A blend containing docosahexaenoic acid, arachidonic acid, vitamin B12, vitamin B9, iron and sphingomyelin promotes myelination in an in vitro model.

During the development of the central nervous system, oligodendrocytes (OLs) are responsible for myelination, the formation of the myelin sheath around axons. This process enhances neuronal connectivity and supports the maturation of emerging cognitive functions. In humans, recent evidence suggests that early life nutrition may affect myelination. In the present study, we investigated the impact of a blend containing docosahexaenoic acid, arachidonic acid, vitamin B12, vitamin B9, iron and sphingomyelin, or each of these nutrients individually, on oligodendrocyte precursor cells (OPCs) proliferation and maturation into OLs as well as their myelinating properties. By using an in vitro model, developed to study each step of myelination, we found that the nutrient blend increased the number of OPCs and promoted their differentiation and maturation into OLs, as measured by quantifying A2B5 positive cells, myelin-associated glycoprotein (MAG) positive cells and area, myelin binding protein (MBP) positive cells and area, respectively. Moreover, measuring myelination by quantifying the overlapping signal between neurofilament and either MAG or MBP revealed a positive effect of the blend on OLs myelinating properties. In contrast, treatment with each individual nutrient resulted in differential effects on the various readouts. This work suggests that dietary intake of these nutrients during early life, might be beneficial for myelination.

The beneficial role of functional food components in mitigating ultraviolet-induced skin damage.

Excessive exposure to ultraviolet (UV) radiation can chemically alter biological molecules and is one of the major environmental health risks with potential to damage the structure and function of the skin. Numerous dietary supplements are known to optimize the skin's defenses against radiation exposure. Several studies in which the beneficial roles of functional food components, that can protect against UV-induced skin damage, have been demonstrated. Supplemental dietary sphingomyelin maintains covalently bound ω-hydroxy ceramides to avert skin barrier defects after UVB irradiation. The oral administration of collagen hydrolysates has been shown to limit decreases in skin elasticity via increases in the dermal hyaluronic acid content. Milk fermented with lactic acid bacteria has been shown to augment DNA repair mechanisms and improve skin immunity in the aftermath of UVB damage. Furthermore, long-term ingestion of fermented milk containing lactic acid bacteria, collagen hydrolysates and sphingomyelin increases the minimal erythema dose (MED) in human subjects with moderate sunburn or redness and tanned skin after exposure to UV solar radiation. Thus, products containing these functional food components are one means by which the adverse effects of UV radiation on the skin can be mitigated.

Intravesical liposome therapy for interstitial cystitis.

Over the past two decades, there has been lot of interest in the use of liposomes as lipid-based biocompatible carriers for drugs administered by the intravesical route. The lipidic bilayer structure of liposomes facilitates their adherence to the apical membrane surface of luminal cells in the bladder, and their vesicular shape allows them to co-opt the endocytosis machinery for bladder uptake after instillation. Liposomes have been shown to enhance the penetration of both water-soluble and insoluble drugs, toxins, and oligonucleotides across the bladder epithelium. Empty liposomes composed entirely of the endogenous phospholipid, sphingomyelin, could counter mucosal inflammation and promote wound healing in patients suffering from interstitial cystitis. Recent clinical studies have tested multilamellar liposomes composed entirely of sphingomyelin as a novel intravesical therapy for interstitial cystitis. In addition, liposomes have been used as a delivery platform for the instillation of botulinum toxin in overactive bladder patients. The present review discusses the properties of liposomes that are important for their intrinsic therapeutic effect, summarizes the recently completed clinical studies with intravesical liposomes and covers the latest developments in this field.

Ameliorating Effects of Sphingomyelin-Based Liposomes on Sarcopenia in Senescence-Accelerated Mice.

The effects of orally administered sphingomyelin-based liposomes (SM-lipo) on muscle function were investigated in senescence-accelerated mice prone 1 (SAMP1) for the purpose of protection against or treatment of sarcopenia. SM-lipo were prepared by thin lipid-film hydration followed by extrusion. Their spherical shape was observed by transmission electron microscopy. The obtained liposomes were stable in gastric liquid and intestinal fluid models as well as in water. In in vitro tests liposomalization of sphingomyelin significantly increased its transport into human intestinal epithelial Caco-2 cells. In addition, SM-lipo upregulated the proliferation of murine C2C12 myoblasts compared with free sphingomyelin or phosphatidylcholine-based liposomes (PC-lipo). Finally, SM-lipo orally administered to SAMP1 for 10 weeks significantly increased quadriceps femoris weight and extended swimming time until fatigue compared with PC-lipo. In conclusion, these findings indicate that SM-lipo are well absorbed into the body and improve muscle weakness caused by senescence.

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.

Effects of Milk Fat Globule Membrane Supplementation Following Exercise Training on Physical Performance in Healthy Young Adults: A Randomized Double-Blind, Placebo-Controlled Pilot Trial.

The purpose of this study was to examine whether 4 wk of daily ingestion of milk fat globule membrane (MFGM) combined with exercise training improves physical performance-muscle strength, agility and muscle power-in healthy young adults. The study was designed as a randomized, double-blind, and placebo-controlled trial. Twenty healthy young adults received either an MFGM powder containing 1.6 g of fat and 160 mg of sphingomyelin or an isocaloric placebo powder daily throughout 4 wk of power or agility training. Physical performance tests and body composition measurements were conducted before and after the 4-wk intervention. Ingestion of MFGM did not affect isometric or isokinetic muscle strength, but it was associated with a greater increase in vertical jump peak power compared with placebo. There were no significant changes in body weight or lean body mass during the intervention period in either group, and no significant differences between groups. We conclude that daily MFGM supplementation combined with exercise training has the potential to improve physical performance in young adults; however, further studies with larger sample sizes should be conducted to obtain more evidence supporting achievement of improved physical performance through MFGM supplementation.

Increase in ceramide level after application of various sizes of sphingomyelin liposomes to a cultured human skin model.

Sphingomyelin-based liposomes (SPM-L) that were sized (or not) by extrusion through a filter with pores of 100, 200, or 400 nm were applied to a three-dimensional cultured human skin model in order to evaluate which size of SPM-L was most effective at increasing its ceramide level. The diameters of the SPM-L in PBS were 102.7, 181.0, 224.0, and 380.1 nm. The diameters of the liposomes in the culture medium were 117.5, 199.2, 242.1, and 749.8 nm. The diameter of the small liposomes (<200 nm in diameter) did not change much, at least for 7 days. SPM-L in saline or culture medium were applied to the basal layer side or stratum corneum side of the cultured skin model, and ceramide II, III, V, and VI were then extracted from it. The extracted ceramide molecules were separated by HPTLC, and the concentration of each type of ceramide was quantified using a densitometer. When the small SPM-L (110 or 190 nm in diameter) were applied to the basal layer side, the levels of ceramide III and V were increased. When they were applied to the stratum corneum side, the levels of ceramide II, III, V, and VI were significantly increased compared to those of the PBS group, especially after the application of the small SPM-L (110 nm in diameter). Thus, the application of small SPM-L was useful for increasing the ceramide II, III, V, and VI levels of a cultured human skin model.

A soft anti-virulence liposome realizing the explosive release of antibiotics at an infectious site to improve antimicrobial therapy.

Pore-forming toxins (PFTs), the most common virulence proteins, are promising therapeutic keys in bacterial infections. CAL02, consisting of sphingomyelin (Sm) and containing a maximum ratio of cholesterol (Ch), has been applied to sequester PFTs. However, Sm, a saturated phospholipid, leads to structural rigidity of the liposome, which does not benefit PFT combination. Therefore, in order to decrease the membrane rigidity and improve the fluidity of liposomes, we have introduced an unsaturated phospholipid, phosphatidylcholine (Pc), to the saturated Sm. In this report, a soft nanoliposome (called CSPL), composed of Ch, Sm and Pc, was artificially prepared. In order to further improve its antibacterial effect, vancomycin (Van) was loaded into the hydrophilic core of CSPL, where Van can be released radically at the infectious site through transmembrane pores formed by the PFTs in CSPL. This soft Van@CSPL nanoliposome with detoxification/drug release was able to inhibit the possibility of antibiotic resistance and could play a better role in treating severe invasive infections in mice.

Cow's milk polar lipids reduce atherogenic lipoprotein cholesterol, modulate gut microbiota and attenuate atherosclerosis development in LDL-receptor knockout mice fed a Western-type diet.

Milk sphingomyelin (SM), a polar lipid (PL) component of milk fat globule membranes, is protective against dyslipidemia. However, it is unclear whether ingestion of milk PLs protect against atherosclerosis. To determine this, male LDLr-/- mice (age 6 weeks) were fed ad libitum either a high-fat, added-cholesterol diet (CTL; 45% kcal from fat, 0.2% cholesterol by weight; n=15) or the same diet supplemented with 1% milk PL (1% MPL; n=15) or 2% milk PL (2% MPL; n=15) added by weight from butter serum. After 14 weeks on diets, mice fed 2% MPL had significantly lower serum cholesterol (-51%) compared to CTL (P<.01), with dose-dependent effects in lowering VLDL- and LDL-cholesterol. Mice fed 2% MPL displayed lower inflammatory markers in the serum, liver, adipose and aorta. Notably, milk PLs reduced atherosclerosis development in both the thoracic aorta and the aortic root, with 2% MPL-fed mice having significantly lower neutral lipid plaque size by 59% (P<.01) and 71% (P<.02) compared to CTL, respectively. Additionally, the 2% MPL-fed mice had greater relative abundance of Bacteroidetes, Actinobacteria and Bifidobacterium, and lower Firmicutes in cecal feces compared to CTL. Milk PL feeding resulted in significantly different microbial communities as demonstrated by altered beta diversity indices. In summary, 2% MPL strongly reduced atherogenic lipoprotein cholesterol, modulated gut microbiota, lowered inflammation and attenuated atherosclerosis development. Thus, milk PL content may be important to consider when choosing dairy products as foods for cardiovascular disease prevention.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Acute effects of milk polar lipids on intestinal tight junction expression: towards an impact of sphingomyelin through the regulation of IL-8 secretion?

Milk polar lipids (MPL) are specifically rich in milk sphingomyelin (MSM) which represents 24% of MPL. Beneficial effects of MPL or MSM have been reported on lipid metabolism, but information on gut physiology is scarce. Here we assessed whether MPL and MSM can impact tight junction expression. Human epithelial intestinal Caco-2/TC7 cells were incubated with mixed lipid micelles devoid of MSM (Control) or with 0.2 or 0.4 mM of MSM via pure MSM or via total MPL. C57Bl/6 mice received 5 or 10 mg of MSM via MSM or via MPL (oral gavage); small intestinal segments were collected after 4 h. Impacts on tight junction and cytokine expressions were assessed by qPCR; IL-8 and IL-8 murine homologs (Cxcl1, Cxcl2) were analyzed. In vitro, MSM increased tight junction expression (Occludin, ZO-1) vs Control, unlike MPL. However, no differences were observed in permeability assays (FITC-dextran, Lucifer yellow). MSM increased the secretion and gene expression of IL-8 but not of other inflammatory cytokines. Moreover, cell incubation with IL-8 induced an overexpression of tight junction proteins. In mice, mRNA level of Cxcl1 and Cxcl2 in the ileum were increased after gavage with MSM vs NaCl but not with MPL. Altogether, these results suggest a specific action of MSM on intestinal tight junction expression, possibly mediated by IL-8. Our study provides clues to shed light on the beneficial effects of MPL on intestinal functions and supports the need for further mechanistic exploration of the direct vs indirect effects of MSM and IL-8 on the gut barrier.

Characterization of apolipoprotein A-I peptide phospholipid interaction and its effect on HDL nanodisc assembly.

Background: Synthetic HDLs (sHDLs), small nanodiscs of apolipoprotein mimetic peptides surrounding lipid bilayers, were developed clinically for atheroma regression in cardiovascular patients. Formation of HDL involves interaction of apolipoprotein A-I (ApoA-I) with phospholipid bilayers and assembly into lipid-protein nanodiscs. Purpose: The objective of this study is to improve understanding of physico-chemical aspects of HDL biogenesis such as the thermodynamics of ApoA-I-peptide membrane insertion, lipid binding, and HDL self-assembly to improve our ability to form homogeneous sHDL nanodiscs that are suitable for clinical administration. Methods: The ApoA-I-mimetic peptide, 22A, was combined with either egg sphingomyelin (eSM) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid vesicles to form sHDL. The sHDL assembly process was investigated through lipid vehicle solubilization assays and characterization of purity, size, and morphology of resulting nanoparticles via gel permeation chromatography (GPC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Peptide-lipid interactions involved were further probed by sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). The pharmacokinetics of eSM-sHDL and POPC-sHDL nanodiscs were investigated in Sprague Dawley rats. Results: sHDL formation was temperature-dependent, with spontaneous formation of sHDL nanoparticles occurring only at temperatures exceeding lipid transition temperatures as evidenced by DLS, GPC, and TEM characterization. SFG and ATR-FTIR spectroscopy findings support a change in peptide-lipid bilayer interactions at temperatures above the lipid transition temperature. Lipid-22A interactions were stronger with eSM than with POPC, which resulted in the formation of more homogeneous sHDL nanoparticles with longer in vivo circulation time as evidenced the PK study. Conclusion: Physico-chemical characteristics of sHDL are in part determined by phospholipid composition. Optimization of phospholipid composition may be utilized to improve the stability and homogeneity of sHDL.

Protective properties of milk sphingomyelin against dysfunctional lipid metabolism, gut dysbiosis, and inflammation.

The identification of natural bioactive compounds aimed at promoting optimal gut health and improving lipid metabolism is paramount in the prevention of chronic disease. In this review, we summarize basic science and clinical research examining the protective properties of milk sphingomyelin (SM) against dysfunctional lipid metabolism, gut dysbiosis, and inflammation. Dietary SM dose-dependently reduces the intestinal absorption of cholesterol, triglycerides, and fatty acids in cell culture and rodent studies. Overall, rodent feeding studies show dietary milk SM, milk polar lipid mixtures, and milk fat globule membrane reduce serum and hepatic lipid concentrations. Furthermore, these hypolipidemic effects are observed in some supplementation studies in humans, although the extent of reductions in serum cholesterol is typically smaller and only one trial was conducted with purified SM. Dietary milk SM has been reported to affect the gut microbiota in rodent studies and its hydrolytic product, sphingosine, displays bactericidal activity in vitro. Milk SM may also improve gut barrier function to prevent the translocation of inflammatory gut bacteria-derived molecules. Current evidence from pre-clinical studies indicates that dietary milk SM has protective properties against dysfunctional lipid metabolism, gut dysbiosis, and inflammation. The hypolipidemic effects of milk SM observed in animal studies have been reported in some human studies, although the magnitude of such effects is typically smaller. More research is warranted to clearly define how dietary milk SM influences lipid metabolism, gut microbiota, and inflammation in humans.

Dietary sphingomyelin inhibits colonic tumorigenesis with an up-regulation of alkaline sphingomyelinase expression in ICR mice.

BACKGROUND: Sphingomyelin (SM) hydrolysis generates biologically active products regulating cell growth, differentiation and apoptosis. Dietary SM has been found to inhibit colonic tumorigenesis. Alkaline sphingomyelinase (alk-SMase) is the key enzyme responsible for sphingomyelin digestion in the gut. Whether or not dietary sphingomyelin affects alk-SMase expression was examined in a colon cancer animal model. MATERIALS AND METHODS: Imprinting control region (ICR) mice were injected with 1,2-dimethylhydrazine (DMH) and then fed a diet with or without SM (0.5 g/kg in diet) for 22 weeks. The colonic tumorigenesis and alk-SMase activity were determined and alk-SMase expression was examined by Western blot and PCR. RESULTS: Dietary SM inhibited the tumorigenesis and increased the alk-SMase activity in the colon by 65%. The increased activity was associated with increased enzyme protein and mRNA expression. No changes of acid and neutral sphingomyelinase activities were found. CONCLUSION: Long-term supplementation with dietary sphingomyelin up-regulates colonic alk-SMase expression, which may contribute to the inhibitory effects of sphingomyelin against colonic carcinogenesis.

Dietary sphingomyelin alleviates experimental inflammatory bowel disease in mice.

The effect of dietary sphingomyelin (SM) on inflammatory bowel disease (IBD) induced with dextran sodium sulfate (DSS) was examined in mice. Although the severity of IBD as expressed by the disease activity index (DAI) markedly increased with DSS administration, feeding a diet containing SM lowered the DAI value significantly. Myeloperoxidase (MPO) activity in colonic tissue also increased with DSS administration, suggesting the development of inflammation. Because simultaneous administration of SM with DSS prevented the MPO activity increase, we concluded that SM could suppress the development of inflammation. These results provide novel evidence that dietary supplementation with SM can alleviate the symptoms of IBD in mice. Dietary SM also increased the amount of IgA in the large intestine, suggesting that SM promotes IgA secretion into the large intestine. These results suggest that the mechanism of IBD mitigation by SM is complex and involves the immune system.

Liposomal Therapy Attenuates Dermonecrosis Induced by Community-Associated Methicillin-Resistant Staphylococcus aureus by Targeting α-Type Phenol-Soluble Modulins and α-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases.

Effect of long-term dietary sphingomyelin supplementation on atherosclerosis in mice.

Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE-/- mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE-/- mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE-/- mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE-/- mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE-/- mice, but it is anti-atherogenic in chow-fed apoE-/- mice.

Milk Fermented by Lactic Acid Bacteria Enhances the Absorption of Dietary Sphingomyelin in Rats.

Supplementation with sphingomyelin has been reported to prevent disease and maintain good health. However, intact sphingomyelin and ceramides are poorly absorbed compared with glycerolipids. Therefore, if the bioavailability of dietary sphingomyelin can be increased, supplementation would be more effective at lower doses. The aim of this study in rats was to evaluate the effect of fermented milk on the bioavailability of dietary sphingomyelin in rats. After the rats had fasted for 15 h, test solutions were administrated orally. Blood samples were collected from the tail vein before and 90, 180, 270, and 360 min after administration. Compared with sphingomyelin/milk phospholipids concentrate (MPL) alone, co-ingestion of sphingomyelin/MPL with fermented milk caused an approximate twofold significant increase in serum ceramides containing d16:1 sphingosine with 16:0, 22:0, 23:0 and 24:0 fatty acids, which was derived from the ingested sphingomyelin. While nonfat milk also increased the serum levels of these ceramides, fermented milk was more effective. Co-ingestion of the upper layer of fermented milk or exopolysaccharide concentrate prepared from fermented milk significantly increased serum ceramide levels. X-ray diffraction analysis also showed addition of fermented milk or EPS concentrate to sphingomyelin eliminated the characteristic peak of sphingomyelin. This study demonstrated for the first time that co-ingestion of dietary sphingomyelin and fermented milk, compared with ingestion of dietary sphingomyelin alone, caused a significant increase in the absorption of sphingomyelin. Our results indicate exopolysaccharides in fermented milk may contribute to inhibition of sphingomyelin crystallization, resulting in enhanced absorption of dietary sphingomyelin in rats.

Dietary sphingolipids suppress a subset of preneoplastic rat liver lesions exhibiting high PTEN, low phospho-Akt and high levels of ceramide species.

Rat liver glutathione-S-transferase Pi-(GST-P)-positive enzyme-altered foci (EAF) are preneoplastic lesions that develop in response to carcinogenic stress. They are often used as endpoints in e.g. chemopreventive studies. In this study we characterize a pAkt-negative/ceramide-positive (pAkt-/cer+) EAF phenotype, as defined by immunohistochemistry for pAkt and ceramide species, in diethylnitrosamine(DEN)-, phenobarbital- or aflatoxinB1-treated rats. There was a close to 100% overlap for the pAkt and the ceramide marker. Furthermore, serial sections stained for PTEN indicated a close correlation between PTEN-positive and pAkt-negative lesions in DEN-treated rats. Experiments with DEN-treated rats given sphingomyelin in the diet suggested that sphingomyelin selectively targeted these lesions. In in vitro experiments sphingosine rapidly decreased pAkt levels in hepatocytes, and in experiments with hepatocytes from DEN-treated rats sphingosine selectively killed EAF cells. Furthermore, pretreatment with antisense Akt oligonucleotides in vitro sensitized non-EAF hepatocytes, so that EAF and non-EAF cells became equally sensitive to sphingosine. It is concluded that rat liver, in response to carcinogenic stress, develops a distinct EAF phenotype exhibiting low pAkt levels and concomitant alterations in sphingolipid metabolism. Our data also suggest that pAkt-/cer+ EAF are selectively targeted by sphingolipids in the diet and that lesions with this phenotype should be of particular interest for future studies on chemopreventive effects that may affect sphingolipid metabolism.

Sphingomyelin consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: implications for dietary sphingolipids and colon carcinogenesis.

Sphingolipids are hydrolyzed in the gastrointestinal tract to ceramide, sphingosine, and other metabolites that can modulate cell growth, differentiation, and apoptosis. To characterize the effects of dietary sphingolipids on colon carcinogenesis, female CF1 mice were administered 1,2-dimethylhydrazine and then fed an essentially sphingolipid-free diet supplemented with 0 to 0.1% (w/w) sphingomyelin (SM) purified from milk. As was found in a previous pilot study (D. L. Dillehay et al., J. Nutr., 124: 615-620, 1994), SM (@ 0.1%) reduced the number of aberrant colonic crypt foci (by 70%, P < 0.001) and aberrant crypts per focus (by 30%, P < 0.003), which are early indicators of colon carcinogenesis. In longer term studies, SM had no effect on colon tumor incidence or multiplicity; however, up to 31% of the tumors of mice fed SM were adenomas, whereas all of the tumors of mice fed the diet without SM were adenocarcinomas. These findings demonstrate that milk SM suppresses the appearance of more advanced, malignant tumors as well as early markers of colon carcinogenesis. Although the sphingolipid content of foods has not been widely studied, several foods (e.g., milk and soybeans) contain the sphingolipid levels used in these investigations; therefore, this class of compounds could be significant contributors to the cancer preventive effects of some foods.