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Life Sciences 81 (2007) 1479 1488

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Myricetin, a naturally occurring flavonol, ameliorates insulin resistance

induced by a high-fructose diet in rats
I-Min Liu a,, Thing-Fong Tzeng b , Shorong-Shii Liou a , Ting-Wei Lan a
a
Department of Pharmacy, Tajen University, Yen-Pou, Ping Tung Shien, Taiwan, ROC
b
Department of Internal Medicine, Pao Chien Hospital, Ping Tung City, Taiwan, ROC
Received 25 May 2007; accepted 19 August 2007

Abstract

The present study was conducted to explore the effects of myricetin on insulin resistance in rats fed for 6 weeks with a diet containing 60%
fructose. Repeated intravenous (i.v.) injection of myricetin (1 mg/kg per injection, 3 times daily) for 14 days was found to significantly decrease
the high glucose and triglyceride levels in plasma of fructose chow-fed rats. Also, the higher degree of insulin resistance in fructose chow-fed rats
as measured by homeostasis model assessment of basal insulin resistance was significantly decreased by myricetin treatment. Myricetin increased
the whole-body insulin sensitivity in fructose chow-fed rats, as evidenced by the marked elevation of composite whole-body insulin sensitivity
index during the oral glucose tolerance test. Myricetin was found to reverse the defect in expression of insulin receptor substrate-1 (IRS-1) and the
p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in soleus muscle of fructose chow-fed rats under the basal state, despite the
protein expression of insulin receptor (IR). Increased basal phosphorylation of IR and IRS-1 as well as Akt was observed in parallel. The reduced
level of insulin action on phosphorylation of IR, IRS-1 and Akt in soleus muscle of fructose chow-fed rats was reversed by myricetin treatment.
Furthermore, myricetin treatment improved the defective insulin action on the translocation of glucose transporter subtype 4 (GLUT 4) in insulin-
resistant soleus muscle. These findings indicate that myricetin improves insulin sensitivity through the enhancement of insulin action on IRS-1-
associated PI 3-kinase and GLUT 4 activity in soleus muscles of animals exhibiting insulin resistance.
2007 Elsevier Inc. All rights reserved.

Keywords: Fructose; Insulin resistance; Myricetin; Oral glucose tolerance test

Introduction management of obesity and cardiovascular diseases as well as

Insulin resistance is a metabolic disorder whose prevalence is
increasing alarmingly in populations worldwide. It is a condition
wherein the body tissues become resistant to insulin, resulting in a
marked decrease of glucose metabolism in response to insulin.
Recent studies suggest that insulin resistance results from
complex interactions between genetic and environmental factors
and is associated with common diseases such as type 2 diabetes,
hypertension, obesity, and coronary heart disease (Grundy, 2007).
It has been demonstrated that the use of pharmacological
intervention in combination with lifestyle modifications that
include diet and moderate exercise is particularly useful in the
Corresponding author. Department of Pharmacy, Tajen University, Yanpu
Shiang, Ping Tung Shien, Taiwan 90701, ROC. Tel.: +886 7 346 0961; fax:
+886 8 762 5308.
E-mail address: iml@mail.tajen.edu.tw (I.-M. Liu).
0024-3205/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.08.045
prevention of insulin resistance and type 2 diabetes mellitus (Hu
et al., 2006). Thiazolidinediones (TZD), agonists of the
peroxisome proliferator-activated receptor , are a class of oral
antidiabetic agents that enhance insulin sensitivity and improve
metabolic control in patients with type 2 diabetes (Gervois et al.,
2007). Despite their proven efficacy, a number of deleterious side
effects such as weight gain and the documented aggravation of
advanced heart failure through fluid retention have been noted
with the use of TZDs, including rosiglitazone and pioglitazone
(Granberry et al., 2007). Accordingly, a significant research effort
in laboratories worldwide is being directed towards generating
modulators that retain the beneficial clinical effects of TZDs while
avoiding their adverse side effects. It seems, therefore, that
developing new agents without side effects would definitely be
helpful in the therapy of diabetic patients with insulin resistance.
Currently, there is also an enormous increase in the use of
herbs and other alternative medicines in the treatment of
1480 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
diabetic disorders. There is growing interest in the therapeutic
applications of bioflavonoids and other naturally occurring
polyphenols for the treatment and prevention of diseases in
humans. Myricetin (3, 5, 7, 3, 4, 5-hexahydroxyflavone) is a
naturally occurring flavonoid that is commonly found in tea,
berries, fruits, vegetables and the medicinal herb Abelmoschus
moschatus (Miean and Mohamed, 2001; Harnly et al., 2006).
Myricetin is reported to have many therapeutic applications,
such as anticarcinogenic action (Ko et al., 2005), antiviral and
antimicrobial effects (Ono et al., 1990), and the prevention of
platelet aggregation (Landolfi et al., 1984; Tzeng et al., 1991). It
has also been demonstrated that myricetin possesses antiox-
idative properties (Robak and Gryglewski, 1988; Robak et al.,
1988) and cytoprotective capacity (Mira et al., 2002; Dajas
et al., 2003). The therapeutic benefit of myricetin in
cardiovascular diseases associated with diabetes mellitus has
also been reported (Ong and Khoo, 1997). Additionally, an
insulinomimetic effect of myricetin on lipogenesis and glucose
transport in adipocytes of rats with noninsulin-dependent dia-
betes mellitus has been observed (Ong and Khoo, 1996). We
have observed that myricetin has the ability to improve glucose
utilization for the lowering of plasma glucose in streptozotocin-
induced diabetic rats, the type 1 diabetes-like animal model (Liu
et al., 2005, 2006). Studies indicate that myricetin plays a role in
glucose homeostasis, the mechanism of which is not clearly
understood at present. Additionally insufficient information is
available regarding the effect of myricetin on insulin resistance.
The present study was undertaken to clarify whether myricetin
is effective in improving insulin resistance in rats. The con-
sumption of fructose worldwide has substantially increased, due
to an increased consumption of soft drinks and other beverages
that are high in fructose, and due to the consumption of breakfast
cereals, baked goods, and prepared desserts sweetened with high-
fructose corn syrup (Basciano et al., 2005; Le and Tappy, 2006).
Studies in rats have demonstrated that a high intake of fructose
produced a decline of insulin sensitivity in the liver and peripheral
tissues (Basciano et al., 2005; Le and Tappy, 2006). Hence
fructose has been implicated as a useful tool in inducing insulin
resistance in animals. Thus for the present study, insulin resistance
in rats was induced by a diet containing 60% fructose.
Materials and methods
Materials
Standard rat chow containing 60% vegetable starch, 5% fat
and 18% protein (Cat. #2018) and fructose-rich rat chow
containing 60% fructose, 5% fat, and 18% protein (Cat. # TD
89247) were obtained from Harlan Teklad (Madison, WI, USA).
Myricetin and protein A-Sepharose beads were purchased from
Sigma-Aldrich, Inc. (St. Louis, MO, USA). Rosiglitazone
maleate (Avandia) was supplied by GlaxoSmithKline (Research
Triangle Park, NC, USA). The diagnostic kits for determination
of plasma levels of glucose (Cat. #COD12503), cholesterol (Cat.
#COD11539) and triglyceride (Cat. #COD11529) were pur-
chased from BioSystem (Costa Brava, Barcelona, Spain). Rat
insulin enzyme-linked immunosorbent assay (ELISA) kit was
obtained from LINCO Research, Inc. (St. Charles, MO, USA; Cat.
#EZRMI-13K). The kit for protein assay was purchased from
Bio-Rad Laboratories (Hercules, CA, USA). Anti-insulin
receptor (IR) -subunit antibodies (Cat. #MS-634 for immuno-
precipitation; Cat. #MS-636 for Western blotting), anti-insulin
receptor substrate (IRS)-1 antibody (Cat. #MS-630), anti-p85
regulatory subunit phosphatidylinositol 3-kinase antibody (Cat.
#RB1625) and anti-phosphotyrosine antibody (Cat. #MS-445)
were obtained from NeoMarkers (Fremont, CA, USA). Anti-
phosphoserine (Ser473) Akt antibody was from Cell Signaling
Technology, Inc. (Beverly, MA, USA; Cat. #9271). Anti-glucose
transporter subtype 4 (GLUT 4) antibody was purchased from
R&D Systems Inc. (Minneapolis, MN, USA; Cat. #BAM1262).
ECL Western Blotting Systems were obtained from Amer-
sham Corp. (Braunschweig, Germany). Human recombinant
insulin (Humulin R) was obtained from Eli Lilly and Company
(Taiwan), Inc. Bovine insulin was obtained from Novo Nordisk
(Bagsvaerd, Denmark). All other reagents were obtained from
standard sources.
Animal models
Male Wistar rats aged 8 weeks were obtained from the Animal
Center of National Cheng Kung University Medical College.
They were maintained in a temperature-controlled room (25
1 C) and kept on a 12:12 lightdark cycle (light on at 06:00 h).
Food and water were available ad libitum. The rats were divided
into two experimental groups. One group of rats was randomly
assigned to receive the fructose chow for 6 additional weeks to
induce insulin resistance (Hyakukoku et al., 2003). The remaining
group receiving standard chow during the 6-week period was
designated as the control group. All animal procedures were
performed according to the Guide for the Care and Use of Labo-
ratory Animals of the National Institutes of Health, as well as the
guidelines of the Animal Welfare Act.
The homeostasis model assessment of basal insulin resis-
tance (HOMA-IR) was used to calculate an index from the
product of the fasting concentrations of plasma glucose (mmol/
l) and plasma insulin (U/ml) divided by 22.5 (Matthews et al.,
1985). Lower HOMA-IR values indicated greater insulin
sensitivity, whereas higher HOMA-IR values indicated lower
insulin sensitivity (insulin resistance).
Repeat treatment of fructose chow-fed rats with myricetin or
rosiglitazone
The rats fed with fructose chow for six weeks were used as
insulin-resistant animals. It is well documented that a fraction of
orally-administered myricetin is absorbed by the gastrointestinal
tract, whereas the remainder is degraded by gastrointestinal micro-
flora (Griffiths and Smith, 1972).Hence, in order to circumvent
these pharmacokinetic considerations, myricetin was injected into
the lateral tail vein of the fructose chow-fed rats every 8 h, 3 times
daily (at 06:00, 14:00 and 22:00 h) at the desired doses. The
injection volume was controlled such that 1 ml/kg was routinely
administered. The injection was given slowly to avoid as much
pain and shock as possible and thereby exclude non-predictive
 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
outcomes. The effect of myricetin was determined for 14
consecutive days in subsequent experiments. Another group of
rats was treated similarly but with the same volume of vehicle
(70% ethanol: saline = 1:19) as was used to dissolve myricetin (Liu
et al., 2005, 2006). Myricetin- or vehicle-treated rats continued to
be fed with fructose chow during the 2-week treatment period.
Additionally, rosiglitazone was given by oral gavage (4 mg/kg per
day) for 14 days to separate groups of fructose chow-fed rats. This
dose was selected since it was comparable to the dose found to
rapidly induce PPAR-dependent genes (Pearson et al., 1996). The
rats were maintained on a fructose chow diet during the 2-week
rosiglitazone treatment period. Water was available ad libitum
throughout the experiment.
Oral glucose tolerance test
An oral glucose tolerance test (OGTT) was performed using an
oral dose of glucose (1 g/kg) for 2 h after 14 consecutive days of
myricetin or rosiglitazone treatment. Animals were food-
restricted and were given only water to drink throughout the
night prior to the OGTT procedure. Plasma glucose and insulin
concentrations were measured before and 30, 60, 90, and 120 min
after the glucose load.
Insulin sensitivity was calculated using the composite whole-
body insulin sensitivity index (ISIcomp) during the OGTT
(Matsuda and DeFronzo, 1999). ISIcomp was estimated with
the following formula: ISIcomp = 10,000/square root of [(mean
plasma insulin mean plasma glucose during OGTT) (fasting
plasma glucose fasting plasma insulin)].
Plasma analysis
Blood samples were collected from the lateral tail vein of rats
anesthetized with sodium pentobarbital (30 mg/kg) administered
intraperitoneally (i.p.). Samples were centrifuged at 2000 g for
10 min at 4 C; aliquots of plasma were removed for the respective
analytical determinations. Plasma glucose concentration was
measured by glucose oxidase method (Hitachi 717 autoanalyzer;
Hitachi Ltd., Tokyo, Japan). Levels of cholesterol and triglycer-
ides in total plasma were analyzed enzymatically (Hitachi 717
autoanalyzer). The ELISA technique was employed to quantify
plasma levels of insulin using a commercially available kit. The
test compounds used in the present study did not affect the
binding of peptide with antibodies. All samples were analyzed in
triplicate.
Measurement of glycogen synthesis in hepatocytes
Hepatocytes were isolated from rats at the end of the 2-week
treatment period by collagenase perfusion of the liver (Agius et al.,
1996). The hepatocytes were suspended in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, 10 mmol/
l glucose, 100 nmol/l insulin, 100 nmol/l dexamethasone and
inoculated in 24-well plates at a density of 5 104 cells/cm2. They
were cultured at 37 C equilibrated with 5% CO2, 95% air.
Approximately 7 h was allowed for cell attachment in the serum-
containing medium (Agius et al., 1996). For determination of
1481
glycogen, hepatocytes were transferred to fresh incubation flasks
containing [U-14C]-glucose (0.25 Ci/ml) after the 30 min pre-
incubation period in KrebsRinger bicarbonate buffer (KRBB) at
37 C, and then incubated with 1.0 nmol/l bovine insulin at 37 C
for 1 h under continuous shaking. Glycogen was precipitated with
70% ethanol overnight on ice. Precipitated glycogen was
centrifuged at 10,000 g for 10 min. Pellets were washed once
with 70% ethanol, resuspended in 0.5 ml water, and counted by
scintillation counting. The incorporation into glycogen was
expressed as nmol/mg of cell protein in 1 h. Protein content was
determined using the Bio-Rad protein dye binding assay.
In vivo insulin receptor activation
To assess the effect of myricetin on insulin receptor activation
in vivo, rats in the fed state were anesthetized with sodium
pentobarbital at the end of the 2-week treatment period. Then, a
bolus of insulin (10 units/kg) was injected into portal vein of rats,
as described previously (Li et al., 2005). Approximately 120 s
after insulin injection, rats were sacrificed and the soleus muscle
was immediately extirpated, washed with cold phosphate buffer,
and cut into 200300 mg portions, which were then stored
separately at 70 C for subsequent immunoprecipitation and
immunoblot analyses.
Muscle processing
Cytosol and membrane fractions were prepared according to a
previously described method (Rodriguez et al., 2004). Briefly,
muscles used for measuring insulin signaling were weighed while
still frozen and then homogenized (Polytron, Brinkmann Instru-
ments, Inc., Westbury, NY, USA) in 0.4 ml homogenizing buffer
containing 250 mmol/l sucrose, 20 mmol/l Tris (pH 7.5), 2 mmol/
l EDTA, 0.5 mmol/l EGTA, 20 g/ml leupeptin, 10 g/ml
aprotinin, 174.2 g/ml phenylmethylsulfonyl fluoride, and
20 mmol/l dithiothreitol. The homogenate was centrifuged at
100,000 g for 1 h at 4 C. The supernatant (cytosolic extract) was
transferred to a tube kept on ice, whereas the pellet was resus-
pended in 0.45 ml homogenizing buffer containing 5% Triton X-
100. The resuspended pellet fraction was then centrifuged at
14,000 g for 5 min at 4 C, and the pellet was discarded. The
supernatant from this spin constitutes the membrane extract.
Protein concentrations were determined by the Bio-Rad protein
dye binding assay. The supernatant was stored at 80C until used
in immunoprecipitation and Western immunoblotting.
Immunoprecipitation and Western immunoblotting
Muscle homogenates (1 mg protein) were subjected to
immunoprecipitation with anti-IR -subunit antibody or anti-
IRS-1 antibody at 4 C overnight, followed by shaking with
protein A-Sepharose beads for 1 h. The bead-Protein A-
antibodyantigen complexes were precipitated by brief centri-
fugation. The pellets were washed three times in ice-cold buffer
(0.5% Triton X-100, 100 mmol/l Tris, pH 7.4, 10 mmol/l EDTA
and 2 mmol/l sodium vanadate, resuspended in Laemmli sample
buffer, and boiled for 5 min. The sepharose beads were
1482 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488

precipitated by brief centrifugation and the supernatant prepared Results

for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE, 10% acrylamide gel) using a Bio-Rad Mini-
Protein II system (55 V and 130 V during the stacking and
separation phases, respectively). Protein was transferred to a
polyvinylidene difluoride (PVDF) membrane using the Bio-Rad
Trans-Blot system (2 h at 20 V in 25 mmol/l Tris, 192 mmol/
l glyceine and 20% MeOH). Following transfer, the membrane
was probed with anti-IR -subunit antibody, anti-IRS-1
antibody or anti-phosphotyrosine antibody according to the
manufacturers' instructions.
For detection of the p85 regulatory subunit of PI3-kinase,
Akt serine (Ser473) phosphorylation, and GLUT 4 content,
equal amounts (50 g) of protein were prepared from muscle
homogenates, subjected to SDS-PAGE, transferred to PVDF
membrane as described above, and blotted with anti-PI3-
kinase p85 subunit antibody, anti-phosphoserine (Ser473) Akt
antibody or anti-GLUT4 antibody according to the manufac-
turer's instructions. After three 5-minute washes in TBST
(20 mmol/l TrisHCl (pH 7.5), 150 mmol/l NaCl, and 0.05%
Tween 20), membranes were incubated with the appropriate
peroxidase-conjugated secondary antibodies. The membranes
were then washed three times in TBST and visualized on X-ray
film using the enhanced chemiluminescence detection system.
Densities of the obtained immunoblots were quantified using a
laser densitometer. The mean value for samples from vehicle-
treated lean rats on each immunoblot, expressed in densitom-
etry units, was adjusted to a value of 1.0. All experimental
sample values were then expressed relative to this adjusted
mean value.
Statistical analysis
Data are expressed as the mean SEM for each group of
animals at the number indicated in tables. Statistical differences
among groups were determined by using two-way repeated-
measures ANOVA. The Dunnett range post-hoc comparisons
were used to determine the source of significant differences
where appropriate. A p-value b 0.05 was considered statistically
significant.
General characteristics of fructose chow-fed rats repeatedly
treated with myricetin or rosiglitazone
At the end of 6-week high-fructose diet feeding, body
weights were similar between standard chow-fed and fructose
chow-fed rats (Table 1). However, animals maintained on the 6-
week high-fructose diet exhibited significant hyperglycemia
compared with the standard chow-fed group for the same
feeding period (Table 1). Meanwhile, the plasma concentrations
of insulin and triglycerides were significantly higher in the
group eating high-fructose diet than in the standard chow-fed
group (Table 1). Conversely, the plasma cholesterol level was
not different between standard chow and fructose chow-fed
animals (Table 1).
Following the injection of myricetin (1 mg/kg per i.v.
injection; 3 injections per day) for 14 consecutive days, plasma
glucose levels in fructose chow-fed rats fell to a value
significantly lower than the vehicle-treated counterparts
(P b 0.05), showing a plasma glucose-lowering activity of
16.5 3.1% (Table 1). Similarly, plasma glucose levels in
fructose chow-fed rats in rosiglitazone (4 mg/kg per day)-
treated rats were significantly decreased to near those observed
in the standard chow-fed group after 2 weeks of treatment
(Table 1). In addition, fructose chow-fed rats that received the 2-
week rosiglitazone treatment exhibited significantly lower
plasma insulin levels as compared with their vehicle-treated
counterparts. Actually, myricetin influences neither the plasma
insulin levels nor body weight of fructose chow-fed rats;
whereas the rosiglitazone-treated animals gained more weight
compared with all other groups at the end of the 2-week
treatment period (Table 1).
Plasma levels of triglyceride were also significantly reduced
in fructose chow-fed rats after 14 days of treatment with
myricetin (1 mg/kg per i.v. injection; 3 injections per day);
similar results were obtained in fructose chow-fed rats that
received a 2-week treatment with rosiglitazone (4 mg/kg per
day; Table 1). At the termination of 2-week treatment, the total
plasma cholesterol in fructose chow-fed rats tended to be

Table 1
General characteristics of fructose chow-fed rats after 14 consecutive days of myricetin or rosiglitazone treatment
Standard chow-fed Fructose chow-fed
Vehicle Vehicle Myricetin (mg/kg per injection) Rosiglitazone
0.1 0.5 1.0
Body weight (g/rat) 286.4 10.8c 290.2 9.6 288.3 11.2 287.9 12.3 288.6 10.7 326.4 11.3a,c
Plasma glucose (mg/dl) 93.8 5.7c 134.5 6.2a 127.4 5.8a 120.3 4.9a 112.4 5.2b,c 106.7 4.7b,c
Plasma insulin (U/ml) 23.2 5.6d 53.4 5.4b 52.7 6.3b 50.4 4.9b 49.6 6.1b 30.6 5.9d
Plasma triglyceride (mg/dl) 106.4 11.2d 351.2 14.3b 307.2 11.8b,c 256.7 13.6b,d 214.5 12.7b,d 176.4 10.2b,d
Plasma cholesterol (mg/dl) 81.3 6.1 84.6 5.3 82.4 4.9 80.5 5.7 79.5 5.2 76.8 6.4
HOMA-IR score 4.9 0.8d 18.1 1.7b 16.8 2.1b 14.8 2.3b,c 12.6 1.9b,c 8.6 1.4a,d
After 6 weeks of standard chow or fructose chow feeding, rats received i.v. injection of myricetin at the indicated dosage, 3 times daily for 14 days. Rosiglitazone was
also given by oral gavage (4 mg/kg per day) for 14 days to separate groups of the fructose chow-fed rats. The vehicle used to dissolve the tested drugs was given at the
same volume. Values (mean SEM) were obtained from 7 rats. aP b 0.05 and bP b 0.01 compared to the values of standard chow-fed rats treated with vehicle,
respectively. cP b 0.05 and dP b 0.01 compared to the values of fructose chow-fed rats treated with vehicle, respectively.
 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
reduced by myricetin or rosiglitazone, although no significant
differences were found between any treatment and those in
standard chow-fed group.
Additionally, the HOMA score in fructose chow-fed rats was
higher by 3.6-fold times that of the standard chow-fed group.
Following 14 days of rosiglitazone (4 mg/kg per day) treatment,
1483
the HOMA score in fructose chow-fed rats markedly fell to 50%
of that in their vehicle-treated counterparts (Table 1). Although
the effect was not as large as in rosiglitazone-treated animals,
the HOMA score in fructose chow-fed rats that received
2 weeks of myricetin treatment showed a significant decrease
(approximately 70% of the score observed in the vehicle-treated
counterparts) (Table 1).
Effects of repeated myricetin or rosiglitazone treatment on the
insulin sensitivity of fructose chow-fed rats
The response to an oral glucose tolerance test at the
termination of 2-week treatment is shown in Fig. 1. Plasma
glucose levels were significantly elevated during the OGTT in
fructose chow-fed rats compared to the standard chow-fed
group at all time points tested (Fig. 1A). In the vehicle-treated
fructose chow-fed rats, plasma glucose concentrations increased
from fasting levels of 132.5 4.3 mg/dl to nearly 212.6
5.9 mg/dl by 30 min and were still greatly increased over
baseline levels 2 h after the oral glucose challenge. The fructose
chow-fed rats treated for two weeks with myricetin (1 mg/kg per
i.v. injection, 3 times daily) showed a significant elevation in
plasma glucose concentrations at 30 min but returned to basal
levels by 2 h after the oral glucose administration; similar results
were obtained in the group treated with rosiglitazone (4 mg/kg
per day) (Fig. 1A).
Also, fasting plasma insulin concentrations were significant-
ly greater in fructose chow-fed rats compared to the standard
chow-fed group and remained higher at all time points
throughout the OGTT study period (Fig. 1B). The plasma
insulin concentrations in both myricetin- and rosiglitazone-
treated fructose chow-fed rats increased 30 min after the oral
glucose challenge, but returned to their respective fasting levels
by 2 h (Fig. 1B).
The ISIcomp was also significantly lower in the fructose
chow-fed group as compared to that in standard chow-fed
group. Repeated myricetin (1 mg/kg per i.v. injection, 3 times
daily) treatment for 2 weeks increased ISIcomp of fructose
chow-fed rats to 1.5-fold of the value in their counterparts
receiving vehicle (Fig. 1C). Additionally, 2-week rosiglitazone
(4 mg/kg per day) treatment raised ISIcomp in fructose chow-
fed rats to nearly 2.4-fold that of their vehicle-treated counter-
parts (Fig. 1C).
Fig. 1. A: Plasma glucose responses during an oral glucose (1 g/kg) tolerance
test (OGTT) in fructose chow-fed rats receiving an i.v. injection of myricetin at
0.1 mg/kg (), 0.5 mg/kg () or 1.0 mg/kg ( ), 3 times daily for 14 days. B:
Plasma insulin responses during OGTT determined in these rats. C: Insulin
sensitivity was calculated using the composite whole-body insulin sensitivity
index (ISIcomp) after a 2-h OGTT. Rosiglitazone was given by oral gavage
(4 mg/kg per day) for 14 days to the separate groups of the fructose chow-fed
rats (). The vehicle used to dissolve the tested drugs was given at the same
volume. Values (mean SEM) were obtained from each group of 7 animals.
a
P b 0.05 and bP b 0.01 compared to the values of vehicle-treated standard chow-
fed rats () at the indicated times, respectively. cP b 0.05 and dP b 0.01 compared
to the values of vehicle-treated fructose chow-fed rats () at the indicated times,
respectively.
1484 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
Effects of repeated myricetin or rosiglitazone treatment on the
hepatocytic glycogen synthesis of fructose chow-fed rats
At the termination of the 2-week treatment, insulin
(1.0 nmol/l) markedly enhanced glycogen synthesis in isolated
hepatocytes of the vehicle-treated standard chow-fed rats to 3.3
0.2 nmol/mg protein/h (Fig. 2). In isolated hepatocytes of
fructose chow-fed rats receiving 2-week rosiglitazone (4 mg/kg
per day) treatment, insulin (1.0 nmol/l) increased the level of
[14C]-glucose incorporation into glycogen (2.8 0.2 nmol/mg
protein/h) to about 2.4-fold the value in the vehicle-treated
counterparts (1.4 0.1 nmol/mg protein/h) (Fig. 2). Also, insulin
(1.0 nmol/l) increased the glycogen synthesis in isolated
hepatocytes of 2-week myricetin (1 mg/kg per i.v. injection, 3
times daily)-treated fructose chow-fed rats to nearly 1.7-fold the
value of the vehicle-treated counterparts (Fig. 2).
Effects of repeated myricetin or rosiglitazone treatment on the
protein levels and the degree of tyrosine phosphorylation of
insulin receptor (IR) in soleus muscle of fructose chow-fed rats
Following 2-week treatment, there were no differences in the
expression of IR protein in soleus muscle of standard chow-fed
and fructose chow-fed rats. Additionally, the expression of IR
protein in soleus muscle was similar in fructose chow-fed rats
and standard chow-fed rats after stimulation with insulin;
treatment with rosiglitazone (4 mg/kg per day) or myricetin
(1 mg/kg per i.v. injection, 3 times daily) for 2 weeks did not
modify these values.
Furthermore, the data indicated that not just the basal level,
but the degree of insulin-stimulated increment in tyrosine
phosphorylation of IR in soleus muscle was clearly lowered in
fructose chow-fed rats relative to muscle from standard chow-
fed group (Fig. 3). Under insulin stimulation, 2-week
Fig. 2. Insulin-stimulated hepatocytic glycogen synthesis of fructose chow-fed
rats following repeated myricetin or rosiglitazone treatment. The fructose chow-
fed rats received i.v. injection of myricetin (1 mg/kg per injection, 3 times daily)
for 14 days. Rosiglitazone was given by oral gavage (4 mg/kg per day) for
14 days to the separate groups of the fructose chow-fed rats. Values (mean
SEM) were obtained from each group of 10 animals. aP b 0.05 and bP b 0.01
represents the level of significance compared to the values with vehicle-treated
standard chow-fed rats, respectively. cP b 0.05 and dP b 0.01 compared to the
values of vehicle-treated fructose chow-fed rats, respectively.
Fig. 3. Representative immunoblots of protein expression and insulin-stimulated
phosphorylation of insulin receptor-related signaling mediators in the soleus
muscles of fructose chow-fed rats following i.v. injection of myricetin (1 mg/kg
per injection, 3 times daily) for 14 days. Rosiglitazone was given by oral gavage
(4 mg/kg per day) for 14 days to the separate groups of the fructose chow-fed
rats. Rats that did not receive any treatment were given in the same volume of
vehicle used to dissolve the test medications. Findings were reproduced on 4
separate occasions. Quantification of the data is shown in Table 2.
rosiglitazone (4 mg/kg per day) treatment elevated the extent
of tyrosine phosphorylation of IR in soleus muscle of fructose
chow-fed rats, which could further increase to 2.4-fold that in
muscle from their vehicle-treated counterparts (Fig. 3). Also,
the degree of tyrosine phosphorylation of IR in soleus muscle of
fructose chow-fed rats was ameliorated by 2-week myricetin
treatment, while the level of insulin-stimulated tyrosine
phosphorylation of IR in the same group markedly increased
to 2.1-fold that of their vehicle-treated counterparts (Fig. 3).
Quantification of immunoblots is summarized in Table 2.
Effect of repeated myricetin or rosiglitazone treatment on the
protein levels and the degree of tyrosine phosphorylation of
insulin receptor substrate (IRS)-1 in soleus muscle of fructose
chow-fed rats
The expression of IRS-1 protein in soleus muscles of
fructose chow-fed rats was close to 60% of that in standard
chow-fed group (Fig. 3). At the end of the 2-week rosiglitazone
(4 mg/kg per day) treatment, the expression of IRS-1 protein in
soleus muscles of fructose chow-fed rats resembled that of the
standard chow-fed group (Fig. 3). Two-week myricetin (1 mg/
kg per injection, 3 times daily) treatment also raised IRS-1
protein expression in soleus muscles of fructose chow-fed rats
to 1.4-fold of that of their vehicle-treated counterparts.
However, insulin administration did not change the protein
levels of IRS-1 in soleus muscles of any group (Fig. 3).
The data indicated that the degree of IRS-1 tyrosine
phosphorylation increased in soleus muscles of fructose chow-
fed rats after 2-week rosiglitazone (4 mg/kg per day) treatment
when compared to their vehicle-treated counterparts; similar
results were obtained in the group treated with myricetin (1 mg/kg
 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488 1485

Table 2
Quantification of the expression and insulin-dependent phosphorylation of specific insulin signaling proteins in soleus muscles of fructose chow-fed rats receiving 14
consecutive days of myricetin or rosiglitazone treatment
Relative units Non-insulin stimulation Insulin stimulation
Standard chow-fed Fructose chow-fed Standard chow-fed Fructose chow-fed
Vehicle Vehicle Myricetin Rosiglitazone Vehicle Vehicle Myricetin Rosiglitazone
IR 1.01 0.05 0.99 0.05 0.98 0.06 1.01 0.04 1.02 0.04 0.96 0.06 0.97 0.05 1.01 0.06
IR-pTyr 1.02 0.04 0.43 0.06b 0.62 0.06a 0.73 0.05a 1.56 0.05a 0.57 0.07b 1.26 0.05a 1.35 0.04a
IRS-1 1.01 0.03 0.64 0.05a 0.72 0.04a 0.89 0.06 1.01 0.04 0.63 0.06a 0.78 0.06a 0.93 0.07
IRS-1-pTyr 1.03 0.05 0.49 0.04b 0.82 0.07a 0.94 0.04 1.48 0.06a 0.58 0.05b 1.21 0.05a 1.33 0.05a
PI 3k (p85a) 1.02 0.06 0.75 0.07a 0.84 0.06a 0.92 0.06 1.02 0.05 0.80 0.06a 0.88 0.08 0.94 0.06
pAkt (Ser473) 1.01 0.02 0.46 0.07b 0.74 0.04a 0.91 0.06 1.52 0.07a 0.49 0.06b 1.17 0.04a 1.35 0.08a
The fructose chow-fed rats received i.v. injection of myricetin at 1 mg/kg per injection, 3 times daily for 14 days. Rosiglitazone was also given by oral gavage (4 mg/kg
per day) for 14 days to separate groups of the fructose chow-fed rats. All values are presented relative to the average of values from samples of vehicle-treated standard
chow-fed rats without insulin stimulation for each immunoblot. The vehicle used to dissolve test drugs was given at the same volume. Values (mean SEM) were
obtained for each group of 5 animals. aP b 0.05 and bP b 0.01 compared to the values from samples of vehicle-treated standard chow-fed rats in the absence of insulin
stimulation, respectively.

per i.v. injection, 3 times daily) for the same treatment period.
Additionally, insulin-stimulated IRS-1 tyrosine phosphorylation
in soleus muscles of fructose chow-fed rats following rosiglita-
zone treatment returned to levels comparable to those of standard
chow-fed animals (Fig. 3). Insulin-stimulated IRS-1 tyrosine
phosphorylation in soleus muscles of fructose chow-fed rats
receiving 2 weeks of myricetin treatment, increased to 2.1-fold the
value in their vehicle-treated counterparts (Fig. 2). The data are
presented in Table 2.
affect the degree of Akt serine (Ser473) phosphorylation in
soleus muscles of the fructose chow-fed group, the value was
significantly increased following 2-week treatment with
rosiglitazone or myricetin (Fig. 3). Quantification of immuno-
blots data is summarized in Table 2.
Effect of repeated myricetin or rosiglitazone treatment on the
insulin-stimulated translocation of glucose transporter subtype
4 (GLUT 4) in soleus muscle of fructose chow-fed rats

Effect of repeated myricetin or rosiglitazone treatment on the
level of p85 regulatory subunit of PI3-kinase in soleus muscle
of fructose chow-fed rats
The basal level of p85 regulatory subunit of PI3-kinase in
soleus muscle of fructose chow-fed rats was depressed to 75%
of that in standard chow-fed group. However, 2-week treatment
with rosiglitazone (4 mg/kg per day) improved the expression of
p85 regulatory subunit of PI 3-kinase, but no change was
observed by insulin stimulation (Fig. 3). Similarly, the
expression of p85 regulatory subunit of PI 3-kinase increased
after the fructose chow-fed animals received 2-week treatment
with myricetin (1 mg/kg per i.v. injection, 3 times daily). Insulin
stimulation in this case too made no difference (Fig. 3).
Quantification of immunoblots is shown in Table 2.
Effect of repeated myricetin or rosiglitazone treatment on the
degree of Akt serine phosphorylation in soleus muscle of
fructose chow-fed rats
In the absence of insulin stimulation, a marked reduction of
Akt serine (Ser473) phosphorylation, (approximately 46% of
that observed in the standard chow-fed rats), was detected in
soleus muscles of the fructose chow-fed group (Fig. 3). It was
observed that after 2-week treatment with rosiglitazone (4 mg/
kg per day) or myricetin (1 mg/kg per i.v. injection, 3 times
daily), the degree of Akt serine (Ser473) phosphorylation in
soleus muscles of fructose chow-fed animals improved
significantly (Fig. 3). Although insulin administration did not
Under insulin-stimulated conditions, GLUT 4 protein
expression in the membrane fraction of soleus muscle from
fructose chow-fed rats was about 40% of that in standard chow-
fed rats, but the same protein content in the cytosolic fraction of
the same sample was about 170% of that observed in the
standard chow-fed group (Fig. 4). However, in fructose chow-
fed animals under insulin-stimulated conditions that received 2-
week treatment with rosiglitazone (4 mg/kg per day), GLUT 4
protein expression in the membrane fraction of soleus muscles
was increased nearly to the same level as in the standard chow-
fed group, whereas the GLUT 4 protein level in the cytosolic
fraction of same sample remained at 60% of the level of their
vehicle-treated counterparts (Fig. 4). At the termination of 2-
week myricetin (1 mg/kg per i.v. injection, 3 times daily)
treatment, insulin-stimulated GLUT 4 protein expression in the
membrane fraction of soleus muscles from fructose chow-fed
animals increased to about 76% of the level of the standard
chow-fed group, while the protein level in the cytosolic fraction
of the same sample decreased to 75% of that observed in their
vehicle-treated counterparts (Fig. 4).
Discussion
Increasing consumption of dietary fructose could be one of
the factors responsible for the development of obesity and the
accompanying insulin resistance syndrome (Basciano et al.,
2005; Le and Tappy, 2006). It has been established that feeding
rats a high-fructose diet induces insulin resistance, hyperinsu-
linemia, hypertriglyceridemia, and multiple metabolic
1486 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
Fig. 4. The autoradiograph resulting from Western blot analysis of representative
protein levels for glucose transporter subtype 4 (GLUT 4) in the membrane (A)
and cytosolic (B) fraction of soleus muscle from fructose chow-fed rats
responded to insulin stimulation following i.v. injection of myricetin (1 mg/kg
per injection, 3 times daily) for 14 days. Rosiglitazone was given by oral gavage
(4 mg/kg per day) for 14 days to the separate groups of the fructose chow-fed
rats. Rats that did not receive any treatment were given in the same volume of
vehicle used to dissolve the test medications. Similar results were obtained with
an additional 4 replications. Quantification of protein levels expressed as mean
with SEM (n = 5 per group) in each column, is indicated in the lower panel.
a
P b 0.05 and bP b 0.01 represents the level of significance compared to the
values with vehicle-treated standard chow-fed rats, respectively.
syndromes observed in humans (Basciano et al., 2005; Le and
Tappy, 2006). The duration of high-fructose feeding and the
lipid content and type of the diets lead to alterations in the
degree of insulin resistance and in the metabolic parameters. In
the present study, an increase in plasma glucose level associated
with hyperinsulinemia definitively suggests impaired insulin
action in 6-week fructose-fed rats. Additionally, the degree of
insulin resistance was found to be higher in fructose-fed rats as
indicated by a higher HOMA-IR, a marker of insulin resistance
(Matthews et al., 1985). Moreover, insulin's activity to stimu-
late disposal of ingested glucose into peripheral tissue was
markedly reduced in the 6-week fructose-fed rats, as evidenced
by the marked reduction of ISIcomp, an index of insulin
sensitivity obtained from the OGTT (Matsuda and DeFronzo,
1999). Compared to HOMA-IR based on measurements of
basal glucose and insulin, the ISIcomp provides a reasonable
approximation of whole-body insulin sensitivity that represents
a composite of hepatic and peripheral tissues and takes into
consideration insulin sensitivity both in the basal state and after
the ingestion of a glucose load (Matsuda and DeFronzo, 1999).
Thus, rats fed 6-week fructose chow could serve as a reliable
model for the investigation of insulin resistance.
Similar to rosiglitazone treatment, the 2-week treatment
regimen with myricetin (1 mg/kg per i.v. injection, 3 times daily)
was found to significantly decrease the high plasma glucose
concentration in rats fed fructose chow for 6 weeks. Conversely,
the same treatment with myricetin had no effect on plasma glucose
level in standard chow-fed rats, showing the beneficial action of
myricetin in rats with insulin resistance. Although the reduced
HOMA-IR could support the fact that insulin resistance in fructose
chow-fed rats was ameliorated by 14-day myricetin treatment, in
contrast to the effect of rosiglitazone, myricetin treatment made no
difference to hyperinsulinemia in fructose chow-fed rats. These
results imply that the major action of myricetin in insulin-resistant
rats is related mainly to improvement of the whole-body insulin
sensitivity. ISIcomp which is an index of composite whole-body
insulin sensitivity, was measured for the entire OGTT period to
ascertain whether myricetin possessed the capacity to increase
insulin sensitivity in insulin-resistant animals. Myricetin treatment
was found to increase the whole-body insulin sensitivity in
fructose chow-fed rats as evidenced by the marked elevation of
ISIcomp in these animals after repeated i.v. injection, as did
rosiglitazone treatment. Clearly, these results imply that myricetin
has the ability to reverse impaired responsiveness to insulin in
insulin-resistant rats demonstrating the insulin-sensitizing effect of
the drug.
Insulin resistance is a general metabolic disorder attributable to
the inefficient functioning of insulin in skeletal muscle, liver and/
or adipose tissue. A common abnormality associated with
diabetes is hypertriglyceridemia (Schwartz, 2006). Myricetin
has been documented to stimulate lipogenesis and to enhance
insulin-stimulated lipogenesis in isolated rat adipocytes (Ong and
Khoo, 1996). The flavonol has also been found to reduce hyper-
glycemia in diabetic rats, possibly through its ability to increase
hepatic glycogen synthesis and to normalize hypertriglyceridemia
(Ong and Khoo, 2000). We observed myricetin to be as effective
as rosiglitazone in the modification of hypertriglyceridemia and in
enhancing hepatic glycogen synthesis in fructose-induced insulin
resistance. The beneficial effects of myricetin on plasma glucose
regulation are thought to be linked to the observed enhancement
of hepatic glycogen synthesis and control of lipid metabolism
(Ong and Khoo, 1996, 2000). However, it was unclear whether
myricetin influenced the insulin responsiveness of skeletal
muscle. It has been documented that insulin action on glucose
 I.-M. Liu et al. / Life Sciences 81 (2007) 14791488
uptake and metabolism is much greater in skeletal muscle
composed primarily of oxidative fibers (e.g., the soleus) as
compared to glycolytic fibers (e.g., the epitrochlearis and extensor
digitorum longus), even though the soleus muscle represents a
small portion of the total muscle mass (Song et al., 1999).
Actually, the increase in insulin action on skeletal muscle is likely
to be related to increased protein expression and/or functional
activity of several key components of the insulin signal trans-
duction cascade. Defects in the insulin signaling cascade leading
to impaired glucose utilization are believed to play a key role in
the pathogenesis of insulin resistance (Shulman, 2000). It is
conceivable that insulin receptor substrate (IRS)-1 tyrosine phos-
phorylation in response to insulin stimulation generally increases
the association of IRS-1 with the p85 subunit of phosphatidyli-
nositol 3-kinase (PI 3-kinase), resulting in increased PI 3-kinase
activity, which in turn leads to activation of serine/threonine
kinase protein B (PKB or Akt) and, ultimately, to an enhancement
in insulin-stimulated glucose disposal (Carvalho et al., 2000). The
detailed mechanisms for the induction of insulin resistance in
carbohydrate-fed animals are still not clear. It seems that excess
fructose may interfere with insulin action by altering the intrinsic
activity of insulin signals, that are similar to the defect caused in
genetically obese Zucker rats, but not the change of IR protein
content, leading to the impairment of insulin sensitivity in
peripheral tissue (Carvalho et al., 2000; Su et al., 2004). There-
fore, we proposed that any observed increases in IRS-1 related
signals in soleus muscles of fructose chow-fed rats after myricetin
treatment would provide strong evidence for the beneficial effects
of the flavonol on impaired insulin action. With this aim in mind,
soleus muscle samples were prepared from all animals after
insulin stimulation.
Similar to the effect of rosiglitazone treatment, we observed
that 2-week myricetin treatment reversed the defect in expression
of IRS-1 and p85 regulatory subunit of PI 3-kinase in soleus
muscle of fructose chow-fed rats under the basal state, but the
protein expression of IR was not influenced by the same treat-
ment. Additionally, increased basal phosphorylation of IR and
down-stream signaling molecules including IRS-1 and Akt in the
soleus muscle of fructose chow-fed rats has been observed by
repeated treatment with this naturally occurring flavonol. Our data
indicate that repeated myricetin treatment improved the impaired
insulin action on the phosphorylation of IR and IRS-1 as well as
Akt in insulin-resistant soleus muscle. Considering that the
regulation of glucose uptake into muscle cells via GLUT4 is a
fundamental action of insulin, and this process is impaired in type
2 diabetes (Ishiki and Klip, 2005), we focused on the cytosolic
and membrane expression of GLUT 4 in soleus muscle. Our data
show that a definite improvement in the defective insulin action
on GLUT 4 translocation from intracellular vesicles to the plasma
membrane of soleus muscle was achieved in fructose chow-fed
rats by repeated myricetin treatment. Although the mode of action
of myricetin is not clear, it could be postulated that myricetin not
only possesses the potential to increase the expression of genes
involved in the insulin signaling pathway, specifically the IRS-1-
associated PI 3-kinase step. In addition it could also potentially
reverse the abnormal responsiveness of insulin in fructose chow-
fed rats mediated through enhanced phosphorylation of signaling
1487
down-stream of IR that ultimately leads to GLUT 4 translocation
in order to maintain glucose homeostasis in fructose-induced
insulin-resistant states.
An amelioration of the impaired insulin signaling pathway by
myricetin treatment in this nutritional model of insulin resistance
clearly indicates that this flavonol displays the characteristics of
rosiglitazone, retaining its insulin sensitization potential but, un-
like rosiglitazone, does not cause any increase in body weight.
The data suggest that that intravenous administration of myricetin
may be a suitable therapeutic adjunct for the treatment of insulin-
resistant patients and/or patients who are particularly sensitive to
the common TZD-induced side effects of weight gain and edema,
while oral administration of myricetin would be feasible give the
high i.v. dose needed be worthy of valuation. Given the high i.v.
dose of myricetin, the oral route of administration could be
considered but this would require extensive prior evaluation.
While it has been documented that in addition to being an
insulin sensitizer, rosiglitazone can also sensitize AMP-activated
protein kinase (AMPK) mediated glucose disposal in peripheral
tissues in insulin-resistant states (Ye et al., 2006), there is no report
to date of the interaction of myricetin treatment with AMPK
activation. Although further studies are needed to determine
whether the insulin-sensitizing effect of myricetin is mediated
through the action of the AMPK pathway, our findings provide a
new insight to the pharmacological benefits of myricetin, which
could be used as a model substance for the development of new
antidiabetic compounds.
In conclusion, myricetin treatment has the potential to
reverse the inability of insulin to act on soleus muscle of rats
receiving fructose-rich chow. The beneficial effects of myricetin
are associated with amelioration of defective insulin action on
specific post-receptor insulin signaling related to IRS-1-
associated PI 3-kinase IRS-1 step and GLUT 4 translocation.
From this point of view, myricetin and its derivatives could
become a promising category of therapeutic agents for in the
treatment arsenal for insulin resistance and type 2 diabetes.
Acknowledgements
The authors would like to thank Miss S.J. Liao for her kind
assistance with the immunoblotting analyses. The present study
was supported by a grant from the National Science Council
(NSC 94-2320-B-127-005) of Taiwan, the Republic of China.
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