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ABSTRACT Japanese eating
habits are characterized
by the consumption of
various food materials
such as
cereals, vegetables,
fish, shellfish, marine
algae and meat.
Therefore, properties of
functional substances in
food
materials may be
enhanced or lessened by
the combination of
various food materials.
In the present study, we
examined how the
combination of wakame
and fish containing
polyunsaturated fatty
acids, which are typical
Japanese food materials,
affected rat lipid
metabolism. Rats were
fed one of four diets
[control diet (C),
AIN-76 diet
with 5 g/100 g rapeseed
oil; wakame diet (W)
containing 19.1 g/100 g
Undaria pinnatifida
(wakame) dried powder
in the C diet; fish oil
diet (FO), AIN-76 diet
with 4.1 g/100 g fish
oil; wakame-fish oil
diet (W FO), the FO diet
containing 19.1 g/100 g
dried wakame powder] for
4 wk. We measured the
concentration of lipids
in serum and liver
and hepatic activities
of enzymes involved in
fatty acid metabolism.
The W diet, FO diet and
W FO diet
significantly reduced
the concentration of
triacylglycerols in the
serum and liver compared
with the C diet. This
decrease in the
concentration of hepatic
triacylglycerol was
greatest in rats fed the
W FO diet. The activity
of
glucose-6-phosphate
dehydrogenase, which is
involved in fatty acid
synthesis in the liver,
of rats fed the W, FO
and
W FO diets was lower
than that in rats fed
the C diet. However, the
activities of malic
enzyme and fatty acid
synthetase did not
differ among the four
groups. In contrast, the
W diet and W FO diet
increased the serum
concentration of
-hydroxybutyrate.
Further, the activity of
3-hydroxyacyl-CoA
dehydrogenase, which is
involved
in fatty acid -oxidation
in the liver, was
greater in rats fed the
W diet (42%), the FO
diet (154%) and the W FO
diet (381%) than in
those fed the C diet.
Because the decrease in
the concentration of
triacylglycerol in the
liver was
greatest when rats were
fed wakame and fish oil
at the same time (W FO
diet), we conclude that
there was a
synergistic process
affecting fatty acid
-oxidation in the liver.
These results suggest
that the simultaneous
consumption of fish
(fish oil) and wakame
decreases the
concentration of
triacylglycerol in the
serum and liver.
J. Nutr. 132: 742–747,
2002.
KEY WORDS: ● Undaria
pinnatifida (wakame) ●
fish oil ●
3-hydroxyacyl-CoA
dehydrogenase ● fatty
acid
● -oxidation ● rats
Undaria pinnatifida
(wakame), a brown
seaweed that contains
several minerals,
vitamins and dietary
fiber, is a constituent
of traditional Japanese
cuisine. Recently, it
has been
reported that wakame
contains several
elements with biological
activities, such as
coagulation protection
in human blood
and antitumor and
antimutagenic activities
(1–3). Further, we
reported that dietary
wakame decreased the
concentration of
triacylglycerol in rat
serum and liver as a
result of increased
activities of hepatic
enzymes involved in
fatty acid oxidation.
We concluded that wakame
is a useful food
material for the
prevention and treatment
of
hypertriacylglycerolemia
(4).
It is possible that the
changes in the food
habits of Japanese
people, which had been
characterized by the
consumption of
rice, vegetables and
marine products, to a
more European and
American type of diet,
characterized by the
consumption of
meat and dairy products,
have led to an increase
in the
incidence rates of
various diet-related
adult diseases.
Traditional
Japanese eating habits
are characterized by the
consumption
of various food
materials such as
cereals, vegetables,
fish, shellfish and
marine algae, which are
cooked by various
techniques such as
steaming, roasting,
boiling and frying.
Therefore, properties of
the functional
substances in food
materials may be
enhanced or lessened by
the combination of
various food materials,
and the chemical changes
that occur
during the processing
and the cooking steps.
Therefore, it is
necessary to analyze
scientifically the
influence of recent
Japanese
eating habits on health.
Many studies have
indicated that fish oil
rich in eicosapentaenoic
acid (EPA)3 and
docosahexaenoic acid
(DHA) decreases
the triacylglycerol
concentrations in the
serum and liver as a
result of the promotion
of hepatic fatty acid
-oxidation
(5–8).
The typical Japanese
breakfast is often
composed of rice,
baked fish and miso soup
with wakame. It is not
known
whether the
triacylglycerol
metabolism is changed by
consumption
of a diet containing
both wakame and fish oil
at the
same time. To address
this, we fed rats diets
containing
wakame and fish oil and
examined in rats the
influence of
these diets on the
concentration of lipids
in the serum and
liver, and the
activities of fatty acid
metabolizing enzymes in
liver.
MATERIALS AND METHODS
Materials. The dried
wakame powder was
obtained from Riken
Vitamin (Tokyo, Japan),
and fish oil was
obtained from Nippon
Chemical Feed (Hokkaido,
Japan). The constituents
(g/100 g) of
dried wakame powder were
17.2 g protein, 3.7 g
lipid, 40.6 g
carbohydrate,
3.1 g fiber and 5.0 g
minerals (9). The fatty
acid composition
(g/100 g total fatty
acid) of fish oil
determined by gas-liquid
chromatography
(10) was as follows:
16:0, 10.3; 16:1, 8.1;
18:0, 2.6; 18:1,
11.4; 18:2, 1.3;
18:3(n-3), 1.0;
18:4(n-3), 5.1; 20:4,
1.8; 20:5(n-3),
34.2; and 22:6(n-3),
17.3. Palmitoyl-CoA
(16:0-CoA), oleoyl-CoA
(18:1-CoA) and
arachidonoyl-CoA
(20:4-CoA) were prepared
according
to the method of
Kawaguchi et al. (11).
Acetyl-CoA, acetoacetyl-
CoA and malonyl-CoA were
purchased from the Sigma
Chemical (St. Louis,
MO).
Animal and diets. Male
Sprague-Dawley rats (n
28) obtained
from the Charles River
(Kanagawa, Japan) were
kept in an
airconditioned
room (temperature,
20–22°C; humidity,
55–65%; lights
on, 0700–1900 h), and
fed a commercial
nonpurified diet (Type
NMF; Oriental Yeast,
Tokyo, Japan) for 1 wk.
After acclimation to
the housing conditions,
rats were divided into
the following four
dietary groups: control
(C) diet; wakame (W)
diet; fish oil (FO)
diet;
wakame and fish oil (W
FO) diet. The
experimental diets were
prepared according to
the recommendations of
the American Institute
of Nutrition (AIN-76)
[Table 1 (12)]. All of
the diet ingredients
were products of
Oriental Yeast. Nutrient
contents were
standardized
by subtracting the
amounts of nutrients
included in dried wakame
powder from the basic
AIN-76 diet. To
standardize nutrient
contents
among the diets, we
replaced 5 g/100 g diet
of protein in the AIN-76
diet with protein of
dried wakame powder in
the W and W FO
diets. Therefore, dried
wakame powder was added
to these diets at a
level of 19.1 g/100 g
diet. In theWdiet andW
FO diet, 19.1 g/100
g diet of the dried
wakame powder in the
diet contained 0.9 g of
lipids. Therefore, we
adjusted the
concentration of
rapeseed oil and
fish oil to 4.1 g/100 g
diet in the W diet and W
FO diet. Further,
to adjust the fish oil
concentration in the W
FO diet and FO diet,
fish oil at 4.1 g/100 g
diet and rapeseed oil at
0.9 g/100 g diet were
added to the FO diet.
However, mineral
contents likely differed
among the diets because
wakame contains high
levels of various
minerals. In the present
study, we did not adjust
the mineral contents
because it was
impossible to completely
standardize the mineral
concentrations of all
diets.
The care and treatment
of the experimental
animals conformed to
the National Research
Institute of Fisheries
Science guidelines for
the
ethical treatment of
laboratory animals. Rats
were fed these
experimental
diets for 4 wk.
Enzyme assays. At the
end of the experiment,
rats were lightly
anesthetized with
diethyl ether, bled from
the abdominal aorta and
livers were quickly
excised. Liver ( 3 g)
from each rat fed the
experimental diets was
homogenized with 7
volumes of 0.25 mol/L
sucrose and centrifuged
at 500 g for 10 min. The
supernatant was
recentrifuged at 9000 g
for 10 min to isolate
the mitochondria. The
mitochondrial fraction
was washed twice with
0.25 mol/L sucrose
containing 1 mmol/L EDTA
and 3 mmol/L Tris-HCl
(pH7.0) and
finally suspended in the
same medium to give a
protein concentration
of 20–25 g/L.
Glucose-6-phosphate
dehydrogenase (EC
1.1.1.49)
(13), malic enzyme (EC
1.1.40) (14) and fatty
acid synthetase (15)
activities were measured
in the 9000 g
supernatant fraction of
the
liver homogenate (16).
The supernatant fraction
obtained after
centrifugation of the
liver
homogenate at 500 g for
10 min was used for
measurements of the
activities of the fatty
acid oxidation enzymes
except for carnitine
palmitoyltransferase (EC
2.3.1.21) and acyl-CoA
dehydrogenase (EC
1.3.99.3) (17).
Carnitine
palmitoyltransferase
activity was measured
in the isolated
mitochondria solubilized
with Triton X-100
according
to the method described
in Murata et al. (4).
Acyl-CoA dehydrogenase
activity was measured in
isolated mitochondria
according to the
method described by
Dommes and Kunau (18)
and Dommes et al.
(19) except that
phenazine methosulfate
was used as the primary
electron acceptor (4).
Acyl-CoA oxidase (EC
1.3.3.6) activity was
measured in the 500 g
supernatant fraction of
liver homogenates as
described elsewhere
(20,21). Palmitoyl-CoA
was used as a substrate
for the carnitine
palmitoyltransferase
assay and palmitoyl-CoA,
oleoyl-CoA, and
arachidoyl-CoA were used
as substrates for the
acyl-CoA dehydrogenase
and acyl-CoA oxidase
assays. Acetoacetyl-
CoA was used as a
substrate for
3-hydroxyacyl-CoA
dehydrogenase
(EC 1.1.1.35) (22) in
the 500 g supernatant
fraction of the liver
homogenate. The
activities of marker
enzymes for cell
organelles
including succinate
dehydrogenase (EC
1.3.99.1) (mitochondria)
(23) and catalase (EC
1.11.1.6) (peroxisomes)
(24) were determined
in the 500 g supernatant
fraction of liver
homogenates.
Liver carnitine was
determined in the
perchloric acid extracts
from the liver
homogenate as described
by Deufel and Wieland
(25).
Protein in fractions of
the liver homogenate
were determined by the
methods of Lowry et al.
(26)
Lipid analyses. The
lipids in the serum and
liver were extracted
and purified (27). The
concentrations of
triacylglycerol,
phospholipids
and cholesterol in the
extracts were determined
as described by
Hara et al. (28). The
fatty acid composition
of the fish oil was
determined using
gas-liquid
chromatography (10).
-Hydroxybutyrate
in the serum was
measured enzymatically
Statistical analyses.
All values are expressed
as the mean SEM.
Data were analyzed by
two-way ANOVA and
post-hoc Duncan’s
multiple range test
(30,31). Differences of
P 0.05 were considered
significant. The
analyses were performed
by the macro statistics
programs using Microsoft
Excel (Microsoft,
Redmond, WA).
RESULTS
The body weight of rats
was 141.7 8.9 g for the
28 rats at
the beginning of this
experiment. Throughout
the experiment,
food intake did not
differ among groups
(Table 2). Body
weight gain tended to be
less (P 0.067) in rats
fed the
wakame-supplemented
diets compared with rats
fed diets without
wakame. Moreover, rats
fed the
wakame-supplemented
diets had lower relative
liver and adipose tissue
around the
testis weights compared
with rats fed the C diet
(Table 2).
The serum
triacylglycerol
concentration was lower
in rats
fed the W, FO and W FO
diets than in rats fed
the C diet
(Table 3). Moreover, the
FO andW FO diets
decreased the
concentration of
triacylglycerol compared
with the W diet.
The W, FO and W FO diets
decreased the
concentration of
cholesterol in the serum
compared with the C
diet, but there
was no difference among
rats fed W, FO and W FO
diets.
The serum phospholipid
concentration was lower
in rats fed
the W, FO and W FO diets
than in rats fed the C
diet, and
that of rats fed W FO
diet was lowest among
these dietary
groups (Table 3).
The hepatic
triacylglycerol
concentrations of rats
fed the
W, FO and W FO diets
were significantly lower
than in rats
fed the C diet, with the
decrease greatest in
rats fed the W
FO diet (Table 3). The
hepatic cholesterol
concentrations
of rats fed the W and W
FO diets also were
significantly
lower than that in rats
fed the C diet. Liver
phospholipid
levels did not differ
among the four dietary
groups.
The serum
-hydroxybutyrate
concentrations of rats
fed the
W, FO andW FO diets were
greater than that in
rats fed the
C diet, with the
greatest increase in
rats fed the W FO diet
(Table 4).
There were no
differences in the
hepatic activities of
malic
enzyme and fatty acid
synthetase among the
four groups. The
hepatic activities of
glucose-6-phosphate
dehydrogenase in
rats fed the W, FO and W
FO diets were lower than
in rats
fed the C diet, and the
activities in rats fed
the FO diet andW
FO diet were lower than
in rats fed the W diet
(Table 4).
The activity of acyl-CoA
oxidase, which is the
rate-limiting
enzyme for fatty acid
-oxidation in liver
peroxisomes, was
significantly greater in
rats fed the W, FO and W
FO diets
compared with rats fed
the C diet using both
16:0-CoA
(saturated fatty
acid-CoA) and 18:1-CoA
(monosaturated
fatty acid-CoA) as
substrates (Table 4).
There was no differ-ence
in the activity of
acyl-CoA dehydrogenase,
which is the
rate-limiting enzyme for
fatty acid -oxidation in
liver mitochondria,
among the rats fed each
of the experimental
diets
using 16:0-CoA, 18:1-CoA
and 20:4-CoA as
substrates.
Another enzyme involved
in fatty acid -oxidation
in liver
mitochondria, carnitine
palmitoyltransferase,
which regulates
the rate of transport of
fatty acids across the
mitochondrial
membrane, had a greater
activity in rats fed the
FO and theW
FO diets than in rats
fed the W and the C
diets (Table 4).
The activity of
3-hydroxyacyl-CoA
dehydrogenase was
greater
in rats fed the W diet
(42%), the FO diet
(154%) and the W
FO diet (381%) than in
rats fed the C diet
(Table 4).
The hepatic carnitine
concentration, which is
the substrate
for carnitine
acyltransferase, was
greater in rats fed the
W and
W FO diets than in
controls, with the
increase greater in
rats fed the W FO diet
(Table 5).
The concentration of
protein in the liver 500
g supernatant
fraction and
mitochondrial fractions
did not differ
among the four dietary
groups. The specific
activities of succinate
dehydrogenase in the
liver 500 g supernatant
fraction
and catalase in the
liver mitochondrial
fraction also did not
differ (data not shown).
DISCUSSION
Until fairly recently,
fish, shellfish, algae
and vegetables
were the main materials
for food used in
Japanese cuisine.
However, the intakes of
animal foods such as
dairy products
and meats have increased
recently in Japan. Along
with this
change in eating habits,
the incidence rates of
diseases such as
diabetes,
arteriosclerosis,
coronary arteries heart
disease,
thrombosis and allergy
have increased. It is
possible that fish,
shellfish, algae and
vegetables, which are
the main food materials
of Japanese cuisine,
have preventative and
therapeutic effects
against such diseases.
To clarify how
traditional Japanese
eating habits contribute
to the health of the
Japanese, we
examined the influence
of diets composed of
marine food
materials, wakame and
fish oil, on rat lipid
metabolism.
In our previous study
(4), a diet that was
supplemented with
dried wakame powder at
10 g/100 g diet
significantly decreased
the concentration of
triacylglycerol in the
serum and liver of
rats compared with an
unsupplemented diet. In
this study, the
W diet containing 19.1
g/100 g dried wakame
powder also
significantly decreased
the concentration of
triacylglycerol in
the serum and liver
compared with the C
diet. The FO diet
containing EPA and DHA
significantly decreased
the concentration
of triacylglycerol in
the serum and liver, and
these data
were comparable to other
reports (32–34).
When wakame and fish oil
were fed at the same
time (W
FO diet), the serum
concentration of
triacylglycerol was
34% of that in rats fed
the C diet, whereas the
serum concentrations
of triacylglycerol in
rats fed the W and FO
diets were
56 and 41% of that in
rats fed the C diet,
respectively. In
addition, the hepatic
triacylglycerol
concentration in rats
fed
the W FO diet also
decreased to 13% of that
in rats fed the
C diet, whereas the W
and FO diets decreased
the concentration
of triacylglycerol in
the liver to 22 and 36%
of that in rats
fed the C diet,
respectively. Thus,
wakame and fish oil (W
FO diet) synergistically
affected the decrease of
triacylglycerol
in the liver.
Because the absorption
of lipids from the small
intestine
and/or the metabolism of
lipids and fatty acids
in the liver
control the
concentration of
triacylglycerol in the
serum and
liver, it is possible
that the W FO diet
modifies the rates of
synthesis and
degradation of fatty
acids in the liver.
Therefore,
to clarify the
mechanism(s) for the
synergistically effect
on the
decrease of
triacylglycerol in the
liver of rats fed the W
FO
diet, we compared the
influences of the W, FO
and W FO
diets on the activities
of various enzymes
involved in fatty acid
synthesis in the liver.
Although the activity of
glucose-6-phosphate
dehydrogenase
in rats fed the W, FO
and W FO diets decreased
to
50, 20 and 17% of that
in rats fed the C diet,
respectively,
there was no difference
in the activity of this
enzyme between
the FO diet group and
the W FO diet group.
Further, the
activities of fatty acid
synthetase and malic
enzyme of rats fed
the W, FO and W FO diets
were almost the same as
those
of rats fed the C diet.
Therefore, the
difference in the
concentration
of liver
triacylglycerols among
the four diets was
not due to differences
in hepatic fatty acid
synthesis. In contrast,
the W, FO and W FO diets
significantly increased
the
concentration of
-hydroxybutyrate in the
serum by 80, 50
and 100% relative to
that in rats fed the C
diet. Therefore, we
hypothesize that hepatic
fatty acid -oxidation in
rats fed the
W FO diet tended to be
increased; thus, we
measured the
activity of enzymes
involved in hepatic
fatty acid -oxidation
in rats fed the four
diets.
There were no
differences in the
activities of acyl-CoA
dehydrogenase and
acyl-CoA oxidase among
the four diet
groups, and the W FO
diet had no effect on
the specificity
of fatty acyl-CoA for
acyl-CoA oxidase and
acyl-CoA dehydrogenase.
However, the activity of
3-hydroxyacyl-CoA
dehydrogenase
in rats fed the W FO
diet was significantly
increased to 381% of
that in rats fed the C
diet compared with
42% in rats fed the W
diet and 154% in rats
fed the FO diet.
3-Hydroxyacyl-CoA
dehydrogenase catalyzes
the third step in
the mitochondrial fatty
acid -oxidation cycle,
and inhibition
of the activity of this
enzyme would lead to the
inhibition of
activity of 2-enoyl-CoA
accompanied by
accumulation of
3-oxoacyl-CoA esters in
the mitochondrion. The
3-oxoacyl-
CoA would inhibit fatty
acid -oxidation (35).
Further, a
deficiency in the
activity of
3-hydroxyacyl-CoA
dehydrogenase
in pregnant woman leads
to high levels of fat
incorporated
into the liver (36).
Thus, 3-hydroxyacyl-CoA
dehydrogenase
has an important role in
hepatic fatty acid
-oxidation
and it is likely that
the activation of this
enzyme by the W
FO diet would increase
fatty acid -oxidation in
the liver.
The liver carnitine
concentration in rats
fed the W, FO and
W FO diets was increased
to 121 and 68 and 296%
of that
in rats fed the C diet,
respectively. Carnitine
also plays an
important role in fatty
acid -oxidation in the
liver, and
Hoppel (37) reported
that an increase in the
concentration of
carnitine in the liver
correlated with the rate
of hepatic
ketoacid production.
Moreover, McGarry et al.
(38) reported
that carnitine
stimulated ketogenesis
from oleic acid.
Therefore,
the increase in the
concentration of
carnitine in the liver
due to the W FO diet
would lead to an
increase in hepatic
fatty acid -oxidation.
In addition, McGarry et
al. (38) also
reported that increased
fatty acid flux through
the carnitine
acyltransferase reaction
was mediated by an
elevation in liver
carnitine concentration.
However, in the present
study, the
increase in hepatic
carnitine concentration
in rats fed the W
FO diet did not reflect
the activity of
carnitine
acyltransferase.
Because the activity of
carnitine
acyltransferase is
controlled
by the levels of
circulating glucose and
free fatty acids
in the serum, and the
hepatic content of
malonyl-CoA (35,
39), measurement of the
concentration of glucose
and free
fatty acids in the
serum, and the
malonyl-CoA content in
the
liver in rats fed the W
FO diet would be
required to clarify
this point.
Thus, the W FO diet
synergistically
increased the activity
of 3-hydroxyacyl-CoA
dehydrogenase and the
concentration
of carnitine in the
liver, and decreased the
concentration
of triacylglycerol in
the liver and serum.
However, we cannot
explain the mechanism(s)
of the increase in the
concentration
of carnitine in the
liver and in the
activity of
3-hydroxyacyl-
CoA dehydrogenase by the
administration of the W
FO
diet. Therefore,
examination of the
influences of the W FO
diet on hepatic fatty
acid -oxidation in more
detail is necessary
to clarify the mechanism
of the synergistic liver
triacylglycerol-
lowering effect of the W
FO diet.
Although there was no
difference in the food
intake among
the dietary groups, the
relative liver and
adipose tissue around
the testis weight in
rats fed the W FO diet
was significantly
lower than those in rats
fed the C diet in the
present study. We
conclude that this
decrease was due to the
promotion of the
fatty acid -oxidation in
the liver by the W FO
diet.
In our previous study,
the 10 g/100 g diet
wakame diet did
not decrease either the
serum or liver
cholesterol levels but
in
the present study, the W
diet containing 19.1
g/100 g of dried
wakame powder
significantly decreased
the concentration of
serum and liver
cholesterol compared
with the C diet. It has
been reported that
wakame contains the
polysaccharide,
alginate,
at 30 g/100 g, and
dietary alginate has
been shown to
decrease the
concentration of
cholesterol in the serum
and
liver (40,41).
Therefore, it is
probable that an
increase in the
alginate in the W diet
decreased the
concentration of
cholesterol
in the serum and liver.
The Wakame diet
containing 10 g/100 g
dried wakame
powder increased the
activity of acyl-CoA
dehydrogenase, and
the extent of the
increase using 16:0-CoA
as a substrate was
higher than that using
EPA-CoA as substrate in
our previous
study (4). However,
although the activity of
acyl-CoA oxidase
in rats fed the W diet
containing 19.1 g/100 g
dried wakame
powder was higher than
that in rats fed the C
diet using in the
activity of acyl-CoA
dehydrogenase between
rats fed
the W and the C diet in
the present study. It is
necessary to
confirm whether the
difference between these
responses depends
on the amount of dried
wakame powder added to
the
diet.
The dried wakame powder
used in this experiment
contained
3.7 g lipid/100 g of
dried wakame powder and
the fatty
acid composition (g/100
g total fatty acid) was
16:0; 12.8, 18:0;
0.4, 18:1; 4.5, 18:2;
5.8, 18:3(n-3); 12.4,
18:4(n-3); 33.9, 20:4;
11.3, and EPA; 16.1.
Therefore, the EPA
content of the W
FO diet was greater than
that of the FO diet.
However, the
amount of EPA from dried
wakame powder in the W
FO
diet was only 0.14 g/100
g diet. On the other
hand, the FO diet
contained 1.71 g/100 g
EPA and 0.87 g of DHA.
It is unlikely
that the fatty acids
from the dried wakame
powder altered
hepatic fatty acid
-oxidation.
Many researchers are
trying to identify the
functional elements
of food materials.
However, because our
diet is composed
of various food
materials, it is
necessary to consider
that
the properties of
functional substances in
food materials may
be enhanced or lessened
by the combination of
various food
materials. When rats
were fed wakame and fish
oil at the same
time, the concentration
of triacylglycerol was
lower than with
consumption of either of
the components alone,
and this
decrease was due to an
increased rate of fatty
acid oxidation in
the liver. These results
suggest that a diet
composed of fish
(fish oil) and wakame
may be useful in the
prevention of
hyperlipidemia.
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