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

Yao Zhang,Yajun Cheng,Gert H Hansen,Lise-Lotte Niels-Christiansen,Frank Koentgen,Lena Ohlsson,Åke Nilsson,Rui-Dong Duan

doi:10.1194/jlr.m012880

Abstract

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.

Highlights

Alkaline sphingomyelinase hydrolyses sphingomyelin (SM) to ceramide in the gut
Our results show that the processes of SM digestion, ceramide formation, and fatty acid (FA) absorption are severely affected in the KO mice, providing evidence for essential physiological roles of alk-SMase in SM digestion and ceramide generation in the gastrointestinal tract
Heterozygous Enpp7+/Ϫ mice were genotyped by Southern blot with en probe (Fig. 2B)

Summary

Introduction

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. SM in the diet has been shown to inhibit the colonic tumorigenesis in mice treated with chemical carcinogens [2], to promote the development of the intestinal mucosa in new born rats [3], and to inhibit cholesterol absorption in the gut [4, 5] Because most of these effects can be reproduced or linked to ceramide, a hydrolytic product of SM [6, 7], it is important to study the enzymes that hydrolyze SM and generate ceramide in the gut. More than 40 years ago, Nilsson [8] identified a type of enzyme in the intestinal mucosa that hydrolyses SM and generates ceramide at alkaline pH This enzyme was thereafter named alkaline sphingomyelinase (alk-SMase) [9].

Methods

Results

Conclusion