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

• DOI: 10.1194/jlr.m012880
• Publication Date: 2010-12-22T04:36:12Z
• Authors: Yao Zhang; Yajun Cheng; Gert H. Hansen; Lise-Lotte Niels-...; Frank Koentgen; Lena Ohlsson; Åke Nilsson; Rui-Dong Duan
• Keywords: Sphingomyelin phosphodiesterase; Knockout mouse; Digestion; Sphingolipid

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate physiological importance of enzyme, we generated alk-SMase knockout (KO) mice by Cre-recombinase-Locus X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) KO were fed 3H-palmitic acid labeled together with milk gavage. The lipids intestinal content, tissues, serum, liver analyzed TLC. In mice, nondigested 3H-SM content increased 6-fold formation 3H-ceramide decreased markedly, resulting 98% reduction 3H-ceramide/3H-SM ratio 1 h after absorbed portion was 95%. After 3 h, a small increase identified distal intestine mice. feces, 243% 74% also showed significantly radioactivity serum. Furthermore, alkaline phosphatase activity mucosa reduced 50% histological comparison two female littermates preliminarily suggested mucosal hypertrophy This study provides definite proof for crucial roles digestion points possible regulating growth function. 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(N-14CH3) choline-labeled milk-SM (14C-SM) provided Swedish Dairies Association. 3H-palmitoyl-labeled (3H-SM) obtained American Radiolabeled Chemicals, Inc. (St Louis, MO). Ceramide, sphingosine, palmitic acid, triglyceride (TG) used standard substances TLC analysis purchased Sigma Chemical Co. (St. Silica gel 60 plates (0.25 mm) Merck (Darmstadt, Germany). Anti antibody developed AgriSera AB (Vännäs, Sweden) described before (14Duan Targeting vector construction, targeting embryonic stem (ES) cells, injection chimeric initial breeding Ozgene Pty. Ltd. 11 sequence Ensembl Mouse genome database (Ensembl Gene ENSMUSG00000046697). Fig. 1, loxP sites inserted flanking 2 PGK-neo selection cassette downstream 2, within floxed region. Deletion region Cre recombinase frame-shift mutation creates early stop codon 3. contained additional BglII restriction site allow screening targeted clones Southern blot hybridization. electroporated W9.5 (129Sv/J) ES cells. Targeted homologous recombination hybridization using 3′ external probe (primers probe: 5′-CATCTGTAGCCTTGGGTTTCTGACG 5′-AATGGAGGTGGGTGAGGGTTAATC). Chimeric donor blastocysts followed implantation injected pseudopregnant recipient mated C57BL/6 produce heterozygous WT/flox which OzCre (C57BL/6 background) WT/KO (Enpp7+/−) genotype Enpp7+/− confirmed "en probe" en 5′-AATGGCACTGTCCTCGCATGAATC 5′-TGGATGTTCAGAGTCAGACATGGTGG). Heterozygous maintained background backcrossed least five times get inbred strain. then intercrossed homozygous Enpp7−/− (alk-SMase KO) All housed facilities Lund University commercial pellet free access water. Mollegaard Denmark. experimental protocols approved Animal Ethics Committee University. PCR analysis. Genomic DNA extracted tail kit GenEluteTM Mammalian Miniprep Kit (Helsingborg, Sweden), digested BglII, hybridized 13.9 kb fragment WT allele, 4.5 allele. For routine genotyping, genomic amplified forward primer (5′-CTGCCACTTTACACTGGTCAC) reverse (5′-TGGCACTGAGGCGAGAAC) yield 732 bp allele 247 respectively (Fig. 1). locations primers designed arrows. program consisted incubation step 95°C 10 min, 35 cycles 30 s, 55°C 72°C s. Forty microliters (50 Ci/mol, 0.1 mCi/ml) dissolved ethanol taken dryness under nitrogen. Fifteen milligrams unlabeled 16 mixture (Sigma B#8756) ml 0.9% sodium chloride added sonication 5 × 15 suspension mixed skim vortex min. Calculating mean molecular mass 775 SM, concentration emulsion 3.2 µmol per ml. fasting water, 0.3 containing µCi anesthetized Xylazine/Ketamine. Blood samples collected cardiac puncture. Stomach, intestine, colon, removed. divided three parts equal length S1, S2, S3, represent proximal, middle, respectively. segments opened longitudinally each rinsed chloride. solution saved, represented content. Lipids contents tissues below. another same amount emulsion, fecal metabolism cage days total extracted. contents, feces chloroform:methanol:water according Bligh Dyer (30Bligh E.G. W.J. rapid method extraction purification.Can. 1959; 37: 911-918Crossref (42861) whole segment, washout piece starting materials. organic phase collected, evaporated nitrogen gas, 500 µl chloroform:methanol (2:1, v/v). One hundred toluene:methanol (70:30, v/v) separate ceramide. solvent Rf values 0.04, 0.14, 0.57, FA TG, silica petroleum ether:ether:acetic (80:20:1, TG 0.29 0.56, bands visualized iodine specific scraped positions standards. band methanol:H2O (1:1) overnight. Nine milliliters Ready Value (Beckman Coulter) radioactivities 3H-SM, 3H-ceramide, 3H-TG determined liquid scintillation counting. Small pieces littermate excised fixed 4% paraformaldehyde 0.1M phosphate buffer (PB), pH 7.2 4◦C, briefly PB stored 1% PB. washing PB, tissue dehydrated embedded paraffin. Sections µm cut Leica RM 2165 microtome. sections dewaxed stained haematoxyline eosin-phloxine. mounting Vecto mount H-5000 permanent DM 4000 B microscope equipped DC FX camera. Pictures random different widths villi, crypts, thickness muscle layer crypts measured. activities previously (31Duan Sphingolipid hydrolyzing tract.Methods Enzymol. 311: 276-286Crossref (58) assay, 95 50 mM Tris-HCl buffer, 9.0, 0.15 M NaCl, EDTA, 6 taurocholate, 100 0.80 μM [14C]choline final volume µl, 37°C reaction terminated adding 0.4 (2/1, partition, cleaved 14C-phosphocholine upper measured similarly except modifications. Neutral assayed Tris MgCl2, 0.12% Triton 100, 7.5. Acid Tris-maleate NaCl 5.0. SMase, assay Western blotting 20 saline. centrifugation, supernatant concentrated 10-fold ultrafiltration off size membrane being kDa. Fifty subjected 10% SDS-PAGE transferred nitrocellulose membrane. blotted anti-human (1:5000 dilution) reacted anti-rabbit IgG conjugated horseradish peroxidase (1:50000 room temperature. ECL advance reagents emitted light recorded Kodak X-ray film. above longitudinally. Each segment solutions homogenized determined. p-nitrophenylphosphate substrate (32Cheng H.T. Administration ursodeoxycholic increases caspase-3 colon.Scand. 34: 915-920Crossref ceramidase 14C-labeled C16-ceramide (33Lundgren tract.Dig. 2001; 46: 765-772Crossref Bile stimulated lipase (BSSL) p-nitrophenylacetate (34Duan Yang Evidence humans.Lipids. 807-812Crossref aminopeptidase N (APN) Amoscato et al. (35Amoscato A.A. Alexander J.W. Babcock G.F. Surface lymphocytes. Biochemical biologic intact Immunol. 1989; 1245-1252PubMed alanine-p-nitroanilide substrate. Bio-Rad bovine albumin standard. adjusted those sample volume. Data means ± SEM. Statistical two-tail unpaired Student's t-test. P < 0.05 statistically significant. Screening recombinant probe. Bg1II negative yielded corresponding Enpp7, whereas 8.8 2A). clone chimeras subsequently genotyped 2B). kb. backcross, WT, later indicated 1. representative result 2C. (+/+) gave band, (+/−) (−/−) only 2C). further confirm successful knockout, mucosa, assayed. 3, little could mice.Fig. 3Alk-SMase removed (+/+), (+/−), washed out 25 saline lysis buffer. dried, weighed, resuspended 0.5 centrifugating, supernatants saved Results group. Methods section.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Interbreeding heterozygous, offspring expected similar Mendelian ratios appearance lethality mutant sexual (male:female) approximately 1:1 mutant. Intercrossing produced offspring, indicating fertility males females largely unaffected. grew normally obvious abnormalities. They significant changes appearance, behavior, body weight, (data sh