Original Article J. Clin. Biochem. Nutr.

, 42, 150–157, March 2008

Ajcbn2008022
Journal
JCBN
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1880-5086
0912-0009
Kyoto,
Original
10.3164/jcbn.2008022
Society
Mouse Japan
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Clinical
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FreeBiochemistry
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Metabolic Syndrome; Increase in Visceral
Adipose Tissue Precedes the Development of Fatty Liver
and Insulin Resistance in High-Fat Diet-Fed Male KK/Ta Mice

Satomi Akagiri1, Yuji Naito1,2,*, Hiroshi Ichikawa3, Katsura Mizushima1, Tomohisa Takagi4,
Osamu Handa4, Satoshi Kokura4, and Toshikazu Yoshikawa1,2,4
1
Inflammation and Immunology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
2
Medical Proteomics, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
3
Department of Food Sciences and Nutritional Health, The Faculty of Human Environment, Kyoto Prefectural
University, Kyoto 606-8522, Japan
4
Biosafety Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

3008
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1
2
42
Received
;157
50
accepted1.10.2007
6.11.2007

Received 1 October, 2007; Accepted 6 November, 2007

Copyright © 2008 To
Summary JCBN
determine the relative contribution of obesity and visceral white adipose tissue
(WAT) to metabolic syndrome, we developed a model that is susceptible to high-fat diet-induced
obesity and insulin resistance using male KK/Ta mice. The ratio of WAT weight to body weight
was greater in the high-fat diet group compared with the control group in 10-, 14-, and 22-
week-old mice. The increase in visceral WAT preceded development of fatty liver and insulin
resistance. Adiponectin mRNA expression in WAT was markedly decreased before the
decrease in its plasma levels or the development of insulin resistance. Insulin resistance
appeared in association with fatty infiltration and TNF-α expression in the liver in 22-week-
old mice. These data indicate that our mouse model would be useful for future studies that
investigate the role of visceral WAT and its products in the development of metabolic syndrome.

Key Words: metabolic syndrome, high-fat diet, white adipose tissue, fatty liver

suggests that adipose tissue is not simply an inert energy

Introduction storage depot but also functions as a major endocrine organ,
producing and releasing a variety of bioactive substances,
The intake of calorie-rich fast food and sedentary life- especially adiponectin, into the bloodstream [4].
styles in developed countries, including Japan, has sharply Traditionally, leptin-deficient (ob/ob) and leptin receptor-
increased the incidence of obesity [1, 2]. Obesity is not only deficient (db/db) mice have been used for studies of obesity.
a serious health and economic burden, but also predisposes Although the ob/ob and db/db mice have many advantages
a person to a variety of metabolic diseases. Metabolic for obesity research, including studies of insulin resistance
syndrome can be characterized by a group of metabolic and adipocyte dysfunction, their utility in studying meta-
risk factors that includes central obesity, insulin resistance, bolic syndrome is limited. For example, the plasma VLDL
dyslipidemia, increased blood pressure, and nonalcoholic and LDL levels do not increase and the HDL levels are
fatty liver disease (NAFLD) [2, 3]. Accumulating evidence elevated upon high-fat diet feeding, which findings are in
contrast to those often noted in humans [5]. The lipoprotein
*To whom correspondence should be addressed.
profiles of ob/ob and db/db mice, both on a chow and high-
Tel: +81-75-251-5505 Fax: +81-75-252-3721 fat diet, are uncharacteristic of wild-type mice or humans.
E-mail: [email protected] Furthermore, these mice exhibit extremely high plasma

150
 Metabolic Syndrome in High-fat Diet-fed Mice 151

glucose levels at young ages (6–10 weeks old). Table 1. Composition of experimental diet
In the current study, we sought to determine the relative Basal High-fat
contribution of obesity and visceral white adipose tissue
w/w (%)
(WAT) to metabolic syndrome, insulin resistance, and
Milk casein 20.00 24.48
NAFLD. To this end, we developed a model that is
DL-methionine 0.30 0.37
susceptible to high-fat diet-induced obesity and insulin
resistance using male KK/Ta mice. We selected this model Corn starch 14.66 5.93
for the following reasons: i) KK/Ta mice, which are known Granulated sugar 49.09 19.85
as an animal model of human non-insulin-dependent Cellulose 5.00 6.12
diabetes mellitus, have such characteristics as moderate Suet powder 6.25 37.50
obesity, polyuria, glucose intolerance, and hyperinsulinemia. Vitamin mix (AIN-76) 1.00 1.22
These characteristics have been shown to appear sponta- Mineral mix (AIN-76) 3.50 4.28
neously at 3 months of age [6, 7]. ii) Male KK/Ta mice are Choline bitartrate 0.20 0.24
more susceptible to visceral obesity with vascular complica- Calorie (kcal/100 g) 373.62 485.60
tions compared to females. Similarly in humans, metabolic Ratio of fat/total calrie (%) 13.10 56.60
syndrome increases visceral adiposity and cardiovascular
disease risk more markedly in men than women [8]. iii) A
high-animal fat diet is thought to be a major risk factor for Histological analysis
metabolic syndrome. A recent review has concluded that Small pieces of epididymal white adipose tissues and
animal fats and omega-6/omega-9-containing plant oils can liver were removed and rinsed with saline. The tissues were
be used to generate an obese and insulin-resistant phenotype fixed with 10% formalin and embedded in paraffin. Tissue
in rodents, whereas fish oil-fed animals do not develop these sections were cut at a thickness of 2.5 µm and stained with
disorders [9]. The aim of the present study was to determine hematoxylin and eosin. To examine the size of the white
the time-course profiles of several parameters which are adipocytes, the area of each adipocyte was measured in 40
characteristic of metabolic syndrome. cells of representative sections with Image J software
(National Institutes of Health, Bethesda, MD). The mean
Materials and Methods value was designated as an index of the cell size.

Animals and treatment Oral glucose tolerance test and determination of plasma
Male KK/Ta Jcl mice (5 weeks old, 18–20 g body weight) glucose, insulin, lipids, and adiponectin
were purchased from CLEA Japan Inc. (Tokyo, Japan). The The oral glucose tolerance test (OGTT) was performed
mice were individually housed in plastic cages at 24 ± 2°C before and 4-, 8- and 16-weeks after the treatment after
and at 45 ± 5% relatively humidity with a 12/12-h light/dark overnight fasting. The mice received a 20% glucose solution
cycle, and were given free access to food and water (2 g/kg). Blood samples were collected just before and
throughout the experimental period. After acclimation for 1 0.5, 1, and 2 h after glucose loading. Plasma glucose levels
week by feeding of a basal diet, 6-week-old KK/Ta mice were determined with a Glutest Ace R (Sanwa Kagaku
were divided into two groups of six mice each by body Kenkyusho Co., Ltd., Nagoya, Japan). Plasma adiponectin
weight and blood glucose level, and fed a purified powdered levels were measured using the mouse adiponectin enzyme-
basal diet or purified high-fat powdered diet containing suet linked immunosorbent assay (ELISA) kit (Ohtsuka, Ltd.,
powder at 37.5 g/100 g diet (high-fat diet) (Table 1), which Tokyo, Japan). Asparate 2-oxoglutarate aminotransferase
were both purchased from CLEA Japan, Inc. (Tokyo, Japan) (AST), alanine 2-oxoglutarate aminotransferase (ALT), blood
and kept sterile. After treatment with the experimental diet urea nitrogen (BUN), free cholesterol, triglyceride, non-
for 2, 4, 8, or 16 weeks, starved animals were sacrificed esterified fatty acid, and LDL-cholesterol were measured
under anesthesia with intraperitoneal injection of sodium with a Shimadzu CL8000 Clinical Chemistry Analyzer
pentobarbital {Nembutal® (Dainippon Sumitomo Pharma (Shimadzu, Ltd., Tokyo, Japan).
Co., Ltd., Osaka, Japan), 50 mg/kg of body weight} to
collect blood, perirenal, epididymal, mesenteric and inguinal Determination of homeostasis model assessment of insulin
WAT, and liver tissue. All animal experiments and care were resistance
conducted in conformity with the Guidelines of the Animal The homeostasis model assessment of insulin resistance
Care and Use Committee of Kyoto Prefectural University of (HOMA-IR) was calculated using glucose and insulin
Medicine. concentrations obtained after 7 h of food withdrawal, using
the following formula: fasting blood glucose (mg/dl) ×
fasting insulin (µU/ml)/405. Plasma insulin levels were

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152 S. Akagiri et al.

measured with an insulin ELISA kit (Morinaga Institute of values <0.05 were considered statistically significant.
Biological Science, Inc., Yokohama, Japan).
Results
mRNA analysis
Mouse epididymal WAT and liver tissue samples were Body weight gain and white adipose tissue weight
taken and stored in an Isogen solution (Nippon Gene Co., Figure 1A shows the changes in body weight of the basal
Ltd., Tokyo, Japan). The mRNA expression levels of diet (control) group and the high-fat diet group, throughout
adiponectin, tumor necrosis factor-α (TNF-α), and β-actin the experimental period. At the beginning of the experiment,
as an internal control were determined by real-time poly- the average body weight of the 6-week-old mice was
merase chain reaction (PCR). Total RNA was isolated 21.4 ± 0.5 g, and this value increased significantly to
from adipose and liver tissues by the acid guanidinium 50.0 ± 2.1 g after 16 weeks of feeding with a high-fat
phenol chloroform method with an Isogen kit (Nippon Gene diet. There were no differences in the average body weight
Co., Ltd.) and the concentration and the quality of RNA between the two groups until 14 weeks of age, but body
were determined by absorbance at 260 nm and the ratio of weight tended to increase in the high-fat diet group
260 nm to 280 nm, respectively. The isolated RNA was compared to the basal diet group in the latter half of the
stored at −70°C until use in real-time PCR. For the real-time experiment period. Figure 1B shows the changes in the ratio
PCR, 1 µg of extracted RNA was reverse-transcribed into of WAT weight to body weight of the control group and the
first-strand complementary DNA (cDNA) at 42°C for 40 min,
using 200 U of M-MLV reverse-transcriptase (Promega
Corporation, Madison, WI) and 0.5 µg of oligo (dT) 15
primer (Takara Bio Inc., Shiga, Japan) in a 20-µl reaction
mixture.
Real-time PCR for adiponectin and TNF-α was carried
out with a 7300 Real-Time PCR system (Applied
Biosystems, Foster City, CA) using the DNA-binding dye
SYBER Green I for the detection of PCR products. The
reaction mixture contained 12.5 µl Premix Ex Taq and
0.5 µL ROX reference dye (Code RRO41A; Takara Bio
Inc., Shiga, Japan), 1 µl custom-synthesized primers, and
2 µl cDNA (equivalent to 20 ng total RNA) to give a final
reaction volume of 25 µl. The PCR settings were as follows:
an initial denaturation for 15 s at 95°C was followed by 40
cycles of amplification for 15 s at 95°C and 31 s at 60°C,
with subsequent melting curve analysis increasing the
temperature from 60 to 95°C. The primers were designed
using a Primer Express® software (Applied Biosystems) and
had the following sequences: for adiponectin, sense 5'-
CTGGCTTTCTTCTCTTCCATGATAC-3', and antisense
5'-GTGTCGACGTTCCATGATTCTC-3' (82-bp product);
for TNF-α, sense 5'-ATCCGCGACGTGGAACTG-3',
and antisense 5'-ACCGCCTGGAGTTCTGGAA-3' (70-bp
product); and for β-actin, sense 5'-TAGCCACCTTCCAG-
CAGATGT-3', and antisense 5'-AGCTCAGTAACAGTC-
CGCCTA-3' (101-bp product). Relative quantification of
gene expression with real-time PCR data was calculated
relative to β-actin.

Data analysis
Fig. 1. Changes in body weight A) and the ratio of white
All statistical analysis was performed with the Statcel 2 adipose tissue (WAT) weight to body weight (B) of the
software package (OMS Publishing Inc, Saitama, Japan). mice fed a basal diet or a high-fat diet. The data are
We used one-way ANOVA with a Scheffe-type multiple expressed as the means ± SE of 5–6 mice. #p<0.05
comparison test to compare several parameters among four compared with 6-week-old mice and +p<0.05 compared
groups. All data are expressed as the means ± SE, and p with basal diet-fed mice.

J. Clin. Biochem. Nutr.

 Metabolic Syndrome in High-fat Diet-fed Mice 153

Fig. 2. Histological examination of white adipose tissues (A) and the changes in the size of white adipocytes (B) obtained from basal
diet-fed and high-fat diet-fed male mice. The data are expressed as the means ± SE of 5–6 mice. #p<0.05 compared with 6-
week-old mice and +p<0.05 compared with basal diet-fed mice.

Fig. 3. Oral glucose tolerance test throughout the experiment. The oral glucose tolerance test (OGTT) was performed both before and
after the treatment after overnight fasting.

high-fat diet group in 6-, 10-, 14-, and 22-week-old mice. Histological analysis of white adipose tissue
The ratio of WAT weight was greater in the high-fat diet Treatment with a high-fat diet clearly increased the
group compared with the control group. In contrast to the average size of adipocytes in the epididymal WAT in a time-
changes of body weight, the weight of white adipose tissue dependent manner (Fig. 2A and B). Compared to adipocytes
was increased in 10- and 14-week-old mice treated with a at the beginning of the experiment, the average size of
high-fat diet compared with the control group. adipocytes was significantly increased by >500% in mice

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154 S. Akagiri et al.

after 16 weeks of feeding with a high-fat diet. As shown in

Fig. 2B, the average size of adipocytes was significantly
larger in the high-fat diet group at the ages of 8, 10, and 14
weeks compared with the control group.

Oral glucose tolerance test

Figure 3 shows the results of the oral glucose tolerance
test in the basal diet and the high-fat diet groups for 6-, 10-,
14-, and 22-week-old mice. Fasting blood glucose levels
were <100 mg/dl in mice throughout the experiment. Blood
glucose levels at 30 min and 60 min after glucose loading
were significantly higher (>300 mg/dl) in 10-, 14-, and 22-
week-old mice than in 6-week-old mice, indicating the
glucose intolerance. There were no significant differences
between two groups throughout the experiment.

mRNA expression of adiponectin and its plasma level

Because adiponectin is reported to be decreased in
obesity, we investigated the changes in adiponectin expres-
sion in epididymal WAT and the plasma adiponectin levels
in high-fat diet-fed mice (Fig. 4A and B). The treatment
with a high-fat diet markedly decreased the adiponectin
mRNA expression in mice at 14 weeks of age compared to
the mice before treatment, and this decrease continued in
mice 22 weeks of age. There was a significant difference in
the level of adiponectin mRNA at 14 weeks of age. The
plasma levels of adiponectin tended to decrease in mice
treated with high-fat diet 14 weeks and 22 weeks of age Fig. 4. Changes in plasma adiponectin (A) and its mRNA
compared with those in 6-week-old mice. expression in epididymal white adipose tissues (B) from
high-fat diet-fed male mice. Plasma adiponectin was
Plasma insulin concentrations and HOMA-IR measured by an ELISA kit, and the mRNA expression
Figure 5A and B show the changes in fasting insulin levels were analyzed by the real-time PCR method. The
concentration and HOMA-IR. These parameters, which are details of the experiments are described in the Materials
indices of insulin resistance, did not change during the and Methods. The data are expressed as the mean ± SE
of 5–6 mice. #p<0.05 compared with 6-week-old mice
feeding with a high-fat diet for 8 weeks, but were signifi-
and +p<0.05 compared with basal diet-fed mice.
cantly increased in 22-week-old mice fed a high-fat diet
for 18 weeks.

Liver histology and TNF-α mRNA expression Blood biochemistry

Because lipid accumulation in the liver accompanied with A summary of the blood chemistry is shown in Table 2.
hepatic inflammation is reported to cause insulin resistance Serum levels of ALT and AST were significantly increased
in high-fat diet-fed C57BL/6J Jcl mice, we investigated the in 14- and 22-week-old mice fed a basal diet and in 22-
histology of the liver and hepatic expression of TNF-α. As week-old mice fed a high-fat diet compared with those in 6-
shown in Fig. 6A, small unstained vesicular regions in the week-old mice. Serum levels of non-esterified fatty acid and
hepatic cell cytoplasm were increased in 14-week-old mice LDL cholesterol level tended to increase in 10-week-old
fed a high-fat diet. In 22-week-old mice, larger unstained mice and in 22-week-old mice, respectively.
vesicular regions were increased in the zone 3 hepatocytes.
Figure 6B shows the changes in hepatic TNF-α mRNA Discussion
expression during the experiment. This expression was
significantly increased in 22-week-old mice compared to The major finding of this study included the following:
that in 6-week-old mice. (1) the increase in visceral adipose tissue preceded develop-
ment of fatty liver and insulin resistance; (2) the increase in
size of white adipocytes was an early event as the fist step to

J. Clin. Biochem. Nutr.

 Metabolic Syndrome in High-fat Diet-fed Mice 155

Fig. 5. Changes in plasma HOMA-IR (A) and plasma insulin (B) from high-fat diet-fed male mice. Plasma insulin was measured by an
ELISA kit, and HOMA-R was calculated by the fasting plasma glucose and insulin levels. The details of the experiments are
described in the Materials and Methods. The data are expressed as the means ± SE of 5–6 mice. #p<0.05 compared with 6-week-
old mice.

Fig. 6. Histological examination of the liver (A) and changes in TNF-α mRNA expression in hepatic tissues from high-fat diet-fed
male mice (B). Liver tissues were stained with hematoxylin and eosin, and the mRNA expression levels were analyzed by the
real-time PCR method. The details of the experiments are described in the Materials and Methods. The data are expressed as the
means ± SE of 5–6 mice. #p<0.05 compared with 6-week-old mice.

Table 2. Plasma biochemical markers

6 week-old 10 week-old 14 week-old 22 week-old

Basal diet High-fat Basal diet High-fat Basal diet High-fat
AST U/l 46 ± 11 48 ± 19 66 ± 5 106 ± 10# 78 ± 6 125 ± 27# 83 ± 3#
ALT U/l 53 ± 10 62 ± 11 66 ± 2 104 ± 14# 80 ± 6 135 ± 34# 95 ± 3#
BUN mg/dl 16 ± 2 18 ± 1 16 ± 1 23 ± 5 18 ± 1 28 ± 7 19 ± 1
Total cholesterol mg/dl 92 ± 12 110 ± 11 124 ± 4 92 ± 8 98 ± 14 84 ± 7 102 ± 8.5
Free cholesterol mg/dl 14 ± 3 12 ± 2 10 ± 0 10 ± 0 10 ± 0 8±2 20 ± 0
Triglyceride mg/dl 42 ± 11 48 ± 13 54 ± 9 42 ± 22 20 ± 7 20 ± 2 28 ± 5
Non-esterified fatty acid µeq/l 446 ± 73 736 ± 157 612 ± 78 710 ± 95 352 ± 140 562 ± 113 497 ± 102
LDL cholesterol mg/dl 76 ± 16 122 ± 13 104 ± 12 82 ± 14 118 ± 12 90 ± 20 132 ± 25
Each data is the mean ± SD of 5 mice. #p<0.05 as compared with 6 week-old mice.

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156 S. Akagiri et al.

obesity; (3) adiponectin mRNA expression was markedly the possibility that the expression of adiponectin mRNA
decreased before the decrease in its plasma levels or the might be disturbed by the accumulation of fatty acids, and
development of insulin resistance; and (4) insulin resistance more closely related to insulin sensitivity than obesity alone.
appeared in association with fatty infiltration and TNF-α Similar findings were reported by Yamauchi et al. [15–17],
expression in the liver. Although there were no significant who demonstrated that adiponectin is decreased in obesity
differences in several parameters between the basal diet and deficient in mice that are lacking in specific adipose
group and the high-fat diet group at the end of experiment tissues that play a causal role in the development of insulin
(22-week-old mice), several characteristics of the metabolic resistance, and that the replenishment of adiponectin might
syndrome, including visceral adipose tissue accumulation provide a novel treatment modality for insulin resistance,
and decreased adiponectin expression were developed more type 2 diabetes, and atherosclerosis. The present data
earlier in the high-fat diet group compared to the basal diet indicate that these mice will be useful for future studies
group. investigating the role of visceral WAT and its products in the
Several risk factors comprise metabolic syndrome— development of metabolic syndrome.
namely, central obesity, insulin resistance, hypertension, Nonalcoholic steatosis (nonalcoholic fatty liver disease,
inflammation, and dyslipidemia. The present data demon- NAFLD) is now considered a metabolic pathway to
strate that this model exhibits many of these features. In advanced liver disease, cirrhosis and hepatocellular
particular, the marked fat accumulation was observed in carcinoma. Accumulating evidence suggests that NAFLD is
visceral white adipose tissue/cells within a month after the the hepatic expression of metabolic syndrome [2, 3]. In
treatment with high-fat diet, and this accumulation preceded this model, fatty infiltration into zone 3, which is similar to
the development of insulin resistance. In addition, the fat NAFLD, was observed in mice fed a high-fat diet for 16
source of the high-fat diet in this study consisted of saturated weeks, in association with the increases in serum ALT and
fatty acids derived from animal fats. Saturated fatty acids AST. In addition, we confirmed the increased expression of
have been known to have deleterious effects on health, but TNF-α mRNA, an index of inflammation, in the liver
more recently, they have been specifically implicated in associated with the appearance of insulin resistance in
activating toll-like receptor-4, leading to the expression of 22-week-old mice. These data indicate the close association
inflammatory gene products [10, 11]. Although it is still between insulin resistance and hepatic steatosis with
unclear how excess visceral fat triggers metabolic syndrome, inflammation. Cai et al. [18] previously demonstrated that
the present data clearly indicated that the high-fat diet hepatic inflammation induced by lipid accumulation plays a
induced visceral fat accumulation preceding insulin crucial role of in the development of insulin resistance. They
resistance. Coenen et al. [12] have also developed a clearly showed that hepatic activation of IKK-β and NF-κB
model of metabolic syndrome in mice fed a Western diet, in common models of obesity, high-fat diet and genetic
which they defined as a caloric density of 4.8 kcal/g with hyperplasia causes insulin resistance both locally in the liver
42% of the calories from anhydrous milk fat and 0.15% and systemically. Consequently, these mice will be useful
added cholesterol. Their model clearly demonstrated that for future studies of the metabolic syndrome associated with
Western diet-induced obesity contributes to accumulation of NAFLD.
adipose tissue-resident macrophages and its downstream In conclusion, we have developed a mouse model of
pathophysiological consequences, such as inflammation and metabolic syndrome using male KK/Ta mice fed a high-fat
insulin resistance, but that hyperlipidemia does not. diet. Using this model, we plan to more precisely clarify the
Together with our present findings, these data support the mechanisms involved in the development of metabolic
idea that adipocytes have important implications for the syndrome, especially the association between lipid
pathogenesis of diet-induced obesity, even when plasma accumulation-induced dysfunction of visceral adipocytes
lipid abnormalities are not present. and the induction of insulin resistance. In addition, the
In addition to the morphological changes, the function of model presented herein may be useful for the evaluation of
adipocytes in mice fed a high-fat diet may be affected by preventive medicine, including food factors, for obesity-
the intracellular fat deposition. The mRNA expression of induced metabolic syndrome.
adiponectin and its plasma levels are significantly reduced in
obese/diabetic mice [13] and humans [14]. In the present Acknowledgment
study, the mRNA levels of adiponectin in the WAT were
markedly decreased 8 weeks after the treatment with a This study was supported by a Grant for the Research and
high-fat diet, and this decrease preceded the decrease in the Development Program for New Bio-industry Initiatives
plasma adiponectin protein levels. In addition, the decrease from Bio-oriented Technology Research Advancement
in plasma adiponectin levels was correlated with the Institution.
increases in plasma insulin and HOMA-IR. These data raise

J. Clin. Biochem. Nutr.

 Metabolic Syndrome in High-fat Diet-fed Mice 157

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