-
Effect of Oryzanol and Ferulic Acid on the
Glucose Metabolism of Mice Fed with
a
High-Fat Diet
Myoung Jin Son, Catherine
W
. Rico, Seok Hyun Nam, and Mi
Y
oung Kang
Abstract:
The
effects
of
oryzanol
and
ferulic
acid
on
the
glucose
metabolism
of
high-fat-fed
mice
were
investigated.
Male
C57BL/6N
mice
were
randomly
divided
into
4
groups:
NC
group
fed
with
normal
control
diet;
HF
group
fed
with
high-fat
(17%) diet; HF-O group fed with high-
fat diet supplemented with 0.5% oryzanol; and
HF-FA group
fed with
high-fat diet supplemented with 0.5%
ferulic acid. All animals
were allowed
free access
to the
experimental diets
and
water
for 7 wk.
At the end of
the
experimental
period,
the
HF-O
and
HF-FA
groups
exhibited
significantly
lower
blood
glucose
level
and
glucose-6-phosphatase
(G6pase)
and
phosphoenolpyruvate
carboxykinase
(PEPCK)
activities,
and
higher
glycogen
and
insulin
concentrations
and
glucokinase (GK) activity compared
with NC and
HF groups.
The results of this
study
illustrate
that
both
oryzanol
and
ferulic
acid
could
reduce
the
risk
of
high-fat
diet-induced
hyperglycemia
via
regulation
of
insulin
secretion
and
hepatic
glucose-regulating enzyme activities.
Keywords:
diabetes, ferulic
acid, high-fat-fed mice, hypoglycemic effect,
oryzanol
Introduction
Chronic consumption of a high-fat diet
has been associated with the development
of obesity and
type 2
diabetes
mellitus
(Hill
and others 1992; Bray and others 2004).
Scientific studies
have
shown that excessive
intake of dietary
fat results
in
increased
body
weight and poor glucose
regulation
(Alsaif and
Duwaihy2004; Petro and
others
2004;
Messier
and
others
2007).
Diabetes
is
characterized
by
hyperglycemia
that
results
in
the
generation of
free radicals
leading
to oxidative
stress (West 2000).
Due
to changes
in
lifestyle patterns, particularly poor
eating
habit and sedentary
lifestyle,
the
incidence of diabetes
has
rapidly
increased
in
epidemic proportions. Around 171
million cases of diabetes
worldwide
were reported
in 2001 and
it was projected
that
by
2030,
366
million
people
will
have
diabetes
(Wild
and
others
2004).
With
this
increasing global prevalence of
diabetes, the need for therapeutic measures
against the
disease
has
become
stronger
and
more
urgent.
A
wide
range
of
oral
medicines
are
currently being
used
for
treating
diabetes. However,
various adverse
effects
and
high
rates
of
secondary
failures
have
been
associated
with
the
available
antidiabetic
medicines
(Inzucchi
2002).
Thus,
finding
natural
drugs
with
hypoglycemic
activity
has now become the
focus of scientists and researchers.
At
present,
there
is
a
considerable
public
and
scientific
interest
in
utilizing
phytochemicals
for
the
treatment
and
prevention
of
various
diseases.
Naturally
occurring phenolic
compounds, such as oryzanol and
ferulic acid, are known
to
have
strong
antioxidant
activities
(Wang
and
others
2002;
Srinivasan
and
others
2007).
Oryzanol is a mixture of ferulic acid
(4-hydroxy-3-methoxycinnamic acid) esters with
phytosterols
(Lerma-Garcia
and others 2009) and primarily extracted
from
rice bran.
Ferulic acid
is commonly
found
in
fruits
and
vegetables,
including
banana, broccoli,
rice bran, and citrus
fruits (Zhao and Moghadasian 2008).
Both oryzanol and
ferulic
acid possess several physiological
proper ties, such as reduction of serum
cholesterol
levels (Wilson and others
2007), inhibition of tumor promotion
(Y
asukawa and others
1998),
and
protective
action
against
liver
injury
(Choti-markorn
and
Ushio
2008).
Oxidative stress
is regarded
as
the key
factor
in
the development of
diabetes and
its
associated
health
disorders.
The
high-fat
diet
fed
C
57BL/6
mouse
model
has
long
been
used
by
researchers
in
investigating
the
pathophysiology
of
impaired
glucose
tolerance and type 2 diabetes and
for
the development of
new treatments (Surwit and
others 1988; Surwit and other s 1991;
Schreyer and others 1998;
Winzell
and
Ahren
2004).
Since diabetes is a free radical mediated disease,
the strong antioxidant activity
of
oryzanol and
ferulic acid
may be
useful
in
preventing the development of diabetic
hyperglycemia
under
a
high-fat
diet.
There
are
limited
reports
on
the
physiological
functions
of
these
phenolic
compounds
in
relation
to
glucose
metabolism
in
animal
models.
Thus, this study was conducted to investigate the
effects of dietary feeding of
oryzanol
and ferulic acid on the glucose metabolism in
high-fat-fed C57BL/6 mice.
1. Materials
and Methods
1.1 Animals and diets
Twenty-four male C57BL/6N mice of 4 wk
of age, weighing 12 g, were obtained
from
Orient
Inc.
(Seoul,
Korea).
They
were
individually
housed
in
stainless
steel
cages in a room
maintained at 25
?
C with 50%
relative humidity and 12/12 h light/dark
cycle and
fed
with a pelletized chow diet
for 2
wk after arrival. The
mice
were
then
randomly divided into 4 dietary groups
(n = 6). The 1st and 2nd groups were fed with
a
normal and
high-fat (17%,
w/w) diets,
respectively,
while the
other 2
groups were
fed
with
high-fat
diet
supplemented
with
either
0.5%
oryzanol
or
0.5%
ferulic
acid
(>98% pure, Tsuno, Osaka, Japan). The
composition of the experimental diet (Table 1)
was
based
on
the
AIN-76
semisynthetic
diet.
The mice were fed for 7 wk
and allowed free access to food and water during
the
experimental period.
The
body
weight
gain
was
measured
weekly
. At
the
end of
the
experimental
period,
the
mice
were
anaesthetized
with
60-
μL
Ketamine
-HCl
following a 12 h
fast and
sacrificed. Blood samples were collected and
centrifuged at
1000 ×
g for
15 min at 4
?
C to obtain the
plasma. The livers were removed, rinsed with
physiological
saline,
and
stored
at
?70?C
until
analysis.
The
current
study
protocol
was approved by the
Ethics Committee of Kyungpook Natl. Univ. for
anima studies.
1.2 Measurement of blood
glucose level
The
blood
glucose
level
in
mice
was
measured
using
Accu-Chek
Active
Blood
Glucose
Test
Strips
(Roche
Diagnostics
GmbH,
Germany).
Blood
samples
were
drawn
from
the
tail
vein
of
the
mice
before
and
after
3
and
7
wk
of
feeding
the
animals with experimental
diets.
1.3
Determination of glycogen and insulin levels
The
glycogen concentration
in
liver
was
determined
using
the
method described
by Seifter
and others
(1950)
。
Fresh
liver (100
mg)
was
mixed
with 30% KOH and
heated at
100
?
C for 30
min.
The
mixture was then added
with 1.5
mL ethanol (95%)
and kept over
night at
4
?
C. The pellet was
mixed with 4 mL
distilled
water. A 500 μL
of
the
mixture
was added with 0.2%
anthrone (in 95%
H
2
SO
4
)
and the absorbance of
the sample
solution was measured at 620 nm. The results were
calculated on the basis
of
a
standard
calibration
curve
of
glucose.
The
insulin
content
was
measured
using
enzyme-linked
immunosorbent
assay
(ELISA)
kits
(TMB
Mouse
Insulin
ELISA
kit,
Sibayagi, Japan).
1.4 Measurement of hepatic glucose-
regulating enzyme activities
The
hepatic enzyme source
was
prepared according to the
method
developed by
Hulcher
and
Oleson
(1973).
The
glucokinase
(GK)
activity
was
determined
based
from
the
method
of Davidson and Arion (1987)
with
slight
modification.
A 0.98
mL
of the
reaction mixture containing 50 mM Hepes-NaOH(pH
7.4), 100 mM KCl, 7.5 m
M
MgCl
2
,
2.5
mM
dithioerythritol,
10
mg/mL
albumin,
10
mM
glucose,
4
units
of
glucose-6-phosphate ( G6pase)
dehydrogenase, 50 mM NAD
+
,
and 10
μL
cytosol was
preincubated at
37
?
C for 10 min. The
reaction was
initiated with the
addition of 10 μL
of 5 mM ATP and the
mixture was incubated at
37
?
C for 10 min. The G6pase
activity
was
measured
using the
method described
by
Alegre and others (1988). The
reaction
mixture contained 765 μL of
131.58
mM
Hepes
-NaOH
(pH
6.5), 100 μL of 18
mM
EDTA
(
pH
6.5),
100
μL
of
265
mM
G6pase,
10
μL
of
0.2
M
NADP
+
,
0.6
IU/mL
mutarotase,
and 0.6 IU/mL
glucose dehydrogenase.
the
mixture
was added
with 5 μL
microsome and
incubated at 37
?
C for 4 min.
The change in absorbance at 340 nm was
measured. The phosphoenolpyruvate
carboxykinase (PEPCK) activity was determined
based
from the
method developed by
Bentle
and Lardy (1976).
The
reaction
mixture
consisted
of
72.92
mM
sodium
Hepes
(pH
7.0),
10
mM
dithiothreitol,
500
mM
NaHCO
3
,
10
mM
MnCl
2
,
25
mM
NADH,
100
mM
IDP,
200
mM
PEP,
7.2
unit
of
malic
dehydrogenase,
and
10μL
cytosol.
The
enzyme
activity
was
determined
based
from the decrease in
the absorbance of the mixture at 350 nm at 25
?
C.
在
25
?
C 350nm
。
1.5 Statistical analysis
All
data
are
presented
as
the
mean
±
SE.
The
data
were
evaluated
by
1-way
ANOV
A using
a Statistical Package for Social Sciences software
program (SPSS Inc.,
Chicago,
Ill.,
U.S.A.)
and
the
differences
between
the
means
we
reassessed
using
Duncan’s
multiple range test. Statistical significance was
considered at P <0.05.
2.
Results
2.1 Body weight gain
There was no significant difference in
the body weight among the animal groups
prior to feeding the mice with the
experimental diets (Table 2).
。