Alkaline sphingomyelinase (NPP7) impacts the homeostasis of intestinal T lymphocyte populations.

Background and aim: Alkaline sphingomyelinase (NPP7) is expressed by intestinal epithelial cells and is crucial for the digestion of dietary sphingomyelin. NPP7 also inactivates proinflammatory mediators including platelet-activating factor and lysophosphatidylcholine. The aim of this study was to examine a potential role for NPP7 in the homeostasis of the intestinal immune system. Methods: We quantified the numbers of B-lymphocytes, plasma cells, T-lymphocytes including regulatory T-lymphocytes (Tregs), natural killer cells, dendritic cells, macrophages, and neutrophils, in the small and large intestines, the mesenteric lymph nodes and the spleens of heterozygous and homozygous NPP7 knockout (KO) and wildtype (WT) mice. Tissues were examined by immunohistochemistry and stainings quantified using computerized image analysis. Results: The numbers of both small and large intestinal CD3ε+, CD4+, and CD8α+ T-lymphocytes were significantly higher in NPP7 KO compared to WT mice (with a dose-response relationship in the large intestine), whereas Treg numbers were unchanged, and dendritic cell numbers reduced. In contrast, the numbers of CD3ε+ and CD4+ T-lymphocytes in mesenteric lymph nodes were significantly reduced in NPP7 KO mice, while no differences were observed in spleens. The numbers of B-lymphocytes, plasma cells, natural killer cells, macrophages, and neutrophils were similar between genotypes. Conclusion: NPP7 contributes to the regulation of dendritic cell and T-lymphocyte numbers in mesenteric lymph nodes and both the small and large intestines, thus playing a role in the homeostasis of gut immunity. Although it is likely that the downstream effects of NPP7 activity involve the sphingomyelin metabolites ceramide and spingosine-1-phosphate, the exact mechanisms behind this regulatory function of NPP7 need to be addressed in future studies.

Digestion and Absorption of Milk Phospholipids in Newborns and Adults.

Milk polar lipids provide choline, ethanolamine, and polyunsaturated fatty acids, which are needed for the growth and plasticity of the tissues in a suckling child. They may also inhibit cholesterol absorption by interacting with cholesterol during micelle formation. They may also have beneficial luminal, mucosal, and metabolic effects in both the neonate and the adult. The milk fat globule membrane contains large proportions of sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and some phosphatidylserine (PS), phosphatidylinositol (PI), and glycosphingolipids. Large-scale technical procedures are available for the enrichment of milk fat globule membrane (MFGM) in milk replacement formulations and food additives. Pancreatic phospholipase A2 (PLA2) and mucosal phospholipase B digest glycero-phospholipids in the adult. In the neonate, where these enzymes may be poorly expressed, pancreatic lipase-related protein 2 probably has a more important role. Mucosal alkaline SM-ase and ceramidase catalyze the digestion of SM in both the neonate and the adult. In the mucosa, the sphingosine is converted into sphingosine-1-phosphate, which is both an intermediate in the conversion to palmitic acid and a signaling molecule. This reaction sequence also generates ethanolamine. Here, we summarize the pathways by which digestion and absorption may be linked to the biological effects of milk polar lipids. In addition to the inhibition of cholesterol absorption and the generation of lipid signals in the gut, the utilization of absorbed choline and ethanolamine for mucosal and hepatic phospholipid synthesis and the acylation of absorbed lyso-PC with polyunsaturated fatty acids to chylomicron and mucosal phospholipids are important.

Pancreatic and mucosal enzymes in choline phospholipid digestion.

The digestion of choline phospholipids is important for choline homeostasis, lipid signaling, postprandial lipid and energy metabolism, and interaction with intestinal bacteria. The digestion is mediated by the combined action of pancreatic and mucosal enzymes. In the proximal small intestine, hydrolysis of phosphatidylcholine (PC) to 1-lyso-PC and free fatty acid (FFA) by the pancreatic phospholipase A2 IB coincides with the digestion of the dietary triacylglycerols by lipases, but part of the PC digestion is extended and must be mediated by other enzymes as the jejunoileal brush-border phospholipase B/lipase and mucosal secreted phospholipase A2 X. Absorbed 1-lyso-PC is partitioned in the mucosal cells between degradation and reacylation into chyle PC. Reutilization of choline for hepatic bile PC synthesis, and the reacylation of 1-lyso-PC into chylomicron PC by the lyso-PC-acyl-CoA-acyltransferase 3 are important features of choline recycling and postprandial lipid metabolism. The role of mucosal enzymes is emphasized by sphingomyelin (SM) being sequentially hydrolyzed by brush-border alkaline sphingomyelinase (alk-SMase) and neutral ceramidase to sphingosine and FFA, which are well absorbed. Ceramide and sphingosine-1-phosphate are generated and are both metabolic intermediates and important lipid messengers. Alk-SMase has anti-inflammatory effects that counteract gut inflammation and tumorigenesis. These may be mediated by multiple mechanisms including generation of sphingolipid metabolites and suppression of autotaxin induction and lyso-phosphatidic acid formation. Here we summarize current knowledge on the roles of pancreatic and mucosal enzymes in PC and SM digestion, and its implications in intestinal and liver diseases, bacterial choline metabolism in the gut, and cholesterol absorption.

Deficiency of alkaline SMase enhances dextran sulfate sodium-induced colitis in mice with upregulation of autotaxin.

Intestinal alkaline SMase (Alk-SMase) cleaves phosphocholine from SM, platelet-activating factor (PAF), and lysophosphatidylcholine. We recently found that colitis-associated colon cancer was 4- to 5-fold enhanced in Alk-SMase KO mice. Here, we further studied the pathogenesis of colitis induced by dextran sulfate sodium (DSS) in WT and KO mice. Compared with WT mice, KO mice demonstrated greater body weight loss, more severe bloody diarrhea, broader inflammatory cell infiltration, and more serious epithelial injury. Higher levels of PAF and lower levels of interleukin (IL)10 were identified in KO mice 2 days after DSS treatment. A greater and progressive increase of lysophosphatidic acid (LPA) was identified. The change was associated with increased autotaxin expression in both small intestine and colon, which was identified by immunohistochemistry study, Western blot, and sandwich ELISA. The upregulation of autotaxin coincided with an early increase of PAF. IL6 and TNFα were increased in both WT and KO mice. At the later stage (day 8), significant decreases in IL6, IL10, and PAF were identified, and the decreases were greater in KO mice. In conclusion, deficiency of Alk-SMase enhances DSS-induced colitis by mechanisms related to increased autotaxin expression and LPA formation. The early increase of PAF might be a trigger for such reactions.

Molecular Insights into Covalently Stained Carious Dentine Using Solid-State NMR and ToF-SIMS.

Dyes currently used to stain carious dentine have a limited capacity to discriminate normal dentine from carious dentine, which may result in overexcavation. Consequently, finding a selective dye is still a challenge. However, there is evidence that hydrazine-based dyes, via covalent bonds to functional groups, bind specifically to carious dentine. The aim of this study was to investigate the possible formation of covalent bonds between carious dentine and 15N2-hydrazine and the hydrazine-based dye, 15N2-labelled Lucifer Yellow, respectively. Powdered dentine from extracted carious and normal teeth was exposed to the dyes, and the staining reactions were analysed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), solid-state 13C-labelled nuclear magnetic resonance (NMR) and 15N-NMR spectroscopy. The results showed that 15N2-hydrazine and 15N2-labelled Lucifer Yellow both bind to carious dentine but not to normal dentine. It can thus be concluded that hydrazine-based dyes can be used to stain carious dentine and leave normal dentine unstained.

Role of Sphingolipids in Infant Gut Health and Immunity.

Sphingomyelin (SM), glycosphingolipids, and gangliosides are important polar lipids in the milk fat globule membrane but are not found in standard milk replacement formulas. Because digestion and absorption of SM and glycosphingolipids generate the bioactive metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P), and because intact gangliosides may have beneficial effects in the gut, this may be important for gut integrity and immune maturation in the neonate. The brush border enzymes that hydrolyze milk SM, alkaline sphingomyelinase (nucleotide phosphodiesterase pyrophosphatase 7), and neutral ceramidase are expressed at birth in both term and preterm infants. Released sphingosine is absorbed, phosphorylated to S1P, and converted to palmitic acid via S1P-lyase in the gut mucosa. Hypothetically, S1P also may be released from absorptive cells and exert important paracrine actions favoring epithelial integrity and renewal, as well as immune function, including secretory IgA production and migration of T lymphocyte subpopulations. Gluco-, galacto-, and lactosylceramide are hydrolyzed to ceramide by lactase-phlorizin hydrolase, which also hydrolyzes lactose. Gangliosides may adhere to the brush border and is internalized, modified, and possibly transported into blood, and may exert protective functions by their interactions with bacteria, bacterial toxins, and the brush border.

Enhanced colonic tumorigenesis in alkaline sphingomyelinase (NPP7) knockout mice.

Intestinal alkaline sphingomyelinase (alk-SMase) generates ceramide and inactivates platelet-activating factor (PAF) and was previously suggested to have anticancer properties. The direct evidence is still lacking. We studied colonic tumorigenesis in alk-SMase knockout (KO) mice. Formation of aberrant crypt foci (ACF) was examined after azoxymethane (AOM) injection. Tumor was induced by AOM alone, a conventional AOM/dextran sulfate sodium (DSS) treatment, and an enhanced AOM/DSS method. ß-Catenin was determined by immunohistochemistry, PAF levels by ELISA, and sphingomyelin metabolites by mass spectrometry. Without treatment, spontaneous tumorigenesis was not identified but the intestinal mucosa appeared thicker in KO than in wild-type (WT) littermates. AOM alone induced more ACF in KO mice but no tumors 28 weeks after injection. However, combination of AOM/DSS treatments induced colonic tumors and the incidence was significantly higher in KO than in WT mice. By the enhanced AOM/DSS method, tumor number per mouse increased 4.5 times and tumor size 1.8 times in KO compared with WT mice. Although all tumors were adenomas in WT mice, 32% were adenocarcinomas in KO mice. Compared with WT mice, cytosol expression of ß-catenin was significantly increased and nuclear translocation in tumors was more pronounced in KO mice. Lipid analysis showed decreased ceramide in small intestine and increased sphingosine-1-phosphate (S1P) in both small intestine and colon in nontreated KO mice. PAF levels in feces were significantly higher in the KO mice after AOM/DSS treatment. In conclusion, lack of alk-SMase markedly increases AOM/DSS-induced colonic tumorigenesis associated with decreased ceramide and increased S1P and PAF levels.

Analysis of carious dentine using FTIR and ToF-SIMS.

Apart from the Maillard reaction, other processes, such as esterification, take place in carious tissue. The aim of the present study was to analyse sound and carious dentine in terms of ester groups and their reaction with hydrazine derivate using Fourier Transform Infrared Spectroscopy (FTIR) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Carious and sound dentine from human premolars were excavated in three series (Experimental Parts I-III) and separated into inner and outer layers of carious dentine. The excavated tooth material was analysed with FTIR (Part I). Carious and sound dentine were also exposed to different chemical treatments and analysed with FTIR-Attenuated Total Reflectance (FTIR-ATR; Part II) and ToF-SIMS (Part III). The FTIR absorption spectra showed that the carious tissue contained ester groups, not detected in sound dentine. The results also indicated a higher occurrence of ester groups in the inner dental caries layer than in the outer ones. Potential binding to these ester groups by hydrazine derivative was observed after different chemical treatments with both FTIR-ATR and ToF-SIMS. The results of the present study revealed ester groups unique to the carious dentine which, after reaction with hydrazine derivative, form a covalent bond not found in sound dentine. The staining of carious unique groups would be clinically helpful in detection and prevention unnecessary removal of sound dentine.

Changes of activity and isoforms of alkaline sphingomyelinase (nucleotide pyrophosphatase phosphodiesterase 7) in bile from patients undergoing endoscopic retrograde cholangiopancreatography.

BACKGROUND: Alkaline sphingomyelinase (NPP7) is an ecto-enzyme expressed in intestinal mucosa, which hydrolyses sphingomyelin (SM) to ceramide and inactivates platelet activating factor. It is also expressed in human liver and released in the bile. The enzyme may have anti-tumour and anti-inflammatory effects in colon and its levels are decreased in patients with colon cancer and ulcerative colitis. Active NPP7 is translated from a transcript of 1.4 kb, whereas an inactive form from a 1.2 kb mRNA was found in colon and liver cancer cell lines. While the roles of NPP7 in colon cancer have been intensively studied, less is known about the function and implications of NPP7 in the bile. The present study examines the changes of NPP7 in bile of patients with various hepatobiliary diseases. METHODS: Bile samples were obtained at endoscopic retrograde cholangiopancreatography (ERCP) in 59 patients with gallstone, other benign disease, tumour, and primary sclerosing cholangitis (PSC). The NPP7 activity was determined. The appearance of the 1.4 and 1.2 kb products in the bile was examined by Western blot. The results were correlated to the diseases and also plasma bilirubin and alkaline phosphatase. RESULTS: NPP7 activity in the tumour group was significantly lower than in the gallstone group (p < 0.05). The activity in the tumour plus PSC group was also lower than in gallstone plus other benign disease group (p < 0.05). Within the tumour group NPP7 activity was lowest in cholangiocarcinoma patients, being only 19% of that in gallstone patients. Bilirubin correlated inversely to NPP7 and was higher in the tumour than in the gallstone group. Western blot identified both the 1.4 kb and the 1.2 kb products in most bile samples. The density ratio for the 1.4/1.2 kb products correlated to NPP7 activity significantly. Two patients (one PSC and one cholangiocarcinoma) lacking NPP7 activity had only the 1.2 kb form in bile. CONCLUSION: NPP7 activity and the ratio of 1.4/1.2 kb products in bile are significantly decreased in malignancy, particularly in cholangiocarcinoma. The implications of the finding in diagnosis of cholangiocarcinoma and 1.2 kb product in hepatobiliary diseases require further investigation.

Alkaline sphingomyelinase (NPP7) promotes cholesterol absorption by affecting sphingomyelin levels in the gut: A study with NPP7 knockout mice.

We previously showed that dietary sphingomyelin (SM) inhibited cholesterol absorption in animals. The key enzyme hydrolyzing SM in the gut is alkaline sphingomyelinase (alk-SMase, nucleotide pyrophosphatase/phosphodiesterase 7). Here using the fecal dual-isotope ratio method we compared cholesterol absorption in the wild-type (WT) and alk-SMase knockout (KO) mice. The animals were fed an emulsion containing [(14)C]cholesterol and [(3)H]sitosterol. The radioactivities in the lipids of the fecal samples collected 4, 8, and 24 h thereafter were determined, and the ratio of (14)C/(3)H was calculated. We found that the fecal [(14)C]cholesterol recovery in the KO mice was significantly higher than in the WT mice. A maximal 92% increase occurred 8 h after feeding. Recovery of [(3)H]sitosterol did not differ between the two groups. Accordingly, the (14)C-to-(3)H ratio of fecal lipids was 133% higher at 8 h and 75% higher at 24 h in the KO than in the WT mice. Decreased [(14)C]cholesterol was also found in the serum of the KO mice 4 h after feeding. Supplement of SM in the emulsion reduced the differences in fecal [(14)C]cholesterol recovery between the WT and KO mice because of a greater increase of [(14)C]cholesterol recovery in the WT mice. Without treatment, the KO mice had significantly higher SM levels in the intestinal content and feces, but not in the intestinal mucosa or serum. The expression of Niemann-Pick C1 like 1 protein in the small intestine was not changed. In conclusion, alk-SMase is a physiological factor promoting cholesterol absorption by reducing SM levels in the intestinal lumen.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly, resulting in 98% reduction of ³H-ceramide/³H-SM ratio 1 h after gavage. The absorbed ³H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in ³H-ceramide was identified in distal intestine in KO mice. In feces, ³H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

Generating ceramide from sphingomyelin by alkaline sphingomyelinase in the gut enhances sphingomyelin-induced inhibition of cholesterol uptake in Caco-2 cells.

Background Sphingomyelin (SM) is present in dietary products and cell plasma membranes. We previously showed that dietary SM inhibited cholesterol absorption in rats. In the intestinal tract, SM is mainly hydrolyzed by alkaline sphingomyelinase (alk-SMase) to ceramide.Aims We investigated the influence of SM and its hydrolytic products ceramide and sphingosine on cholesterol uptake in intestinal Caco-2 cells.Methods Micelles containing bile salt, monoolein, and (14)C-cholesterol were prepared with or without SM, ceramide,or sphingosine. The micelles were incubated with Caco-2 cells, and uptake of radioactive cholesterol was quantified.Results We found that confluent monolayer Caco-2 cells expressed NPC1L1, and the uptake of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. Incorporation of SM in the cholesterol micelles inhibited cholesterol uptake dose-dependently; 38% inhibition occurred at an equal mole ratio of SM and cholesterol.The inhibition was further enhanced to 45% by pretreating the cholesterol/SM micelles with recombinant alk-SMase, which hydrolyzed SM in the micelles by 85%, indicating ceramide has stronger inhibitory effects on cholesterol uptake. To confirm this, we further replaced SM in the micelles with ceramide and sphingosine, and found that at equal mole ratio to cholesterol, ceramide exhibited stronger inhibitory effect (50% vs 38%) on cholesterol uptake than SM, whereas sphingosine only had a weak effect at high concentrations.Conclusion Both SM and ceramide inhibit cholesterol uptake, the effect of ceramide being stronger than that of SM. Alk-SMase enhances SM-induced inhibition of cholesterol uptake by generating ceramide in the intestinal lumen.

Sphingolipids in human ileostomy content after meals containing milk sphingomyelin.

BACKGROUND: Sphingomyelin occurs in modest amounts in the diet, in sloughed mucosal cells, and in bile. It is digested by the mucosal enzymes alkaline sphingomyelinase and ceramidase. In humans, alkaline sphingomyelinase is also secreted in bile. The digestion of sphingomyelin is slow and incomplete, which has been linked to the inhibition of cholesterol absorption and colonic carcinogenesis. OBJECTIVE: We evaluated whether the supply of moderate amounts of milk sphingomyelin increases the exposure of the colon to sphingomyelin and its metabolites. DESIGN: Two experimental series were performed. In experiment A, we measured the content of sphingomyelin and ceramide in human ileostomy content by HPLC during 8 h after consumption of a test meal containing 250 mg milk sphingomyelin. In experiment B, we measured the molecular species of sphingomyelin and ceramide by HPLC-tandem mass spectrometry after doses of 50, 100, or 200 mg sphingomyelin. RESULTS: In experiment A, the average increase in ileostomy content of ceramide plus sphingomyelin amounted to 19% of the fed dose of sphingomyelin. In experiment B, the output of C-22:0-sphingomyelin, C-23:0-sphingomyelin, C-24:0-sphingomyelin, and sphingosine increased significantly, and palmitoyl-sphingomyelin increased proportionally less. Outputs and concentrations of palmitoyl-ceramide and sphingosine showed great individual variation, and stearoyl-sphingomyelin and stearoyl-ceramide did not increase after the meals. Although the output of long-chain sphingomyelin species increased significantly, the data indicated that >81% of all measured sphingomyelin species had been digested. CONCLUSIONS: Humans digest and absorb most of the sphingomyelin in normal diets. The amount of sphingolipid metabolites to which the colon is exposed can, however, be influenced by realistic amounts of dietary sphingomyelin.

Long term effects on human plasma lipoproteins of a formulation enriched in butter milk polar lipid.

BACKGROUND: Sphingolipids (SL), in particular sphingomyelin (SM) are important components of milk fat polar lipids. Dietary SM inhibits cholesterol absorption in rats (Nyberg et al. J Nutr Biochem. 2000) and SLs decrease both cholesterol and TG concentrations in lipid- and cholesterol fed APOE*3Leiden mice (Duivenvoorden et al. Am J Clin Nutr. 2006). This human study examines effects of a butter milk formulation enriched in milk fat globule membrane material, and thereby in SLs, on blood lipids in healthy volunteers. In a four week parallel group study with 33 men and 15 women we examined the effects of an SL-enriched butter milk formulation (A) and an equivalent control formulation (B) on plasma lipid levels. Plasma concentrations of HDL and LDL cholesterol, triacylglycerols (TG), apolipoproteins AI and B, and lipoprotein (a) were measured. The daily dose of SL in A was 975 mg of which 700 mg was SM. The participants registered food and drink intake four days before introducing the test formula and the last four days of the test period. RESULTS: A daily increase of SL intake did not significantly influence fasting plasma lipids or lipoproteins. In group B TG, cholesterol, LDL, HDL and apolipoprotein B concentrations increased, however, but not in group A after four weeks. The difference in LDL cholesterol was seen primarily in women and difference in TG primarily in men. No significant side effects were observed. CONCLUSION: The study did not show any significant decrease on plasma lipids or lipoprotein levels of an SL-enriched formulation containing 2-3 times more SL than the normal dietary intake on cholesterol, other plasma lipids or on energy intake. The formulation A may, however, have counteracted the trend towards increased blood lipid concentrations caused by increased energy intake that was seen with the B formulation.

Ezetimibe inhibits expression of acid sphingomyelinase in liver and intestine.

Ezetimibe inhibits cholesterol absorption in the intestine. Sphingomyelin has strong interactions with cholesterol. We investigated the effects of ezetimibe on Sphingomyelinase (SMase) expression in intestine and liver. After feeding rats with ezetimibe (5 mg/kg per day) for 14 days, acid SMase activities in the liver and in the proximal part of small intestine were reduced by 34 and 25%, respectively. Alkaline SMase (alk-SMase) was increased in the proximal part of the small intestine. Administration of lower doses of ezetimibe reduced acid SMase only in the liver by 14% (P < 0.05). In cell culture studies, ezetimibe decreased acid SMase activity in Hep G2 and Caco-2 cells dose-dependently. The reductions were more rapid for Hep G2 cells than for Caco-2 cells. Western blot showed that acid SMase protein was decreased in both Hep G2 and Caco-2 cells by 100 microM ezetimibe. The SM content was increased in Hep G2 cells but not Caco-2 cells, and total cholesterol content was increased in both cell lines 24 h after stimulation with 100 microM ezetimibe. Mevastatin, the inhibitor of cholesterol synthesis, induced a mild increase in acid SMase activity in Hep G2 cells but not Caco-2 cells. Following the reduction of acid SMase, ezetimibe at high dose slightly increased alk-SMase activity. In conclusion, the study demonstrates an inhibitory effect of ezetimibe on acid SMase activity and expression in both liver and intestine.

Metabolism of sphingolipids in the gut and its relation to inflammation and cancer development.

Sphingolipids are abundant in the microvillar membrane of intestinal epithelial cells where they are essential for structural integrity and may act as receptors for toxins, virus and bacteria. Metabolism of dietary and membrane sphingolipids in the intestine generates ceramide, sphingosine, sphingosine-1-phosphate, and ceramide-1-phosphate, via the action of alkaline sphingomyelinase, neutral ceramidase, sphingosine-1-kinase, and ceramide-1-kinase. These intermediary metabolites act as bioactive lipid messengers, influencing numerous cellular functions including growth, differentiation and apoptosis of both epithelial and immunocompetent cells in the gastrointestinal tract, and also the progress of inflammation and responsiveness of the mucosal cells to pathogens. This review summarizes background and recent progress in the metabolism of dietary and endogenous sphingolipids in the gut and its pathophysiological implications.

Expression of intestinal and lung alkaline sphingomyelinase and neutral ceramidase in cystic fibrosis f508del transgenic mice.

OBJECTIVES: The intestinal brush border enzymes alkaline sphingomyelinase (alk-SMase) and neutral ceramidase (CDase) digest milk sphingomyelin in suckling neonates. In addition, alk-SMase, CDase, and acid sphingomyelinase (acid-SMase) have been implicated in sphingolipid signaling, which exhibits abnormalities in cystic fibrosis (CF). In this study, we tested the hypothesis that the expression of these enzymes is different in CF. MATERIALS AND METHODS: We used mice with F508del (Cftr) mutation, a CF mouse model with well-characterized intestinal pathology. Enzyme activities were measured using radiolabeled sphingolipid substrates incubated with tissue homogenates from different organs and intestinal contents of wild-type mice, homozygous, and heterozygous F508del mice. RESULTS: No difference was found in levels of CDase and alk-SMase in the small intestinal mucosa or in their longitudinal distribution. Acid-SMase activity was significantly lower in the mucosa of the distal half of the small intestine of F508del compared with wild-type mice. Despite a lower body weight of F508del mice, length and weight of the small intestine and weight per centimeter of colon were larger than in wild-type. Neutral CDase and alk-SMase activities in lungs were lower than in the gut, whereas acid-SMase activity was comparable in both organs. CDase activity in the spleen was significantly higher in F508del than in wild-type mice. CONCLUSIONS: Alk-SMase and neutral CDase are normally expressed in F508del CF mice, whereas activity of acid-SMase in the distal small intestine is decreased. We found no differences in activity of these enzymes in lungs in this mouse model.