Glycobiology vol. 11 no. 2 pp. 165–174, 2001
© 2001 Oxford University Press
Ectopic expression of α1,6 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
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, and7Division 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
The α1,6 fucosyltransferase (α1,6 FucT) catalyzes the
transfer of a fucose from GDP-fucose to the innermost
GlcNAc residue of N-linked glycans via an α1,6 linkage.
α1,6 FucT was overexpressed in transgenic mice under the
control of a combined cytomegalovirus and chicken β-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 lyso-
somes. 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 trans-
genic and nontransgenic mice examined, the activity of
liver LAL was negatively correlated with the level of α1,6
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 lyso-
somes. 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 α1,6 FucT transgenic mice.
Key words: carbohydrate function/fucosyltransferase/
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, 1995). N-glycans
have a common core structure, and their branching patterns are
determined by glycosyltransferases, such as N-acetylglucos-
aminyltransferases and fucosyltransferases.
ferase (α1,6 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, α1,6
fucosylated oligosaccharides, are widely distributed in
mammalian tissues. It is generally believedthatα1,6fucosylation
plays an important role in fetal development (Bakkers et al.,
1997). Under some pathological conditions, the expression of
α1,6 FucT and the extent of fucosylation are altered. For
example, the level of α1,6 FucT is elevated in both liver and
serum during the process of hepatocarcinogenesis (Hutchinson
et al., 1991). The presence of fucosylated α-fetoprotein is a
good marker for distinguishing patients with hepatocarcinoma
from those with chronic hepatitis and liver cirrhosis (Sato et al.,
1993; Taketa et al., 1993).
Followed by the development of convenient assay method
for the enzyme activity (Uozumi et al., 1996), α1,6 FucT was
homogeneously purified, and its cDNA was cloned from
porcine brain and human gastric tumor cells in our laboratory
(Uozumi et al., 1996; Yanagidani et al., 1997). The α1,6 FucT
gene was found to be expressed in most rat organs (Miyoshi et
al., 1997). 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 α1,6 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-acetyl-
glucosaminyltransferase III (GnT III) genes, in an attempt to
elucidate thebiological roles of the bisectingGlcNAc inN-linked
glycans (Ihara et al., 1998). 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 α1,6 FucT gene, in an attempt to study the biological
roles of the core α1,6 fucose residue in N-linked glycans. The
α1,6 FucT transgenic mice showed a unique phenotype of
1These authors contributed equally to this work.
2To whom correspondence should be addressed
W. Wang et al.
Expression of human α1,6 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, α1,6 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.
mg) in the case of the transgenic mice, compared to the normal
mice (100–200 pmol/h/mg). In contrast, α1,6 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 α1,6 FucT transgenic mice
To determine the manner in which the glycans of proteins are
fucosylated in the α1,6 FucT transgenic mice, a liver extract
was subjected to aleuria aurantia lectin (AAL) blot analysis to
detect the α1,6 fucose in N-linked glycans (Fukumori et al.,
1989). 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 α1,6 fucose structures
were detected (Figure 2B). Immunoblot with a monoclonal
antibody CAB4, which recognizes the α1,6 fucose structure in
the coreof N-glycans (Srikrishna et al.,1997),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 α1,6 FucT transgenic mice compared to
the control mice (data not shown). These observations indicate
that the introduced α1,6 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.
Histological changes in α1,6 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 trans-
genic 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 α1,6
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 wereoftenfilledwith 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
Fig. 1. Expression of human α1,6 FucT in the liver of the transgenic mice.
(A) Northernblot ofthelivers ofwildmice (control)andα1,6FucT-transgenic
mice (FucT-1 and FucT-2). Twenty micrograms of total RNA from the livers
was electrophoresed, blotted, and probed with cDNA of human α1,6 FucT.
(B) Western blot of liver proteins with a mouse monoclonal antibody 15C6,
which recognizes human α1,6 FucT. (C) α1,6 FucT activity levels in the livers
of the transgenic mice. All data were reproducible.
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.
Expression of α1,6 fucosyltransferase in mice
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 baso-
lateral 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-
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 metab-
olism of these entities (Figure 6B). No apparent ultrastructural
changes were detected in the glomeruli or the other part of
Serum lipid level in α1,6 FucT transgenic mice
As a reason for liver steatosis, dysfunction of secretion is a
possibility. If this is the case, abnormalities in serum lipo-
proteins would be expected. Actually, GnT III transgenic mice
developed a fatty liver as the result of aberrant apolipoprotein
B secretion (Ihara et al., 1998). 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
Fig. 3. Microscopic observation of the livers of the transgenic mice. (A) and
(C), normal mice; (B) and (D), α1,6 FucT transgenic mice. (A) and (B),
paraffin section, hematoxylin/eosin staining; (C) and (D), frozen section, Oil
red O staining (original magnification: 200×).
Fig. 4. Microscopic observation of the kidneys of the transgenic mice. (A) and (C), normal mice; (B) and (D), α1,6 FucT transgenic mice. (A) and (B), paraffin
section, hematoxylin/eosin staining; (C) and (D), frozen section, Sudan III stainin. (original magnification: 400×).
W. Wang et al.
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 find-
ings suggest that no abnormality in serum lipoproteins exists in
the transgenic mice.
Accumulated lipids in α1,6 FucT transgenic mice
To determine which step is damagedinthe 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). Further-
more, thin-layer chromatography revealed that the amount of
triglyceride and cholesterol ester increased significantly in the
transgenic mice,comparedwiththeirwildlittermates(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).
Fig. 5. Electron microscopic photographs of hepatocytes of a control and α1,6 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: 4700×, bar = 4 µm). (B) In a hepatocyte of α1,6 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: 4700×, 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: 20,000×, 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: 77,000×, bar = 200 nm).
Table I. Lipid levels in the livers of normal control and α1,6 FucT transgenic
*Expressed as content per gram of wet liver.
Free Fatty Acid
5.10 ± 0.72
7.65 ± 1.19
Control6 27.77 ± 3.19
35.09 ± 4.54
3.40 ± 0.52
4.02 ± 0.39
p = 0.009 p = 0.042p = 0.0012
Expression of α1,6 fucosyltransferase in mice
Expression of lipid-metabolizing proteins in α1,6 FucT
In an attempt to determine the mechanism of lipid accumulation,
we examined the activities of microsomal triglyceride transfer
protein (Wetterau et al., 1992), cholesterol and free fatty acid
synthesis (Shapiro et al., 1969), lysosomal acid lipase (LAL,
EC 126.96.36.199) (Ishii et al., 1995; Merkel et al., 1999), and both
mitochondrial and peroxisomal β-oxidation (Otto and Ontko,
1978; Lazarow, 1981) 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 α1,6 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 α1,6 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).
Fig. 6. Electron microscopic photographs ofthe proximal renal tubularcells of acontrol and α1,6 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: 4200×, bar = 4 µm).
(B) In proximal tubular cells of α1,6 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: 4200×, bar = 4 µm).
Table II. Lipid-metabolizing protein activities in livers of normal control and
α1,6 FucT transgenic mice
*Microsomal triglyceride transfer protein (MTP) activity is expressed as
percent triglyceride transferred per h.
Micen LAL Activity
Control 11365.5 ± 43.9
243.3 ± 56.1
7.8 ± 0.8
7.0 ± 0.7
76.5 ± 3.3
74.9 ± 5.5
p < 0.01
Fig. 7. High-performance thin-layer chromatography of liverlipids. 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.
W. Wang et al.
Effects on specific activity and fucosylation of lysosomal acid
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 α-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.
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 α1,6 FucT and prompted us to investigate whether
the N-glycan attached to LAL is more highly fucosylatedin the
transgenic mice. Lectin and immunoblot analysis of LAL
using AAL lectin and CAB4 monoclonal antibody, which
recognize the α1,6 fucose residue in the core of N-glycans,
revealed that LAL in normal mouse liver is slightly fucosylated
but in the α1,6 FucT transgenic mice it is even more fuco-
sylated (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 α1,6 FucT in transgenic mice.
The apparent Kmvalues 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
The overexpression of α1,6 FucT in mice caused the accumu-
lation 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 α1,6 FucT transgenic mice may be classified as
a form of microvesicular steatosis (Hautekeete et al., 1990).
There are many diseases that show a fatty liver with this
feature, such as an acute fatty liver during pregnancy (Rolfes
and Ishak, 1986; Sims et al., 1995; Ibdah et al., 1999), Reye’s
syndrome (Kolata, 1980), tetracycline toxicity (Wenk et al.,
1981; Freneaux et al., 1988), defects in urea cycle enzymes
(Weber et al., 1979) and mitochondrial fatty acid oxidation
(Hautekeete et al., 1990), Wolman’s disease (Anderson et al.,
1994; Pagani et al., 1998), cholesteryl ester storage disease
(CESD) (Sloan and Frederickson, 1972; Pagani et al., 1998),
and some varieties of viral hepatitis (Prior et al., 1987),
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
maybe causedbychemical 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., 1995).
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., 1998).
Unlike the GnT III transgenic mice, no significant changes in
serum lipids in α1,6 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 α1,6 FucT
of cholesterol ester andtriglyceride, suggest that the hydrolysis
Fig. 8. Lectin and immunoblot results for LAL. (A) Immunoblot of LAL in
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
Fig. 9. Correlation of LAL and α1,6 FucT activities in the livers of normal
(open circles) and α1,6 FucT transgenic (closed circles) mice. Correlation
coefficient = 0.59 (p < 0.01).
Expression of α1,6 fucosyltransferase in mice
of lipid esters in the lysosomes is abrogated in the transgenic
Lysosomes are important organelles involved in lipid
metabolism (Lusa et al., 1998). 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., 1970). Lyso-
somes with accumulated lipids, called lipolysosomes, are
regarded as a specific feature of Wolman’s disease (Lough et
al., 1970). Lipolysosomes can be occasionally found in some
other liver disorders (Hayashi et al., 1977), 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.,
1983). Wolman’s disease is an autosomal recessive disorder
with an inherited deficiency of LAL (Anderson et al., 1994;
Pagani et al., 1998). LAL catalyzes the hydrolysis of cholesterol
ester and triglycerides in the lysosomes. In the case of the α1,6
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
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, 1985; Ameis et al., 1994). Others have concluded
that glycosylation is important by showing that the activity is
reduced, as the result of the same treatment (Pariyarath et al.,
1996) 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., 1995). 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 α1,6 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 α1,6 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,
1990; Nakagawa et al., 1995). One is Wolman’s disease and
the other is designated CESD, in which only cholesteryl esters
are stored (Sloan and Frederickson, 1972; Pagani et al., 1998).
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., 1994; Pagani et al.,
1998). The LAL-knockout mice share many features of
Wolman’s disease but have a milder phenotype and are fertile,
althoughthey undergomassivecholesterol esterandtriglyceride
storage with complete loss of LAL activity (Du et al., 1998).
We also found lipid accumulation within lysosomes in the
proximal renal tubular cells of our α1,6 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 accumu-
lation. According to the few available reports on the ultra-
structure 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, 1984;Junget al.,1993).Inseveral experimental animal
models ofliversteatosis (Fan et al., 1996; Shimano et al., 1996;
Reue and Doolittle, 1996; Hashimoto et al., 1999) no accumu-
lation 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 lyso-
somes. 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., 1981; Gorin et al., 1982), and a physiological
detergent, such as saposins (Bierfreund et al., 2000), 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 α1,6 FucT
transgenic mice. Alternatively, the accumulated inactive LAL
mayhavea dominant negative effect onthe 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 α1,6 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
Human α1,6 FucT cDNA containing the entire open reading
frames (Yanagidani et al., 1997) 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 β-actin promoters (Nitta et al.,
1998). The DNA fragment that was cut out with Sal I and
BamH I, containing the promoter and α1,6 FucT cDNA
regions, was used for microinjection into fertilized eggs of a
DBF1 mouse strain.
DNA was extracted from tails of mice developed from the
above-mentioned fertilized eggs and analyzed by Southern
W. Wang et al.
blotting for the incorporation of human α1,6 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-α1,6
FucT cDNA. Two mouse lines with high levels of expression
of α1,6FucT wereestablished. 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., 1996). The
CAB4 monoclonal antibody that recognizes the α1,6 fucose
residue in the core of N-linked glycans was kindly provided by
Dr. Freeze (Srikrishna et al., 1997). A monoclonal antibody
15C6 against human α1,6 FucT was obtained from Fujirebio
Inc. (Japan). Goat polyclonal anti-human cathepsin D anti-
bodies 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., 1997; Ihara et al.,
α1,6 FucT activity assay
α1,6FucT activity was assayed by the method of Uozumi et al.
(1996). 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 α1,6 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
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 homo-
genized 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 Amer-
sham, 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 extractedwith10 vol ofchloroform/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 × 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.
Fresh tissues were fixed in a 10% formaldehyde in 0.1 M phos-
phate buffer (pH 7.4). Paraffin sections and frozen sections
were prepared for hematoxylin-eosin staining andfor 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., 1995).
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 ultra-
microtome, 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., 1995; Merkel et al., 1999) 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 lysole-
cithin 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, andthe 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
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 × 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
Expression of α1,6 fucosyltransferase in mice
released by 1 mg of protein per h. α-Fucosidase activity was
assayed as described previously (Lovell et al., 1994; Prasad
and Pullarkat, 1996).
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.
α1,6 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.
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.
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.
Bakkers, J., Semino, C.E., Stroband, H., Kijne, J.W., Robbins, P.W., and
Spaink, H.P. (1997) An important developmental role for oligosaccha-
rides during early embryogenesis of cyprinid fish. Proc. Natl Acad. Sci.
USA, 94, 7982–7986.
Bierfreund, U., Kolter, T., Sandhoff, K. (2000) Sphingolipid hydrolases and
activator proteins. Methods Enzymol., 311, 255–276.
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.
Du, H., Duanmu, M., Witte, D., and Grabowski, G.A. (1998) Targeted-
disruption of the mouse lysosomal acid lipase gene: long-term survival
with massive cholesteryl ester and triglyceride storage. Hum. Mol. Genet.,
Dwek, R.A. (1995) Glycobiology: more functions for oligosaccharides.
Science, 269, 1234–1235.
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) Hepato-
cellular and hepatic peroxisomal alterations in mice with a disrupted
peroxisomal fatty acyl-coenzyme A oxidase gene. J. Biol. Chem., 271,
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–
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.
Gorin, E., Gonen, H., and Dickbuch, S. (1982) A serum protein inhibitor of
acid lipase and its possible role in lipid accumulation in cultured fibro-
blasts. Biochem. J., 204, 221–227.
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 perox-
isome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA
oxidase. Genotype correlation with fatty liver phenotype. J. Biol. Chem.,
Hautekeete, M.L., Degott, C., and Benhamou, J.P. (1990) Microvesicular
steatosis of the liver. Acta. Clin. Belg., 45, 311–326.
Hayashi, H., Winship, DH., and Sternlieb, I. (1977) Lipolysosomes in human
liver: distribution in livers with fatty infiltration. Gastroenterology, 73,
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,
Hutchinson, W.L., Du, M.Q., Johnson, P.J., and Williams, R. (1991) Fucosyl-
transferases: differential plasma and tissue alterations in hepatocellular
carcinoma and cirrhosis. Hepatology, 13, 683–688.
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.
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-acetylglucosaminyl-
transferase III in transgenic hepatocytes disrupts apolipoprotein B
secretion and induces aberrant cellular morphology with lipid storage.
Proc. Natl Acad. Sci. USA, 95, 2526–2530.
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.
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.
Kolata, G.B. (1980) Reye’s syndrome: a medical mystery. Science, 207, 1453–
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 β-oxidation of fatty acids. Methods
Enzymol., 72, 315–319.
Lough, J., Fawcett, J., and Wiegensberg, B. (1970) Wolman’s disease. An
electron microscopic, histochemical, and biochemical study. Arch.
Pathol., 89, 103–110.
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.
Lusa, S., Tanhuanpaa, K., Ezra, T., and Somerharju, P. (1998) Direct observation
of lipoprotein cholesterol ester degradation in lysosomes. Biochem. J.,
Merkel, M., Tilkorn, A.C., Greten, H., and Ameis, D. (1999) Lysosomal acid
lipase. Assay and purification. Methods Mol. Biol., 109, 95–107.
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.
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.
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.
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.
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.
Otto, D.A. and Ontko, J.A. (1978) Activation of mitochondrial fatty acid
oxidation by calcium. Conversion to the energized state. J. Biol. Chem.,
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.
W. Wang et al.
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.
Prasad, V.V. and Pullarkat, R.K. (1996) Brain lysosomal hydrolases in neuro-
nal ceroid-lipofuscinoses. Mol. Chem. Neuropathol., 29, 169–179.
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,
Reue, K. and Doolittle, M.H. (1996) Naturally occurring mutations in mice
affecting lipid transport and metabolism. J. Lipid Res., 37, 1387–1405.
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.
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.
Shapiro, D.J., Imblum, R.L., and Rodwell, V.W. (1969) Thin-layer chromato-
graphic assay for HMG-CoA reductase and mevalonic acid. Anal.
Biochem., 31, 383–390.
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.
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.
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 associ-
ated with maternal acute fatty liver of pregnancy. Proc. Natl Acad. Sci.
USA, 92, 841–845.
Slater, D.N. and Hague, W.M. (1984) Renal morphological changes in
idiopathic acute fatty liver of pregnancy. Histopathology, 8, 567–581.
Sloan, H.R. and Frederickson, D.S. (1972) Enzyme deficiency in choleteryl
ester storage disease. J. Clin. Invest., 51, 1923–1926.
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
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.
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 fluores-
cent assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha
1-6 fucosyltransferase activity, involving high performance liquid chroma-
tography. J. Biochem., 120, 385–392.
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.
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.
(1981) Tetracycline-associated fatty liver of pregnancy, including possible
pregnancy risk after chronic dermatologic use of tetracycline. J. Reprod.
Med., 26, 135–141.
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.
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.
Yoshida, H. and Kuriyama, M. (1990) Genetic lipid storage disease with lyso-
somal acid lipase deficiency in rats. Lab. Anim. Sci., 40, 486–489.