Ectopic expression of {alpha}1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase

Wenge Wang1,3, Wei Li1,,3, Yoshitaka Ikeda3, Jun-Ichiro Miyagawa4, Masako Taniguchi5, Eiji Miyoshi3, Yin Sheng3, Atsuko Ekuni3, Jeong Heon Ko3, Yorihiro Yamamoto6, Taizo Sugimoto4, Shizuya Yamashita4, Yuji Matsuzawa4, Gregory A. Grabowski7, Koichi Honke3 and Naoyuki  Taniguchi2,3

3Department of Biochemistry, Osaka University Medical School, Osaka 565-0871, Japan, 4Department of Internal Medicine and Molecular Science, Osaka University Medical School, Osaka 565-0871, Japan, 5Osaka International University for Women, Osaka 570-0014, Japan, 6Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan, and 7Division of Human Genetics, Children’s Hospital Research Foundation, Cincinnati, OH 45229-3039, USA

Received on July 25, 2000; revised on September 29, 2000; accepted on September 29, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The {alpha}1,6 fucosyltransferase ({alpha}1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an {alpha}1,6 linkage. {alpha}1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken ß-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of {alpha}1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the {alpha}1,6 FucT transgenic mice.

Key words: carbohydrate function/fucosyltransferase/glycosyltransferase/N-glycan/transgenic mice


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
N-glycans are essential for a variety of biological events by virtue of contributing to the folding, stability, and physiological activity of relevant glycoproteins (Dwek, 1995Go). N-glycans have a common core structure, and their branching patterns are determined by glycosyltransferases, such as N-acetylglucosaminyltransferases and fucosyltransferases.

GDP-L-Fuc:N-acetyl-ß-D-glucosaminide {alpha}1,6 fucosyltransferase ({alpha}1,6 FucT) catalyzes the transfer of a fucose residue from GDP-fucose to the position 6 of the innermost GlcNAc residue of N-glycans and is involved in the biosynthesis of hybrid and complex types of N-linked oligosaccharides in glycoproteins. The reaction products of this enzyme, {alpha}1,6 fucosylated oligosaccharides, are widely distributed in mammalian tissues. It is generally believed that {alpha}1,6 fucosylation plays an important role in fetal development (Bakkers et al., 1997Go). Under some pathological conditions, the expression of {alpha}1,6 FucT and the extent of fucosylation are altered. For example, the level of {alpha}1,6 FucT is elevated in both liver and serum during the process of hepatocarcinogenesis (Hutchinson et al., 1991Go). The presence of fucosylated {alpha}-fetoprotein is a good marker for distinguishing patients with hepatocarcinoma from those with chronic hepatitis and liver cirrhosis (Sato et al., 1993Go; Taketa et al., 1993Go).

Followed by the development of convenient assay method for the enzyme activity (Uozumi et al., 1996Go), {alpha}1,6 FucT was homogeneously purified, and its cDNA was cloned from porcine brain and human gastric tumor cells in our laboratory (Uozumi et al., 1996Go; Yanagidani et al., 1997Go). The {alpha}1,6 FucT gene was found to be expressed in most rat organs (Miyoshi et al., 1997Go). A relatively high level of expression was observed in brain and small intestine, but only trace levels were found in liver. The molecular cloning of the {alpha}1,6 FucT gene enabled us to manipulate the gene and to remodel the N-linked glycans in individual cells and some animal models. We previously produced transgenic mice that overexpressed the N-acetylglucosaminyltransferase III (GnT III) genes, in an attempt to elucidate the biological roles of the bisecting GlcNAc in N-linked glycans (Ihara et al., 1998Go). The N-linked glycans that were attached to apolipoprotein B in the liver of GnT III transgenic mice underwent a change, and the mice developed a fatty liver due to aberrant apolipoprotein B secretion. In the present paper, we report on a study of transgenic mice that overexpress human {alpha}1,6 FucT gene, in an attempt to study the biological roles of the core {alpha}1,6 fucose residue in N-linked glycans. The {alpha}1,6 FucT transgenic mice showed a unique phenotype of steatosis.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Expression of human {alpha}1,6 FucT in the transgenic mice
Of the 30 mice developed from the microinjected fertilized eggs, 6 were found to contain the transgene, as evidenced by Southern blotting. To detect the expression of the introduced gene, Northern blotting, {alpha}1,6 FucT activity, and lectin blotting were performed (Figure 1). The transgene was found to be highly expressed in two mouse lines. These two lines, designated as FucT-1 and FucT-2, were used for further experiments. All the following data were reproducible in these two lines.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 1. Expression of human {alpha}1,6 FucT in the liver of the transgenic mice. (A) Northern blot of the livers of wild mice (control) and {alpha}1,6 FucT-transgenic mice (FucT-1 and FucT-2). Twenty micrograms of total RNA from the livers was electrophoresed, blotted, and probed with cDNA of human {alpha}1,6 FucT. (B) Western blot of liver proteins with a mouse monoclonal antibody 15C6, which recognizes human {alpha}1,6 FucT. (C) {alpha}1,6 FucT activity levels in the livers of the transgenic mice. All data were reproducible.

 
When {alpha}1,6 FucT activity was measured in various organs, liver and kidney showed a 10 times higher activity (1500–3000 pmol/h/mg) in the case of the transgenic mice, compared to the normal mice (100–200 pmol/h/mg). In contrast, {alpha}1,6 FucT activity in the spleen, thymus, small intestine, and adrenal glands was, at most, only twice as high in the transgenic mice, compared with the normal littermates (data not shown).

Changes of N-linked glycans in {alpha}1,6 FucT transgenic mice
To determine the manner in which the glycans of proteins are fucosylated in the {alpha}1,6 FucT transgenic mice, a liver extract was subjected to aleuria aurantia lectin (AAL) blot analysis to detect the {alpha}1,6 fucose in N-linked glycans (Fukumori et al., 1989Go). When whole homogenates were used, the observed difference between the transgenic mice and wild mice was very slight. However, significant changes were found after isolation of the light mitochondrial and microsomal fractions (Figure 2A). When the light mitochondrial fraction was subjected to two-dimensional electrophoresis and stained with AAL, additional specific bands with {alpha}1,6 fucose structures were detected (Figure 2B). Immunoblot with a monoclonal antibody CAB4, which recognizes the {alpha}1,6 fucose structure in the core of N-glycans (Srikrishna et al., 1997Go), showed patterns similar to the AAL blots (data not shown). AAL blots of serum and kidney proteins also showed additional components and stronger signals in the {alpha}1,6 FucT transgenic mice compared to the control mice (data not shown). These observations indicate that the introduced {alpha}1,6 FucT in fact catalyzes the addition of fucose residues to the core of N-linked oligosaccharides of a large number of glycoproteins in liver and kidney.



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 2. AAL lectin blot of proteins from the liver. (A) AAL lectin blot of liver proteins of normal mice (–) and FucT transgenic mice (+). Liver homogenates were fractionated and subjected to lectin blot following SDS–PAGE as described in Materials and methods. Whole: whole homogenates, LM: light mitochondrial fraction, MS: microsomal fraction. (B) AAL blot of liver light mitochondrial fraction subjected to two-dimensional electrophoresis. Upper, control mice. Lower, FucT mice.

 
Histological changes in {alpha}1,6 FucT transgenic mice
When organs from the transgenic mice, including brain, lung, thymus, heart, liver, spleen, kidney, stomach, intestine, colon, and skeletal muscle, were examined by hematoxylin-eosin stain, no significant change was found except for in the liver and kidney. Hepatocytes of the transgenic mice were found to have numerous small vacuoles and appeared to be slightly larger than those of normal mice (Figure 3). Oil red O staining revealed that the vacuoles stored neutral lipids. Lipid vacuoles were also found in the proximal renal tubular cells of the transgenic mice (Figure 4). Interestingly, most of the vacuoles were located in the basolateral compartments of the epithelial cells. Consistent with the light microscopic observations, electron microscopic observation revealed that hepatocytes of {alpha}1,6 FucT transgenic mice were larger than normal mice and contained many lipid droplets of various sizes (Figure 5). Most of the lipid droplets were spherical and were sometimes fused with one another to make larger ones. The number of lysosomes appeared to be increased and the glycogen particles were decreased compared to the control littermates. The secondary lysosomes were often filled with electron-lucent lipid materials (Figure 5C). Small lipid droplets were membrane-bound, although the trilaminar structure of the limiting membrane could not be clearly recognized (Figure 5D). The accumulation of lipids was not observed in the endoplasmic reticulum or the Golgi complex. Lipid droplets were also evident in the Kupffer cells in the transgenic mice (data not shown). In the kidney, numerous lipid droplets of various sizes were found in the proximal tubular cells of the transgenic mice, but only several small lipid droplets were observed in the case of the wild mice (Figure 6). The lipid droplets were largely located in the basolateral compartments of the epithelial cells. The small lipid droplets were membrane-bound, but the limiting membranes around the large lipid droplets were observed with difficulty. However, the large lipid droplets often possessed an electron-dense marginal rim and/or a polar matrix, suggesting that they had already interacted with the lysosomes or that they originated from the lysosomes themselves. In addition, numerous secondary lysosomes with various amounts of lipid materials were observed, mainly in the apical portion of the cells, suggesting the presence of a disturbed lysosomal function in lipid metabolism of these entities (Figure 6B). No apparent ultrastructural changes were detected in the glomeruli or the other part of kidney.



View larger version (165K):
[in this window]
[in a new window]
 
Fig. 3. Microscopic observation of the livers of the transgenic mice. (A) and (C), normal mice; (B) and (D), {alpha}1,6 FucT transgenic mice. (A) and (B), paraffin section, hematoxylin/eosin staining; (C) and (D), frozen section, Oil red O staining (original magnification: 200x).

 


View larger version (134K):
[in this window]
[in a new window]
 
Fig. 4. Microscopic observation of the kidneys of the transgenic mice. (A) and (C), normal mice; (B) and (D), {alpha}1,6 FucT transgenic mice. (A) and (B), paraffin section, hematoxylin/eosin staining; (C) and (D), frozen section, Sudan III stainin. (original magnification: 400x).

 


View larger version (177K):
[in this window]
[in a new window]
 
Fig. 5. Electron microscopic photographs of hepatocytes of a control and {alpha}1,6 FucT transgenic mice (10-week-old, male). (A) A hepatocyte of a negative littermate showed a normal appearance with numerous mitochondria, with moderately developed rough endoplasmic reticulum. Electron-dense small lysosomes scattered in the cytoplasm and glycogen areas (*) were also observed. N: nucleus (original magnification: 4700x, bar = 4 µm). (B) In a hepatocyte of {alpha}1,6 FucT transgenic mouse, many lipid droplets of various sizes were recognized (L). Some of these were juxtaposed or gathered together in the cytoplasm (original magnification: 4700x, bar = 4 µm). (C) In a hepatocyte from a transgenic mouse, electron-lucent lipid materials can be seen in the secondary lysosomes (arrowhead), and these lysosomes, when filled with lipid materials, became lower in electron density (arrow) (original magnification: 20,000x, bar = 1 µm). (D) A limiting membrane (arrowheads) could be recognized in a small lipid droplet (L). The content of this lipid droplet was homogeneous and slightly osmiophilic (original magnification: 77,000x, bar = 200 nm).

 


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 6. Electron microscopic photographs of the proximal renal tubular cells of a control and {alpha}1,6 FucT transgenic mice (10-week-old, male). (A) Normal proximal tubular cells of a negative control littermate had a small number of lysosomes and a few small lipid droplets. N: nucleus (original magnification: 4200x, bar = 4 µm). (B) In proximal tubular cells of {alpha}1,6 FucT transgenic mouse, increased number of secondary lysosomes could be seen, and some of them contained electron-lucent lipid materials (arrowheads). In the basolateral cytoplasm, various sized lipid droplets (L) could be recognized, many of which were rimmed with thin electron-dense structure or their surfaces were dotted with electron-dense substance, which derived presumably from lysosomes (original magnification: 4200x, bar = 4 µm).

 
Serum lipid level in {alpha}1,6 FucT transgenic mice
As a reason for liver steatosis, dysfunction of secretion is a possibility. If this is the case, abnormalities in serum lipoproteins would be expected. Actually, GnT III transgenic mice developed a fatty liver as the result of aberrant apolipoprotein B secretion (Ihara et al., 1998Go). We therefore investigated the levels of serum lipoproteins by electrophoresis. However, no differences in the levels of very low density lipoproteins (VLDLs) and high-density lipoproteins (HDL) were found between the transgenic mice and wild mice (data not shown). In addition, no significant changes were found in the levels of serum triglycerides, total cholesterol, and free fatty acids (data not shown). Although we also determined the detailed levels of individual free fatty acids, including C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:4, and C22:6, no differences except for C18:1 were detected (data not shown). These findings suggest that no abnormality in serum lipoproteins exists in the transgenic mice.

Accumulated lipids in {alpha}1,6 FucT transgenic mice
To determine which step is damaged in the transgenic mice, we attempted to characterize the accumulated lipids. Triglycerides, total cholesterol, and free fatty acid levels were found to be increased in the liver of the transgenic mice (Table I). Furthermore, thin-layer chromatography revealed that the amount of triglyceride and cholesterol ester increased significantly in the transgenic mice, compared with their wild littermates (Figure 7). These findings indicate that triglycerides and cholesterol ester had accumulated in the transgenic mice. A similar storage pattern of triglycerides and cholesterol ester was observed in the kidney (data not shown).


View this table:
[in this window]
[in a new window]
 
Table I. Lipid levels in the livers of normal control and {alpha}1,6 FucT transgenic mice
 


View larger version (78K):
[in this window]
[in a new window]
 
Fig. 7. High-performance thin-layer chromatography of liver lipids. Lanes 1–3, controls; 4–7, FucT Mice. CE, cholesterol esters; TG, triglycerides; FFA, free fatty acids; Cho, free cholesterol; Ori., original place of sample.

 
Expression of lipid-metabolizing proteins in {alpha}1,6 FucT transgenic mice
In an attempt to determine the mechanism of lipid accumulation, we examined the activities of microsomal triglyceride transfer protein (Wetterau et al., 1992Go), cholesterol and free fatty acid synthesis (Shapiro et al., 1969Go), lysosomal acid lipase (LAL, EC 3.1.1.13) (Ishii et al., 1995Go; Merkel et al., 1999Go), and both mitochondrial and peroxisomal ß-oxidation (Otto and Ontko, 1978Go; Lazarow, 1981Go) in the livers of the transgenic mice, compared with their wild littermates. No significant changes were found except that LAL activity was significantly decreased (p < 0.01, Table II) in the case of the transgenic mice. When {alpha}1,6 FucT was overexpressed in Hep 3B hepatoma cells and WiDr colon carcinoma cells, LAL activity was also reduced by 10–20% (data not shown). These results suggest that the overexpression of {alpha}1,6 FucT leads to an inhibition of lysosomal lipase activity. Furthermore, gene expression (as assessed by Northern blotting) of LAL and other proteins involved in lipid metabolism, including carnitine palmitoyltransferase I, HMG-CoA reductase, free fatty acid synthase, and acyl-CoA oxidase, was found to be at a normal level (data not shown).


View this table:
[in this window]
[in a new window]
 
Table II. Lipid-metabolizing protein activities in livers of normal control and {alpha}1,6 FucT transgenic mice
 
Effects on specific activity and fucosylation of lysosomal acid lipase
LAL activity was 30–40% lower in the transgenic mice than in their wild-type littermates (Table II). To examine the issue of whether the reduction of LAL activity is due to a decrease in the level of LAL protein, immunoblotting analysis was carried out. Unexpectedly, the immunoreactive signals toward LAL in the transgenic mice were stronger than those of control mice (Figure 8A), indicating that LAL protein levels were increased in the transgenic mice. No differences in the activity of lysosomal {alpha}-fucosidase or in the protein level of lysosomal cathepsin D were detectable between transgenic and wild mice (data not shown), suggesting that the effects on LAL activity and protein level are specific phenomena.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 8. Lectin and immunoblot results for LAL. (A) Immunoblot of LAL in liver light mitochondrial fraction of the transgenic mice (upper). Immunoblot of cathepsin D is done as control (lower). Lanes 1–4, control mice; 5–8, FucT transgenic mice. (B) Blots of immunoprecipitated LAL with an anti-LAL antibody, AAL, and CAB4. Control mice are indicated by (–) and (+) for the transgenic mice.

 
Considering the fact that the activity of LAL was decreased but that its protein level was increased, its specific activity should be greatly reduced in the case of the transgenic mice. These findings suggest that LAL is directly affected by the introduced {alpha}1,6 FucT and prompted us to investigate whether the N-glycan attached to LAL is more highly fucosylated in the transgenic mice. Lectin and immunoblot analysis of LAL using AAL lectin and CAB4 monoclonal antibody, which recognize the {alpha}1,6 fucose residue in the core of N-glycans, revealed that LAL in normal mouse liver is slightly fucosylated but in the {alpha}1,6 FucT transgenic mice it is even more fucosylated (Figure 8B). As shown in Figure 9, of the 10 mice of each control group and each FucT transgenic mouse examined, the activity of LAL was negatively correlated with that of FucT (correlation coefficient = 0.59, p < 0.01), indicating that LAL is likely to be a target protein of {alpha}1,6 FucT in transgenic mice. The apparent Km values for cholesterol oleate of wild and FucT mouse LALs were found to be 220 and 255 µM, respectively, indicating that the fucosylation of LAL does not affect its substrate recognition.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 9. Correlation of LAL and {alpha}1,6 FucT activities in the livers of normal (open circles) and {alpha}1,6 FucT transgenic (closed circles) mice. Correlation coefficient = 0.59 (p < 0.01).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The overexpression of {alpha}1,6 FucT in mice caused the accumulation of lipids in liver and kidney. Based on the number and size of the lipid droplets and the position of the nucleus, the fatty livers in {alpha}1,6 FucT transgenic mice may be classified as a form of microvesicular steatosis (Hautekeete et al., 1990Go). There are many diseases that show a fatty liver with this feature, such as an acute fatty liver during pregnancy (Rolfes and Ishak, 1986Go; Sims et al., 1995Go; Ibdah et al., 1999Go), Reye’s syndrome (Kolata, 1980Go), tetracycline toxicity (Wenk et al., 1981Go; Freneaux et al., 1988Go), defects in urea cycle enzymes (Weber et al., 1979Go) and mitochondrial fatty acid oxidation (Hautekeete et al., 1990Go), Wolman’s disease (Anderson et al., 1994Go; Pagani et al., 1998Go), cholesteryl ester storage disease (CESD) (Sloan and Frederickson, 1972Go; Pagani et al., 1998Go), and some varieties of viral hepatitis (Prior et al., 1987Go), although their mechanisms are different. Many of these results from the dysfunction of one or more enzymes in the lipid metabolism pathway are the result of genetic defects. Others may be caused by chemical or biological inhibitions in the lipid metabolism pathway. In addition, deficiency or inhibition of VLDL assembly or secretion may also cause an accumulation of lipids in liver (Nagayoshi et al., 1995Go).

We previously reported on the ectopic overexpression of GnT III, which resulted in the addition of the bisecting GlcNAc, which regulates the branching of N-glycans, disrupting apolipoprotein B secretion (Ihara et al., 1998Go). Unlike the GnT III transgenic mice, no significant changes in serum lipids in {alpha}1,6 FucT transgenic mice were observed, suggesting that no problem exists in terms of the secretion of lipids out of the hepatocytes. The electron microscopic observation revealed that lipid droplets had accumulated in the lysosomes in hepatocytes and renal tubular cells of {alpha}1,6 FucT transgenic mice. These data, along with that on the accumulation of cholesterol ester and triglyceride, suggest that the hydrolysis of lipid esters in the lysosomes is abrogated in the transgenic mice.

Lysosomes are important organelles involved in lipid metabolism (Lusa et al., 1998Go). Triglycerides and cholesterol ester carried by VLDL and low-density lipoproteins (LDLs) are endocytosed into cells via LDL receptors. The endosomes join with primary lysosomes to become secondary lysosomes, in which triglycerides and cholesterol ester are hydrolyzed by hydrolases. If the balance of load and degradation is disturbed, these lipids may accumulate in the lysosomes and finally form membrane-bound lipid droplets (Lough et al., 1970Go). Lysosomes with accumulated lipids, called lipolysosomes, are regarded as a specific feature of Wolman’s disease (Lough et al., 1970Go). Lipolysosomes can be occasionally found in some other liver disorders (Hayashi et al., 1977Go), but the ratios of membrane-bound to naked lipid droplets were <3.1%, which is much less than in Wolman’s disease and CESD (Hayashi et al., 1983Go). Wolman’s disease is an autosomal recessive disorder with an inherited deficiency of LAL (Anderson et al., 1994Go; Pagani et al., 1998Go). LAL catalyzes the hydrolysis of cholesterol ester and triglycerides in the lysosomes. In the case of the {alpha}1,6 FucT transgenic mice, cholesterol ester and triglycerides had accumulated and LAL activity was significantly reduced, suggesting that a reduced level of hydrolysis is at least partly responsible for the accumulation of such lipids.

Mouse and human LALs contain five conserved potential N-linked glycosylation sites. The potential role of glycosylation of LAL in the formation or maintenance of a catalytically active enzyme has been a controversial issue. Some investigators have suggested that glycosylation might not be essential for catalytic function by demonstrating that enzyme activity, after treatment with endoglycosidase H, was unchanged (Sando and Rosenbaum, 1985Go; Ameis et al., 1994Go). Others have concluded that glycosylation is important by showing that the activity is reduced, as the result of the same treatment (Pariyarath et al., 1996Go) and that tunicamycin treatment led to the production of inactive LAL and that an active form of LAL could not be expressed in a bacterial system (Sheriff et al., 1995Go). In the present study, we found that the specific activity of LAL was greatly reduced when LAL became highly fucosylated via the introduction of the {alpha}1,6 FucT gene. This finding supports the conclusion that the glycosylation of LAL regulates its activity.

Organs affected in Wolman’s disease include mainly liver, spleen, intestine, and the adrenal gland. Unlike Wolman’s disease, lipid accumulation is confined to hepatocytes and proximal renal tubular cells in the {alpha}1,6 FucT transgenic mice. This may reflect the high expression of the transgene in liver and kidney (Figure 1). The deficient state of LAL is expressed in two major phenotypes in the clinic (Yoshida and Kuriyama, 1990Go; Nakagawa et al., 1995Go). One is Wolman’s disease and the other is designated CESD, in which only cholesteryl esters are stored (Sloan and Frederickson, 1972Go; Pagani et al., 1998Go). Wolman’s disease is the more severe form; it is nearly always fatal in the first year of life. CESD is more benign; these patients may survive to adulthood. The molecular basis of the different phenotypes is actually not yet clear and may be due to residual enzyme activity (Anderson et al., 1994Go; Pagani et al., 1998Go). The LAL-knockout mice share many features of Wolman’s disease but have a milder phenotype and are fertile, although they undergo massive cholesterol ester and triglyceride storage with complete loss of LAL activity (Du et al., 1998Go).

We also found lipid accumulation within lysosomes in the proximal renal tubular cells of our {alpha}1,6 FucT transgenic mice. It is unique that the lipid vacuoles are mainly located in the basolateral compartments of the epithelial cells. No previous study has been found that describes this type of lipid accumulation. According to the few available reports on the ultrastructure of kidney of patients with microvesicular fatty liver, lipid accumulation in kidney proximal tubular cells may occasionally be found but in different manners (Slater and Hague, 1984Go; Jung et al., 1993Go). In several experimental animal models of liver steatosis (Fan et al., 1996Go; Shimano et al., 1996Go; Reue and Doolittle, 1996Go; Hashimoto et al., 1999Go) no accumulation of lipid in the lysosomes of renal tubules has been reported. The basis for lipid accumulation in the lysosomes in the basolateral compartments of the epithelial cells might be due to the fact that the nutrition of epithelial cells of the proximal tubule is derived from outside of the basal membrane, where the lipids within VLDL and LDL were endocytosed via the receptors and then fused with nearby lysosomes. Because of the deficiency of hydrolysis, the esterified lipids accumulated in this location. The lysosomes in the apical compartments might be less responsible for lipid metabolism and therefore be less affected.

A number of proteins may be involved in the lysosomal transport and digestion of triglycerides and cholesterol ester. For instance, LAL-inhibitory proteins have been reported (Kubo et al., 1981Go; Gorin et al., 1982Go), and a physiological detergent, such as saposins (Bierfreund et al., 2000Go), could help LAL digest those lipids. Little is known about how the degradation products of lipid hydrolysis exit the lysosomes. Any dysfunction in these processes may lead to an accumulation of lipids in the organelle. Because the lipid storage that is actually observed by microscopic analysis appears to be more severe than that expected by the reduction of LAL activity, other factor(s) in these processes may be blocked in {alpha}1,6 FucT transgenic mice. Alternatively, the accumulated inactive LAL may have a dominant negative effect on the hydrolysis of lipids in the lysosomes.

In conclusion, we report the development of an experimental model mouse with steatosis in the liver and kidney by ectopic expression of {alpha}1,6 FucT. A novel mechanism for lipid storage due to down-regulation of lysosomal acid lipase activity by remodeling of its glycosylation is proposed.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Vector construction
Human {alpha}1,6 FucT cDNA containing the entire open reading frames (Yanagidani et al., 1997Go) was cut out with EcoR I from a pBluescript cloning vector and ligated into a mammalian expression vector, pCAGGS, containing a combination of cytomegarovirus and chicken ß-actin promoters (Nitta et al., 1998Go). The DNA fragment that was cut out with Sal I and BamH I, containing the promoter and {alpha}1,6 FucT cDNA regions, was used for microinjection into fertilized eggs of a DBF1 mouse strain.

Mice
DNA was extracted from tails of mice developed from the above-mentioned fertilized eggs and analyzed by Southern blotting for the incorporation of human {alpha}1,6 FucT cDNA. Six out of 30 mice were found to be positive and were mated with C57BL/6 mice. Northern blot analysis of RNAs from the tail, liver, and kidney and lectin blots of serum proteins were carried out in order to detect the expression of the trans-{alpha}1,6 FucT cDNA. Two mouse lines with high levels of expression of {alpha}1,6 FucT were established. These animals were maintained in 12–12 h light-dark cycles (light from 8 AM to 8 PM) and fed with a chow diet (Oriental Corp, Osaka), which contained 75 mg/kg cholesterol and 3.7 g/kg fat.

Lectin and immunoblotting
Biotin-labeled AAL was obtained from the Honen Corp (Japan). Affinity purified rabbit anti-human LAL IgG, which cross-reacts with mouse LAL, was used for the detection and immunoprecipitation of mouse LAL (Du et al., 1996Go). The CAB4 monoclonal antibody that recognizes the {alpha}1,6 fucose residue in the core of N-linked glycans was kindly provided by Dr. Freeze (Srikrishna et al., 1997Go). A monoclonal antibody 15C6 against human {alpha}1,6 FucT was obtained from Fujirebio Inc. (Japan). Goat polyclonal anti-human cathepsin D antibodies were prepared in our laboratory. This antibody cross-reacts with mouse cathepsin D. Proteins from serum, liver, or other organs were subjected to SDS–PAGE and transferred to PVDF membranes. Western blots and lectin blots were carried out as described previously (Miyoshi et al., 1997Go; Ihara et al., 1998Go).

{alpha}1,6 FucT activity assay
{alpha}1,6 FucT activity was assayed by the method of Uozumi et al. (1996)Go. Briefly, cell homogenates were mixed with the assay buffer in a total volume of 15 µl, containing 10–20 µg protein, 200 mM MES, pH 6.2, 1% Triton X-100, 500 µM GDP-fucose, and 5 µM {alpha}1,6 FucT acceptor. After 1 h of incubation at 37°C, the mixture was boiled for 3 min and centrifuged at high speed for 10 min. Ten microliters of the supernatant were subjected to HPLC. Activity was expressed as pmols of GDP-fucose transferred to the acceptor per h per mg protein.

Preparation of tissue homogenates and subcellular fractionation
The liver from each mouse was perfused through the portal vein with an ice-cold sucrose medium (0.25 M sucrose in 10 mM Tris–Cl buffer, pH 7.4, and 1 mM EDTA) and homogenized in 10 vol of the ice-cold sucrose medium using a Potter-Elvehjem-type homogenizer with six strokes of a loose-fitting Teflon pestle. Subcellular fractions were separated by differential centrifugation using OptiprepTM (Nycomed Amersham, Norway) according to the manufacturer’s instruction. Marker enzymes of each organelle were used for identification of the fractions. Protein concentration was determined with a BCA protein assay kit (Pierce).

Analysis of lipids
Total lipids were extracted with 10 vol of chloroform/methanol (2:1, v/v). After the solvent was evaporated, the residue was dissolved in either a minimum vol of chloroform/methanol (2:1, v/v) for thin-layer chromatography or 1% Triton X-100 for the determination of total cholesterol, triglycerides, and free fatty acids. For the separation of lipids, the samples were applied to a thin-layer plate (10 x 10 cm, silica gel 60, Merck, Germany) and developed with hexane/ether/formic acid (80:20:2 v/v/v). After drying, the plate was submerged in a solution containing 3% copper acetate and 8% phosphoric acid for 5 min and then baked at 200°C for visualization of lipids. For the determination of total cholesterol, triglycerides, and free fatty acids, Monotest kit (Boehringer Mannheim, Germany), TG I kit (Wako, Japan), and NEFA IC kit (Wako, Japan) were used, respectively.

Analysis of serum lipoproteins
Fresh mouse serum in sample buffer was loaded onto a MultiGel-Lipo ready-made acrylamide gel (Daiichi Pure Chemicals Co., Ltd., Japan) for electrophoresis according to the manufacturer’s recommended protocol. The gel was stained with Sudan black to reveal VLDL and HDL components.

Histochemical examination
Fresh tissues were fixed in a 10% formaldehyde in 0.1 M phosphate buffer (pH 7.4). Paraffin sections and frozen sections were prepared for hematoxylin-eosin staining and for Oil red O or Sudan III staining to reveal neutral lipids, respectively.

Electron microscopic observation
Anesthetized mice were perfused via the left ventricle with a 3% glutaraldehyde solution buffered at pH 7.4 with 0.1 M Millonig’s phosphate buffer. The liver and kidney were excised as described previously (Miyagawa et al., 1995Go). Briefly, the liver and kidney were cut into small pieces and fixed in the same fixative for 2 h at 4°C. After a secondary fixation with 1% osmium tetroxide buffered at pH 7.4 with 0.1 M Millonig’s phosphate buffer for 1 h at 4°C, specimens were dehydrated and embedded in Epon (epoxy resin). Ultra-thin sections, cut on a Reichert-Jung Ultracut E ultramicrotome, were doubly stained with aqueous uranyl acetate (3.0%) and Reynolds’s lead citrate and then subjected to electron microscopy using a Hitachi H-7000 apparatus.

Lysosomal enzyme assays
LAL activity was assayed using cholesterol-[1-14C]-oleate (American Radiolabled Chemicals, Inc., USA) as described previously (Ishii et al., 1995Go; Merkel et al., 1999Go) with slight modifications. First, a substrate stock solution was made by mixing 0.57 ml of nonradioactive cholesteryl-oleate (10 mg/ml in hexane) with 50 µCi of cholesteryl-[1-14C]-oleate and adding hexane to 2 ml. For 20 reactions, 100 µl of substrate stock solution was mixed with 100 µl of 18.4 mg/ml lysolecithin in chloroform/methanol (1:1, v/v). After the solution was dried under a stream of nitrogen, 0.8 ml 0.9% NaCl was added, and the resulting mixture was sonicated for 10 min in an ice-water bath. For assays, a 40 µl aliquot of this substrate was mixed with 0.1–1.0 mg of protein and a solution containing 100 mM sodium acetate (pH 5.0) and 1% Triton X-100 in a final volume of 200 µl. The reaction mixture was incubated at 37°C for 30–60 min, and the reaction was stopped by adding 3.25 ml of chloroform/methanol/heptane (1.42:1.25:1.00, v/v/v), followed by vortexing for 10 s. One milliliter of 1 N NaOH was then added, and the samples were vortexed for 30 s. After centrifugation for 10 min at 1000 x g, 1 ml of the upper layer was transferred to a vial, mixed with 5 ml of scintillation fluid, and counted for radioactivity with a liquid scintillation counter. Activity was expressed as pmol of free fatty acid released by 1 mg of protein per h. {alpha}-Fucosidase activity was assayed as described previously (Lovell et al., 1994Go; Prasad and Pullarkat, 1996Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. J. Miyazaki, Osaka University Medical School, and Dr. H. H. Freeze, Burnham Institute, for providing pCAGGS vector and CAB4 antibody, respectively. We are also grateful to Dr. V. D. D’Agati, Columbia University, for his valuable discussion on histological observations. This study was supported by a Grant-in-Aid for Scientific Research on Priority Area No. 10178104 and 10178105 from the Ministry of Education, Science and Culture, Japan.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1,6 FucT, alpha 1,6 fucosyltransferase; LAL, lysosomal acid lipase; AAL, aleuria aurantia lectin; CESD, cholesteryl ester storage disease; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ameis, D., Merkel, M., Eckerskorn, C., and Greten, H. (1994) Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase. Eur. J. Biochem., 219, 905–914.[Abstract]

Anderson, R.A., Byrum, R.S., Coates, P.M., and Sando, G.N. (1994) Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman disease. Proc. Natl Acad. Sci. USA, 91, 2718–2722.[Abstract]

Bakkers, J., Semino, C.E., Stroband, H., Kijne, J.W., Robbins, P.W., and Spaink, H.P. (1997) An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish. Proc. Natl Acad. Sci. USA, 94, 7982–7986.[Abstract/Free Full Text]

Bierfreund, U., Kolter, T., Sandhoff, K. (2000) Sphingolipid hydrolases and activator proteins. Methods Enzymol., 311, 255–276.[ISI][Medline]

Du, H., Witte, D.P., and Grabowski, G.A. (1996) Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein. J. Lipid Res., 37, 937–949.[Abstract]

Du, H., Duanmu, M., Witte, D., and Grabowski, G.A. (1998) Targeteddisruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage. Hum. Mol. Genet., 7, 1347–1354.[Abstract/Free Full Text]

Dwek, R.A. (1995) Glycobiology: more functions for oligosaccharides. Science, 269, 1234–1235.[ISI][Medline]

Fan, C.Y., Pan, J., Chu, R., Lee, D., Kluckman, K.D., Usuda, N., Singh, I., Yeldandi, A.V., Rao, M.S., Maeda, N., and Reddy, J.K. (1996) Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene. J. Biol. Chem., 271, 24698–24710.[Abstract/Free Full Text]

Freneaux, E., Labbe, G., Letteron, P., The, L.D., Degott, C., Geneve, J., Larrey, D., and Pessayre, D. (1988) Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic. Hepatology, 8, 1056–1062.[ISI][Medline]

Fukumori, F., Takeuchi, N., Hagiwara, T., Ito, K., Kochibe, N., Kobata, A., and Nagata, Y. (1989) Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia. FEBS Lett., 250, 153–156.[ISI][Medline]

Gorin, E., Gonen, H., and Dickbuch, S. (1982) A serum protein inhibitor of acid lipase and its possible role in lipid accumulation in cultured fibroblasts. Biochem. J., 204, 221–227.[ISI][Medline]

Hashimoto, T., Fujita, T., Usuda, N., Cook, W., Qi, C., Peters, J.M., Gonzalez, F.J., Yeldandi, A.V., Rao, M.S., and Reddy, J.K. (1999) Peroxisomal and mitochondrial fatty acid beta-oxidation in mice nullizygous for both peroxisome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA oxidase. Genotype correlation with fatty liver phenotype. J. Biol. Chem., 274, 19228–19236.[Abstract/Free Full Text]

Hautekeete, M.L., Degott, C., and Benhamou, J.P. (1990) Microvesicular steatosis of the liver. Acta. Clin. Belg., 45, 311–326.[ISI][Medline]

Hayashi, H., Winship, DH., and Sternlieb, I. (1977) Lipolysosomes in human liver: distribution in livers with fatty infiltration. Gastroenterology, 73, 651–654.[ISI][Medline]

Hayashi, H., Sameshima, Y., Lee, M., Hotta, Y., and Kosaka, T. (1983) Lipolysosomes in human hepatocytes: their increase in number associated with serum level of cholesterol in chronic liver diseases. Hepatology, 3, 221–225.[ISI][Medline]

Hutchinson, W.L., Du, M.Q., Johnson, P.J., and Williams, R. (1991) Fucosyltransferases: differential plasma and tissue alterations in hepatocellular carcinoma and cirrhosis. Hepatology, 13, 683–688.[ISI][Medline]

Ibdah, J.A., Bennett, M.J., Rinaldo, P., Zhao, Y., Gibson, B., Sims, H.F., and Strauss, A.W. (1999) A fetal fatty-acid oxidation disorder as a cause of liver disease in pregnant women. N. Engl. J. Med., 340, 1723–1731.[Abstract/Free Full Text]

Ihara, Y., Yoshimura, M., Miyoshi, E., Nishikawa, A., Sultan, A.S., Toyosawa, S., Ohnishi, A., Suzuki, M., Yamamura, K., Ijuhin, N., and Taniguchi, N. (1998) Ectopic expression of N-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid storage. Proc. Natl Acad. Sci. USA, 95, 2526–2530.[Abstract/Free Full Text]

Ishii, I., Kimuro, T., Saito, Y., and Hirose, S. (1995) Cholesterol metabolism in monocyte-derived macrophages from macrophage colony-stimulating factor administered rabbits. Biochim. Biophys. Acta, 1254, 51–55.[ISI][Medline]

Jung, K.C., Myong, N.H., Chi, J.G., Choi, H.R., Lee, H.S., and Ahn, Y.M.(1993) Leigh’s disease involving multiple organs. J. Korean Med. Sci., 8, 214–220.[Medline]

Kolata, G.B. (1980) Reye’s syndrome: a medical mystery. Science, 207, 1453–1454.[Medline]

Kubo, M., Matsuzawa, Y., Yokoyama, S., Tajima, S., Ishikawa, K., Yamamoto, A., and Tarui, T. (1981) Apo A-I and apo A-II inhibit hepatic triglyceraide lipase from human postheparin plasma. Biochem. Biophys. Res. Commun., 106, 261–266.

Lazarow, P.B. (1981) Assay of peroximal ß-oxidation of fatty acids. Methods Enzymol., 72, 315–319.[Medline]

Lough, J., Fawcett, J., and Wiegensberg, B. (1970) Wolman’s disease. An electron microscopic, histochemical, and biochemical study. Arch. Pathol., 89, 103–110.[ISI][Medline]

Lovell, K.L., Kranich, R.J., and Cavanagh, K.T. (1994) Biochemical and histochemical analysis of lysosomal enzyme activities in caprine beta-mannosidosis. Mol. Chem. Neuropathol., 21, 61–74.[ISI][Medline]

Lusa, S., Tanhuanpaa, K., Ezra, T., and Somerharju, P. (1998) Direct observation of lipoprotein cholesterol ester degradation in lysosomes. Biochem. J., 332, 451–457.[ISI][Medline]

Merkel, M., Tilkorn, A.C., Greten, H., and Ameis, D. (1999) Lysosomal acid lipase. Assay and purification. Methods Mol. Biol., 109, 95–107.[Medline]

Miyagawa, J., Kuwajima, M., Hanafusa, T., Ozaki, K., Fujimura, H., Ono, A., Uenaka, R., Narama, I., Oue, T., Yamamoto, K., and others. (1995) Mitochondrial abnormalities of muscle tissue in mice with juvenile visceral steatosis associated with systemic carnitine deficiency. Virchows Arch., 426, 271–279.[ISI][Medline]

Miyoshi, E., Uozumi, N., Noda, K., Hayashi, N., Hori, M., and Taniguchi, N. (1997) Expression of alpha1-6 fucosyltransferase in rat tissues and human cancer cell lines. Int. J. Cancer, 72, 1117–1121.[ISI][Medline]

Nagayoshi, A., Matsuki, N., Saito, H., Tsukamoto, K., Kaneko, K., Wakashima, M., Kinoshita, M., Yamanaka, M., and Teramoto, T. (1995) Defect in assembly process of very-low-density lipoprotein in suncus liver: an animal model of fatty liver. J. Biochem., 117, 787–793.[Abstract]

Nakagawa, H., Matsubara, S., Kuriyama, M., Yoshidome, H., Fujiyama, J., Yoshida, H., and Osame, M. (1995) Cloning of rat lysosomal acid lipase cDNA and identification of the mutation in the rat model of Wolman’s disease. J. Lipid Res., 36, 2212–2218.[Abstract]

Nitta, Y., Tashiro, F., Tokui, M., Shimada, A., Takei, I., Tabayashi, K., and Miyazaki, J. (1998) Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Hum. Gene Ther., 9, 1701–1707.[ISI][Medline]

Otto, D.A. and Ontko, J.A. (1978) Activation of mitochondrial fatty acid oxidation by calcium. Conversion to the energized state. J. Biol. Chem., 253, 789–799.[ISI][Medline]

Pagani, F., Pariyarath, R., Garcia, R., Stuani, C., Burlina, A.B., Ruotolo, G., Rabusin, M., and Baralle, F.E. (1998) New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease. J. Lipid Res., 39, 1382–1388.[Abstract/Free Full Text]

Pariyarath, R., Pagani, F., Stuani, C., Garcia, R., and Baralle, F.E. (1996) L273S missense substitution in human lysosomal acid lipase creates a new N- glycosylation site. FEBS Lett., 397, 79–82.[ISI][Medline]

Prasad, V.V. and Pullarkat, R.K. (1996) Brain lysosomal hydrolases in neuronal ceroid-lipofuscinoses. Mol. Chem. Neuropathol., 29, 169–179.[ISI][Medline]

Prior, C., Fuchs, D., Hausen, A., Judmaier, G., Reibnegger, G., Werner, E.R., Vogel, W., and Wachter, H. (1987) Potential of urinary neopterin excretion in differentiating chronic non-A, non-B hepatitis from fatty liver. Lancet, 2(8570), 1235–1237.

Reue, K. and Doolittle, M.H. (1996) Naturally occurring mutations in mice affecting lipid transport and metabolism. J. Lipid Res., 37, 1387–1405.[Abstract]

Rolfes, D.B. and Ishak, K.G. (1986) Liver disease in pregnancy. Histopathology, 10, 555–570.[ISI][Medline]

Sando, G.N. and Rosenbaum, L.M. (1985) Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture. J. Biol. Chem., 260, 15186–15193.[Abstract/Free Full Text]

Sato, Y., Nakata, K., Kato, Y., Shima, M., Ishii, N., Koji, T., Taketa, K.,Endo, Y., and Nagataki, S. (1993) Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein. N. Engl. J. Med., 328, 1802–1806.[Abstract/Free Full Text]

Shapiro, D.J., Imblum, R.L., and Rodwell, V.W. (1969) Thin-layer chromatographic assay for HMG-CoA reductase and mevalonic acid. Anal. Biochem., 31, 383–390.[ISI][Medline]

Sheriff, S., Du, H., and Grabowski, G.A. (1995) Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression. J. Biol. Chem., 270, 27766–27772.[Abstract/Free Full Text]

Shimano, H., Horton, J.D., Hammer, R.E., Shimomura, I., Brown, M.S., and Goldstein, J.L. (1996) Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J. Clin. Invest., 98, 1575–1584.[Abstract/Free Full Text]

Sims, H.F., Brackett, J.C., Powell, C.K., Treem, W.R., Hale, D.E., Bennett, M.J., Gibson, B., Shapiro, S., and Strauss, A.W. (1995) The molecular basis of pediatric long chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with maternal acute fatty liver of pregnancy. Proc. Natl Acad. Sci. USA, 92, 841–845.[Abstract]

Slater, D.N. and Hague, W.M. (1984) Renal morphological changes in idiopathic acute fatty liver of pregnancy. Histopathology, 8, 567–581.[ISI][Medline]

Sloan, H.R. and Frederickson, D.S. (1972) Enzyme deficiency in choleteryl ester storage disease. J. Clin. Invest., 51, 1923–1926.[ISI][Medline]

Srikrishna, G., Varki, N.M., Newell, P.C., Varki, A., and Freeze, H.H.(1997) An IgG monoclonal antibody against Dictyostelium discoideumglycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates inhuman tissues. J. Biol. Chem., 272, 25743–25752[Abstract/Free Full Text]

Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., Koji, T., Taga, H., Satomura, S., Matsuura, S., Kawai, T., and Hirai, H. (1993) A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res., 53, 5419–5423.[Abstract]

Uozumi, N., Teshima, T., Yamamoto, T., Nishikawa, A., Gao, Y.E., Miyoshi, E., Gao, C.X., Noda, K., Islam, K.N., Ihara, Y., and others. (1996) A fluorescent assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6 fucosyltransferase activity, involving high performance liquid chromatography. J. Biochem., 120, 385–392.[Abstract]

Uozumi, N., Yanagidani, S., Miyoshi, E., Ihara, Y., Sakuma, T., Gao, C.X., Teshima, T., Fujii, S., Shiba, T., and Taniguchi, N. (1996) Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1->6 fucosyltransferase. J. Biol. Chem., 271, 27810–27817.[Abstract/Free Full Text]

Weber, F.L. Jr., Snodgrass, P.J., Powell, D.E., Rao, P., Huffman, S.L., and Brady, P.G. (1979) Abnormalities of hepatic mitochondrial urea-cycle enzyme activities and hepatic ultrastructure in acute fatty liver of pregnancy. J. Lab. Clin. Med., 94, 27–41.[ISI][Medline]

Wenk, R.E., Gebhardt, F.C., Bhagavan, B.S., Lustgarten, J.A., and McCarthy, E.F. (1981) Tetracycline-associated fatty liver of pregnancy, including possible pregnancy risk after chronic dermatologic use of tetracycline. J. Reprod. Med., 26, 135–141.[ISI][Medline]

Wetterau, J.R., Aggerbeck, L.P., Bouma, M.E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D.J., and Gregg, R.E. (1992) Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia. Science, 258, 999–1001.[ISI][Medline]

Yanagidani, S., Uozumi, N., Ihara, Y., Miyoshi, E., Yamaguchi, N., and Taniguchi, N. (1997) Purification and cDNA cloning of GDP-L-Fuc:N-acetyl- beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT) from human gastric cancer MKN45 cells. J. Biochem., 121, 626–632.[Abstract]

Yoshida, H. and Kuriyama, M. (1990) Genetic lipid storage disease with lysosomal acid lipase deficiency in rats. Lab. Anim. Sci., 40, 486–489.[Medline]