitro amyloid-beta peptide levels and amyloid-beta 42/40 ratios.

Amyloid-beta peptides (Abeta) generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases play an important role in the pathogenesis of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of Abeta peptides into senile plaques, one of the hallmarks of AD. With the aim to clarify the molecular basis of the interaction between Abeta and cellular membranes, we investigated the effects of various phospholipids (PLs) and a PL-rich diet on Abeta production. Here we show that modulation of Abeta production and Abeta42:40 ratio is not limited to individual fatty acids, rather it is the composition of the PLs of the membrane bilayer, that influences the specificity and level of the regulated intramembranous proteolysis of APP by the gamma-secretase complex. We show that Abeta levels in the conditioned media, in response to some of the PL supplements, is increased in the center and decreased on either side of a graph that resembles bell-shaped distribution. This means that the PLs have less of a tendency to produce unusually extreme effects on Abeta production in SP-C99 transfected Cos-7 cultured cells. We proposed a mechanism-based hypothesis to rationalize PLs' effects on Abeta production.

Egg sphingomyelin lowers the lymphatic absorption of cholesterol and alpha-tocopherol in rats.

Evidence indicates that phosphatidylcholine (PC) inhibits the intestinal absorption of cholesterol (CH) in rats. This study was designed to determine whether sphingomyelin (SM), structurally similar to PC, also inhibits the lymphatic absorption of CH. Sprague-Dawley rats with lymph cannulae were infused at 3.0 mL/h for 8 h via a duodenal catheter with a lipid emulsion [33.3 kBq 14C-CH, 20.7 micromol CH, 451.7 micromol triolein, 3.1 micromol alpha-tocopherol (alphaTP), 75.4 nmol retinol and 396.0 micromol sodium taurocholate in 24 mL of PBS (pH, 6.5)], without egg SM (SM0) as control, or with 5.0 micromol/h (SM5) or 10.0 micromol/h (SM10). Egg SM lowered the lymphatic absorption of 14C-CH in a dose dependent manner. Likewise, SM lowered the lymphatic absorptions of alphaTP and fatty acid (oleic acid), whereas it had no effect on retinol absorption. SM at a high dose (SM10) lowered the lymphatic outputs of both PC and SM, whereas there was no such effect at a lower dose (SM5). These results indicate that luminal egg SM has an inhibitory effect on the intestinal absorption of CH and other lipids of relatively high hydrophobicity. Our findings suggest that SM, if ingested in sufficient amounts, may inhibit the intestinal absorption of dietary lipids including cholesterol and alphaTP.

Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner.

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol in the target membrane. Recently, we have demonstrated that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the present paper, we show that the activation of low-pH-dependent fusion of SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo-, and L-threo-ceramide did not support fusion of the virus. Similar results were obtained with the corresponding sphingomyelin stereoisomers. The stereospecificity of SFV fusion activation was confirmed by using an assay based on degradation of the viral capsid protein by trypsin encapsulated in the target liposomes. Fusion mediated by D-erythro-ceramide was not affected by the additional presence in the target liposomes of ceramide stereoisomers incapable of fusion activation. Binding of the virus to the liposomes, as assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes. The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the target membrane represents an essential step in the activation of the fusion capacity of SFV.

Externalization of aminophospholipids in platelet liposome bilayers by supplementation with sphingomyelin.

Negatively charged phospholipids are mainly located on the inner leaflet of platelet liposomes. Using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, we have shown that in large artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of exogenous sphingomyelin, an alteration in the amino-phospholipid topology occurs, with a progressive appearance of these lipid species -in particular phosphatidylserine- in the outer membrane bilayer.

Liposomes in immunology: multilamellar phosphatidylcholine liposomes as a simple, biodegradable and harmless adjuvant without any immunogenic activity of its own.

Multilamellar phosphatidylcholine liposomes were coated with the antigens human serum albumin (HSA) or bovine gamma globulin (BGG) simply by suspending the liposomes in a solution of the antigens. Antigen coated phosphatidylcholine liposomes showed nearly the same adjuvant activity after intravenous injection as liposomes of a more complex phospholipid composition. Since phosphatidylcholine liposomes are biodegradable, harmless, easily obtainable, have no immunogenic activity of their own and may be administered intravenously, they seem to be a promising immunoadjuvant.

The removal of cholesterol from aortic smooth muscle cells in culture and Landschutz ascites cells by fractions of human high-density apolipoprotein.

Ascites cells were labeled by intraperitoneal injection of [3H]cholesterol and aortic smooth muscle cells by addition of [3H]cholesterol to the serum component of the culture medium. The release of cholesterol from cells into a serum-free medium supplemented with the various "acceptors" was studied using ascites cells in suspension and aortic smooth muscle cells in a multilayer culture. Unfractionated human high-density apolipoprotein was somewhat more effective in the removal of labeled cellular free cholesterol, in both cell types, than apolipoprotein derived from rat high-density lipoprotein. Following separation of human high-density apolipoprotein into four fractions by Sephadex chromatography, the effect of each fraction on the removal of cellular cholesterol from ascites cells was studied. The individual fractions had a lower capacity for cholesterol removal than the original unfractionated high-density apolipoprotein and the lowest activity was detected in Fraction II which comprised 75% of the total apolipoprotein. The effectiveness to remove cholesterol could be restored to all the fractions, as well as enhanced, by addition of sonicated suspensions of lecithin or sphingomyelin, which by themselves promoted a more limited removal of cellular cholesterol. Negatively stained preparations of mixtures of the four fractions and sonicated dispersion of lecithin were shown to consist of vesicles and discs of various sizes. Addition of the apolipoprotein fractions (especially Fractions II and IV) to sonicated dispersion of sphingomyelin resulted in a pronounced formation of discs which showed a high tendency towards stack formation. Mixtures of Fraction II and lecithin or sphingomyelin were effective in the release of cellular cholesterol from multilayers of aortic smooth muscle cells in culture. These results show the feasibility of net removal of cholesterol from cells which grow in a form resembling a tissue and thus provide a model to study the role of apolipoprotein-phospholipid mixtures in cholesterol removal from cells and tissues in vivo.