Effects of Dietary Sphingomyelin on Central Nervous System Myelination in Developing Rats

0031-3998/03/5304-0589 PEDIATRIC RESEARCH Copyright © 2003 International Pediatric Research Foundation, Inc. Vol. 53, No. 4, 2003 Printed in U.S.A. Effects of Dietary Sphingomyelin on Central Nervous System Myelination in Developing Rats KYOICHI OSHIDA, TAKASHI SHIMIZU, MITSUNORI TAKASE, YOSHITAKA TAMURA, TOSHIAKI SHIMIZU, AND YUICHIRO YAMASHIRO Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd, Zama, Kanagawa [K.O., Ta.S., M.T., Y.T.] and Department of Pediatrics, Juntendo University School of Medicine, Bunkyo-ku, Tokyo [To.S., Y.Y.], Japan ABSTRACT Human milk contains sphingomyelin (SM) as a major component of the phospholipid fraction. Galactosylceramide (cerebroside), a metabolite of sphingolipids, increases along with CNS myelination, and is generally considered a universal marker of myelination in all vertebrates. L-Cycloserine (LCS) is an inhibitor of serine palmitoyltransferase (SPT), a rate-limiting enzyme for sphingolipid biosynthesis that is reported to show increased activity with development of the rat CNS. The present study examined the effects of dietary SM on CNS myelination during development in LCS-treated rats. From 8 d after birth, Wistar rat pups received a daily s.c. injection (100 mg/kg) of LCS. From 17 d after birth, the animals were fed an 810 mg/100g of bovine SM-supplemented diet (SM-LCS group) or a nonsupplemented diet (LCS group). At 28 d after birth, the animals were killed and subjected to biochemical and morphometric analyses. The myelin dry weight, myelin total lipid content, and cerebroside content were significantly lower in the SM-LCS and LCS groups than in a group not treated with LCS (the non-LCS group). However, these levels were significantly higher in the SM is composed of phosphocholine as the polar head group and sphingosine as the backbone of the molecule, and it is therefore classified as one of the sphingolipids. Recent studies have demonstrated that sphingolipids are found in all eukaryotic and some prokaryotic organisms (1). These molecules are involved in the regulation of cell growth (2), cell differentiation, and diverse other functions, including cell–substratum interactions and intracellular signal transduction (3, 4). Human milk has a lower content of phospholipids compared with triglycerides. Bitman et al. (5) reported that human milk has a total phospholipid content of approximately 15 to 20 mg/dL, with SM accounting for approximately 37% of the phospholipid fraction. Although many foods contain a small amount of Received February 15, 2002; accepted October 4, 2002. Correspondence: Kyoichi Oshida, Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., 1-83, 5-Chome Higashihara, Zama, Kanagawa, 228-8583, Japan; e-mail: DOI: 10.1203/01.PDR.0000054654.73826.AC SM-LCS group than in the LCS group. Morphometric analysis of the optic nerve revealed that the axon diameter, nerve fiber diameter, myelin thickness, and g value (used to compare the relative thickness of myelin sheaths around fibers of different diameter) were significantly lower in the LCS group than in the other groups, but were similar in the SM-LCS and non-LCS groups. These findings suggest that dietary SM contributes to CNS myelination in developing rats with experimental inhibition of activity. (Pediatr Res 53: 589–593, 2003) Abbreviations SM, sphingomyelin LCS, L-cycloserine PC, phosphatidylcholine PE, phosphatidylethanolamine PI, phosphatidylinositol PS, phosphatidylserine SPT, serine palmitoyltransferase TLC, thin-layer chromatography SM (6), its nutritional and physiologic roles have not been fully examined. CNS myelin has a higher lipid content (65– 80%) than that of general cell membranes. SM and sphingolipid metabolites, such as cerebroside and sulfatide, are prominent components of the myelin sheath that surrounds the axons of some neurons. This sheath acts as an insulator for nerve impulses and controls the salutatory mode of conduction via the nodes of Ranvier. Myelination of the human CNS begins from 12 to 14 wk of gestation in the spinal cord (7, 8) and continues into the third decade of life in the intracortical fibers of the cerebral cortex (9), but the most rapid and dramatic changes occur between midgestation and the end of the second postnatal year (10, 11). Myelination accounts for a large part of the more than tripling of brain weight that occurs during this period. Recently, Luberto and Hannun (12) reported on a metabolic pathway for sphingolipids. SPT (EC 2.3.1.50) is the first step and the rate-limiting enzyme in sphingolipid biosynthesis (13, 14), catalyzing the synthesis of 3-ketosphinganine from L- 589 590 OSHIDA ET AL. serine and palmitoyl-CoA (15). This enzyme is located in the endoplasmic reticulum or Golgi apparatus (16). A recent study showed that SPT activity gradually increases from the third prenatal to the third postnatal week in the hypothalamus of rats (17). As myelination begins at the same period in these animals, it is conceivable that an increment of SPT activity may be one of the major factors involved in myelinogenesis. CNS myelin has a high cerebroside content when compared with its level in other tissues (18). Cerebroside is generated from ceramide by ceramide UDP-galactosyltransferase, which is the key enzyme in the biosynthesis of cerebrosides and catalyzes the transfer of galactose from UDP-galactose to ceramide (19). In rats, cerebroside is hardly detectable in the brain before 10 d after birth, but the cerebroside content increases markedly from the second to the third postnatal weeks, especially between d 14 and 23 of life (20). Because the period of maximum cerebroside biosynthesis corresponds with the time of most active myelination (21), cerebroside is generally recognized as a universal marker of CNS myelination (22–25). Ceramides can be generated from L-serine and palmitoylCoA by de novo synthesis of SPT, and from SM by sphingomyelinase. Therefore, during the period of low SPT activity, we hypothesized that cerebroside in CNS myelin of developing rats may be mainly derived from dietary SM ingested in milk that is transformed to ceramide and then to cerebroside. Miller and Denisova (26) reported that LCS caused a decrease of cerebroside in rat CNS myelin by inhibiting SPT activity and therefore could be useful for investigating the role of cerebroside in the formation of myelin. The rat optic nerve has been widely used for correlative morphometric, physiologic, and biochemical studies of the CNS because of its structural and functional homogeneity (27, 28). In particular, morphometric analysis of optic nerve was performed to evaluate the myelin formation, as neonatal rat optic nerves are entirely unmyelinated and almost all of the axons undergo myelination during maturation (27). In the present study, to examine the influence of dietary SM on the maturation of CNS myelin, we created a rat model of low SPT activity by administration of LCS and evaluated the effect (...truncated)