cho2010

Food and Chemical Toxicology 48 (2010) 937–943
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism
in high-fat diet-induced-obese mice
Ae-Sim Cho a, Seon-Min Jeon b, Myung-Joo Kim c, Jiyoung Yeo d, Kwon-Il Seo e, Myung-Sook Choi b,*,
Mi-Kyung Lee e,*
a
Department of Nutrition Education, Graduate School of Education, Sunchon National University, Suncheon 540-742, Republic of Korea
Department of Food Science and Nutrition, Kyungpook National University, Daegu 702-701, Republic of Korea
Department of Food Science and Nutrition, Daegu Polytechnic College, Daegu 706-022, Republic of Korea
d
School of Oriental Medicine, Pusan National University, Busan 626-810, Republic of Korea
e
Department of Food and Nutrition, Sunchon National University, Suncheon 540-742, Republic of Korea
b
c
a r t i c l e
i n f o
Article history:
Received 5 September 2009
Accepted 5 January 2010
Keywords:
Chlorogenic acid
Caffeic acid
High-fat diet
Lipid metabolism
Obesity
a b s t r a c t
This study investigated the efficacy of chlorogenic acid on altering body fat in high-fat diet (37% calories
from fat) induced-obese mice compared to caffeic acid. Caffeic acid or chlorogenic acid was supplemented with high-fat diet at 0.02% (wt/wt) dose. Both caffeic acid and chlorogenic acid significantly lowered body weight, visceral fat mass and plasma leptin and insulin levels compared to the high-fat control
group. They also lowered triglyceride (in plasma, liver and heart) and cholesterol (in plasma, adipose tissue and heart) concentrations. Triglyceride content in adipose tissue was significantly lowered, whereas
the plasma adiponectin level was elevated by chlorogenic acid supplementation compared to the high-fat
control group. Body weight was significantly correlated with plasma leptin (r = 0.894, p < 0.01) and insulin (r = 0.496, p < 0.01) levels, respectively. Caffeic acid and chlorogenic acid significantly inhibited fatty
acid synthase, 3-hydroxy-3-methylglutaryl CoA reductase and acyl-CoA:cholesterol acyltransferase activities, while they increased fatty acid b-oxidation activity and peroxisome proliferator-activated receptors
a expression in the liver compared to the high-fat group. These results suggest that caffeic acid and chlorogenic acid improve body weight, lipid metabolism and obesity-related hormones levels in high-fat fed
mice. Chlorogenic acid seemed to be more potent for body weight reduction and regulation of lipid
metabolism than caffeic acid.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Phenolic acids are secondary metabolites, which are commonly
found in plants. Many epidemiological studies have found that the
consumption of foods and drinks with high phenolic content is
associated with the prevention of coronary disease, cancer and so
on (Hertog et al., 1995; Scalbert and Williamson, 2000). Among
these, hydroxycinnamic acids constitute a major class of phenolic
acids that are widely available in seeds, fruits and vegetables.
The daily intake of hydroxycinnamic acid derivates may easily
reach 0.5–1 g in humans (Radtke et al., 1998; Clifford, 1999). Previous studies revealed that hydroxycinnamic acids (q-coumaric
* Corresponding authors. Addresses: Department of Food Science and Nutrition,
Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu, 702-701,
Korea (M.-S. Choi); Department of Food and Nutrition, Sunchon National University,
413 Jungangno, Suncheon, Jeonnam, 540-742, Republic of Korea (M.-K. Lee). Tel.:
+82 53 950 6232; fax: +82 53 950 6229 (M.-S. Choi), Tel.: +82 61 750 3656; fax: +82
61 752 3657 (M.-K. Lee).
E-mail addresses: mschoi@knu.ac.kr (M.-S. Choi), leemk@sunchon.ac.kr (M.-K.
Lee).
0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.01.003
acid, caffeic acid, ferulic acid) and their derivates efficiently improved hypercholesterolemia and type 2 diabetes (Kim et al.,
2003; Lee et al., 2003; Jung et al., 2006). In particular, caffeic acid
is one of the most abundant hydroxycinnamic acids in the human
diet and may occur in esterified form with ether quinic acid or tartaric acid (Gonthier et al., 2006). Among the quinic acid conjugates,
chlorogenic acid (5-O-caffeoylquinic acid, Fig. 1) is predominant in
plants, fruits and vegetables such as coffee beans, apples, pears,
tomatoes, blueberries, potatoes, peanuts and eggplants (Azuma
et al., 2000).
Chlorogenic acid inhibits carcinogenesis in the colon, liver, and
tongue, and protects against oxidative stress in vivo (Mori et al.,
1986; Tanaka et al., 1993; Tsuchiya et al., 1996). Chlorogenic acid
has been claimed to modulate the glucose-6-phosphatase involved
in glucose metabolism (Hemmerle et al., 1997) and to reduce the
risk cardiovascular disease by decreasing oxidation of low density
lipoprotein (LDL)-cholesterol and total cholesterol (Nardini et al.,
1995). More recently, Hsu et al. (2006) reported that chlorogenic
acid inhibited preadipocyte population growth, which may provide
a proposed mechanism for reducing obesity. Therefore, there is
938
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
2.3. Plasma and tissue lipids
The plasma concentrations of total cholesterol, HDL-cholesterol and triglyceride
(Asan Diagnostics, Seoul, Korea) were determined using an enzymatic method. The
plasma free fatty acid (FFA) concentrations were determined using an enzymatic
colorimetric method (Wako Chemicals, Richmond, VA). The hepatic, adipose tissue
and heart lipids were extracted using the procedure developed by Folch et al. (1957)
and the cholesterol and triglyceride concentrations were analyzed with the same
enzymatic kit as used in the plasma analysis.
2.4. Preparation of samples
Fig. 1. Chemical structures of caffeic acid and chlorogenic acid.
Table 1
Composition of the experimental diets.
a
b
Component
Normal % HF control % HF-caffeic
HF(w/w)
(w/w)
acid % (w/w) chlorogenic
acid % (w/w)
Casein
Corn starch
Sucrose
Cellulose
Corn oil
Beef tallow
AIN-mineral mixturea
AIN-vitamin mixtureb
DL-Methionine
20.0
50.0
15.0
5.0
5.0
–
3.5
1.0
0.3
20.0
34.0
15.0
5.0
–
21.0
3.5
1.0
0.3
20.0
33.98
15.0
5.0
–
21.0
3.5
1.0
0.3
20.0
33.98
15.0
5.0
–
21.0
3.5
1.0
0.3
Choline bitartrate
Caffeic acid
Chlorogenic acid
0.2
–
–
0.2
–
–
0.2
0.02
–
0.2
–
0.02
Total (%)
100
100
100
100
kcal/100 g diet
Calories from fat (%)
421.5
11.0
515.5
37.0
510
37.0
513.5
37.0
Mineral mixture according to AIN-76.
Vitamin mixture according to AIN-76.
The enzyme source fractions in the liver were prepared according to the method
developed by Hulcher and Oleson (1973) with a slight modification. A 20% (w/v)
homogenate was prepared in a buffer containing 0.1 M triethanolamine, 0.02 M
EDTA and 2 mM dithiothreitol (pH 7.0). This homogenate was centrifuged at 600g
for 10 min to discard any cellular debris. The supernatant was then centrifuged at
10,000g for 20 min and then again at 12,000g for 20 min at 4 °C to remove the mitochondrial pellet. Subsequently, the supernatant was ultracentrifuged twice at
100,000g for 60 min at 4 °C to obtain the cytosolic supernatant. The mitochondrial
and microsomal pellets were then redissolved in 800 lL of a homogenization buffer,
and their protein content was determined by the method of Bradford (Bradford,
1976) using bovine serum albumin (BSA) as the standard.
2.5. Hepatic lipid-regulating enzyme activities
The fatty acid synthase (FAS) activity was determined by a spectrophotometric
assay. This assay is based on measuring the malonyl-CoA-dependent oxidation of
NADPH according to the methods by Nepokroeff et al. (1975) with a slight modification. One unit of enzyme activity represented the oxidation of 1 nmol of NADPH
per minute at 37 °C. Fatty acid b-oxidation (b-oxidation) activity was measured
spectrophotometrically by monitoring the reduction of NAD to NADH in the presence of palmitoyl-CoA as described by Lazarow (1981) with a slight modification.
3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase activity was determined in
the microsome with [14C]-HMG-CoA as the substrate based on a modification of
the method of Shapiro et al. (1974). The activity was expressed as the synthesized
mevalonate pmol/min/mg protein. Acyl-CoA:cholesterol acyltransferase (ACAT)
activity was determined by the rate of incorporation of [14C]-oleoyl CoA into cholesterol ester fractions, as described by Erickson et al. (1980) and modified by Gillies
et al. (1986). The activity was expressed as synthesized cholesteryl oleate pmol/
min/mg protein.
2.6. Western blot analysis
increasing interest in an anti-obesity effect of chlorogenic acid
in vivo. In this study, the anti-obesity potential of chlorogenic acid
was investigated using high-fat diet-induced-obese mice by comparing it with that of caffeic acid.
2. Methods and materials
2.1. Animals and diets
Thirty-two male ICR mice (4 weeks old) were obtained from Orient Inc. (Seoul,
Republic of Korea). The mice were all individually housed in polycarbonate cages at
22 ± 2 °C on a 12 h light–dark cycle. All mice were fed pellets of commercial chow
for 1 week after arrival. The mice were randomly divided into four groups (n = 8),
and respectively fed a normal diet (5% corn oil, wt/wt), a high-fat diet containing
37% calories from fat (21% beef tallow, wt/wt), a high-fat diet plus 0.02% caffeic acid
(0.2 g/kg diet, TCI Co., Ltd., Japan) and a high-fat diet plus 0.02% chlorogenic acid
(0.2 g/kg diet, TCI Co., Ltd., Japan). The composition of the experimental diet (Table 1) was based on the AIN-76 semisynthetic diet (American Institute of Nutrition,
1977, 1980). The mice had free access to food and water, and their food consumption was measured daily while their weight gain was measured weekly.
At the end of the experimental period, the mice were anesthetized with ether
after withholding food for 12 h. Blood samples were taken from the inferior vena
cava to determine the plasma biomarkers. After collecting the blood, the liver, adipose tissue and heart were removed, rinsed with a physiological saline solution and
immediately stored at 70 °C. The white adipose tissues (epididymal and perirenal)
were collected and then weighed immediately. All of the mice were treated in strict
accordance with the Sunchon National University guidelines for the care and use of
laboratory animals.
The liver was homogenized with a buffer containing 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol-bis(2-aminoethylethertetraacetic acid (EGTA), 50 mM NaF, 1% triton-X, 1 mM phenylmethylsulfonyl fluoride, 25 lg/mL leupeptin and 2 lg/mL aprotinin. Lysates were centrifuged at 8550g
for 1 h at 4 °C. The supernatant protein content was determined following the
method established by Bradford (1976), using BSA as the standard. Equal amounts
of protein (30 lg per lane) were separated by 7% sodium dodecyl sulfate (SDS)–
polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes,
blocked BSA and incubated overnight with polyclonal rabbit anti-PPAR a (1:200)
(Santa Cruz Biotechnology, Inc., USA). After a washing procedure, the blots were
incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody
(Santa Cruz Biotechnology, Inc., USA) for 1 h at room temperature. The immunoreactive bands were visualized with an enhanced chemiluminescence kit according to
the manufacture’s instructions (Santa Cruz Biotechnology, Inc., USA). The blots were
stripped by treating them two times for 30 min with 200 mM glycine, 0.1% SDS and
1% Tween-20 followed, washed, again incubated overnight at 4 °C with b-actin antibody (1:1000) (Santa Cruz Biotechnology, Inc., USA) and the remaining procedures
as described above were followed. The b-actin was used for loading standardization.
2.7. Statistical analysis
All data are presented as the mean ± S.E. The data were evaluated by a one-way
ANOVA SPSS program and by determining the differences between the means using
the Duncan’s multiple-range test. Correlation analyses utilized the Pearson’s coefficient. Values were considered statistically significant when p < 0.05.
3. Results
2.2. Plasma leptin, insulin and adiponectin levels
3.1. Body weight, food intake, energy intake and visceral fat weight
The plasma leptin (R&D systme, USA), adiponectin (R&D systme, USA) and insulin (Shibaygi Co., Ltd., Japan) levels were determined using a quantitative sandwich
enzyme immunoassay kit.
The final body weight of mice fed the high-fat diet was significantly higher than that of mice fed the normal diet (Table 2). How-
939
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
Table 2
Effect of caffeic acid and chlorogenic acid supplementation on body weight, food intake and daily energy intake in high-fat diet-induced-obese mice.*
Initial body weight (g)
Normal
HF control
HF-caffeic acid
HF-chlorogenic acid
28.49 ± 0.38
28.75 ± 0.42
28.42 ± 0.40
28.53 ± 0.22
Final body weight (g)
a
42.28 ± 0.73
47.47 ± 2.05b
43.53 ± 1.05a
40.05 ± 1.26a
Body weight gain (g)
ab
13.90 ± 0.91
18.50 ± 1.32c
15.45 ± 1.07bc
11.79 ± 0.97a
Food intake (g/day)
Energy intake (kcal/day)
7.07 ± 0.30
6.58 ± 0.30
6.32 ± 0.13
6.28 ± 0.08
29.83 ± 1.26a
33.90 ± 0.38b
32.24 ± 0.46b
32.20 ± 0.49b
abc
*
The means in the column not sharing a common letter are significantly different between groups (p < 0.05).
Values are expressed as means ± S.E.
Table 3
Effect of caffeic acid and chlorogenic acid supplementation on plasma leptin, insulin
and adiponectin concentrations in high-fat diet-induced-obese mice.*
Normal
HF control
HF-caffeic acid
HF-chlorogenic acid
Leptin
(ng/mL)
Insulin
(ng/mL)
Adiponectin
(ug/mL)
3.32 ± 0.30a
5.58 ± 0.50b
3.32 ± 0.34a
2.52 ± 0.15a
5.32 ± 0.21a
6.93 ± 0.25b
5.21 ± 0.33a
4.86 ± 0.45a
7.41 ± 0.41ab
3.92 ± 0.37c
4.24 ± 0.49c
6.02 ± 0.23b
abc
The means in the column not sharing a common letter are significantly different
between groups (p < 0.05).
*
Values are expressed as means ± S.E.
Fig. 2. Effect of caffeic acid and chlorogenic acid supplementation on visceral fat
weight in high-fat diet-induced-obese mice. Values are expressed as means ± S.E.
abc
The means not sharing a common letter are significantly different between
groups (p < 0.05).
ever, the caffeic acid and its ester chlorogenic acid supplements
significantly reduced body weight compared to the high-fat control
group by 8% and 16%, respectively. Among the high-fat fed groups,
body weight gain was significantly lower in the chlorogenic acid
group compared to the high-fat control or caffeic acid supplemented group. Caffeic acid and chlorogenic acid did not affect food
intake and daily energy intake in high-fat feeding mice (Table 2).
Obesity with predominant visceral fat is associated with diabetes mellitus, dyslipidemia and hypertension. As shown in Fig. 2,
visceral fat weight of mice fed high-fat diet for 8 weeks was significantly higher than the normal group. However, the epididymal
white adipose tissues weight was significantly lower in the caffeic
acid and chlorogenic acid groups as compared to the high-fat control group by 22% and 46%, respectively (Fig. 2). In particular, the
perirenal adipose tissue weight of the chlorogenic acid was only
42% of the high-fat control value (Fig. 2).
3.2. Plasma leptin, insulin and adiponectin levels
A high-energy diet generally increases adipose tissue mass and
leptin secretion, which induces leptin resistance, resulting in lipotoxicity (Torre-Villalvazo et al., 2008). To evaluate the effect of
chlorogenic acid on plasma insulin and adipokine levels, we determined the plasma leptin, insulin and adiponectin concentrations
using ELISA assay.
The high-fat control diet caused to increase the plasma leptin
and insulin levels significantly compared to the normal diet fed
mice, while it decreased the plasma adiponectin level. However,
both caffeic acid and chlorogenic acid supplemented groups were
lower in the plasma letpin and insulin levels compared to the
high-fat control group (Table 3). In this study, the leptin level
was positively correlated with the insulin level in plasma
(r = 0.594, p < 0.01). Body weight was positively correlated with
the plasma leptin level (r = 0.894, p < 0.01) and the insulin level
(r = 0.496, p < 0.01), respectively. Epididymal white adipose tissue
weight was positively correlated with the plasma leptin level
(r = 0.791, p < 0.01) and the insulin level (r = 0.462, p < 0.01) after
the 8-week experimental period (Fig. 3). When compared to the
high-fat control group, the plasma adiponectin concentration was
only significantly higher in the chlorogenic acid group (Table 3).
3.3. Lipid contents in plasma, liver, adipose tissue and heart
Supplementation of caffeic acid and chlorogenic acid to a highfat diet significantly lowered the plasma triglyceride and total cholesterol concentrations compared to the high-fat group. Plasma
free fatty acid concentration was significantly lowered by caffeic
acid and chlorogenic acid supplementation. In addition, the caffeic
acid and chlorogenic acid supplementation significantly increased
HDL-cholesterol to total cholesterol ratio compared to the highfat control group (Table 4).
Supplementation with caffeic acid and chlorogenic acid significantly lowered the triglyceride concentrations in the liver and
heart compared to the high-fat control group (Fig. 4). Adipose tissue triglyceride concentration was only decreased by chlorogenic
acid supplementation. The cholesterol concentrations of adipose
tissue and heart were significantly lower in the caffeic acid and
chlorogenic acid groups compared to the high-fat control group
(Fig. 4).
3.4. Activities of hepatic lipid-regulating enzymes
To understand the mechanism involved in the effect of chlorogenic acid on lipid metabolism, hepatic lipid-regulating enzymes
activities such as lipogenesis, fatty acid oxidation and cholesterol
synthesis were investigated.
The hepatic FAS, HMG-CoA reductase and ACAT activities were
significantly higher in the high-fat control group than in the normal group, whereas the b-oxidation activity was significantly lower (Table 5). The caffeic acid and chlorogenic acid lowered hepatic
FAS, HMG-CoA reductase and ACAT activities compared to the
high-fat control group, while they elevated fatty acid b-oxidation
activity. Thus, caffeic acid and chlorogenic acid inhibited fatty acid
and cholesterol synthesis and stimulated fatty acid oxidation in the
liver (Table 5).
940
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
Fig. 3. Effect of caffeic acid and chlorogenic acid supplementation on correlation between parameters in high-fat diet-induced-obese mice.
Table 4
Effect of caffeic acid and chlorogenic acid supplementation on plasma lipid contents in high-fat diet-induced-obese mice.*
Triglyceride (mmol/L)
Normal
HF control
HF-caffeic acid
HF-chlorogenic acid
b
1.65 ± 0.12
1.95 ± 0.26c
1.22 ± 0.16ab
0.91 ± 0.09a
Free fatty acid (mmol/L)
a
0.40 ± 0.03
0.52 ± 0.05b
0.33 ± 0.01a
0.30 ± 0.04a
Total cholesterol (mmol/L)
a
3.23 ± 0.31
5.53 ± 0.47b
4.09 ± 0.28a
3.42 ± 0.27a
HDL-cholesterol (mmol/L)
HTRA (%)
1.76 ± 0.05
1.82 ± 0.06
1.90 ± 0.17
1.86 ± 0.18
52.17 ± 1.72ab
34.73 ± 3.08c
46.15 ± 1.92b
53.73 ± 1.19a
abc
*
The means in the column not sharing a common letter are significantly different between groups (p < 0.05).
Values are expressed as means ± S.E.
HTR = HDL-cholesterol/total cholesterol 100.
A
3.5. Expression of hepatic PPAR a
4. Discussion
PPAR a is one of nuclear transcription factors that act as lipid
sensors and regulate lipid metabolism (Willson and Wahli,
1997). Liver is the major target tissue of PPAR a, and its key
genes include the enzymes involved in the b-oxidation of fatty
acid (Chen et al., 2008). Therefore, we examined the hepatic
PPAR a expression by Western blotting. In comparison with
the high-fat control group, supplementation of caffeic acid and
chlorogenic acid stimulated the expression of PPAR a in the liver
(Fig. 5).
The role of chlorogenic acid in a diet-induced obesity model has
not yet been fully established. Therefore, the effect of chlorogenic
acid has been investigated on body weight, body fat distribution
and lipid metabolism in high-fat diet-induced-obese mice by comparing it with caffeic acid, which is an unesterified structure of
chlorogenic acid.
This study demonstrated that caffeic acid and its ester, chlorogenic acid, significantly reduced body weight, by approximately
8% and 16%, respectively, and epididymal adipose tissue weight,
941
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
Fig. 4. Effect of caffeic acid and chlorogenic acid supplementation on lipid contents of liver (A and B), epididymal adipose tissue (C and D) and heart (E and F) in high-fat dietinduced-obese mice. Values are expressed as means ± S.E. abcThe means not sharing a common letter are significantly different between groups (p < 0.05).
Table 5
Effect of caffeic acid and chlorogenic acid supplementation on hepatic lipid-regulating enzyme activities in high-fat diet-induced-obese mice.*
Normal
HF control
HF-caffeic acid
HF-chlorogenic
acid
Fatty acid synthaseA (nmol/min/mg
protein)
Fatty acid b-oxidation (nmol/min/mg
protein)
HMG-CoA reductase (pmol/min/mg
protein)
ACAT (pmol/min/mg
protein)
2.09 ± 0.17a
3.62 ± 0.08b
2.01 ± 0.26a
1.57 ± 0.11a
7.06 ± 0.49a
3.41 ± 0.54c
5.43 ± 0.65b
7.24 ± 0.47a
220.64 ± 20.89a
462.57 ± 19.33c
352.43 ± 23.30b
348.05 ± 20.21b
169.34 ± 8.91a
250.60 ± 10.58c
210.87 ± 16.39b
207.40 ± 6.70b
abc
The means in the column not sharing a common letter are significantly different between groups (p < 0.05).
Values are expressed as means ± S.E.
A
FAS, fatty acid synthase; b-oxidation, free fatty acid b-oxidation; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase; ACAT, acyl-coenzyme A:cholesterol
acyltransferase.
*
by approximately 22% and 46%, respectively, compared to the highfat control group without changing daily food or energy intake. Under a normal diet feeding condition, the oral administration of
chlorogenic acid (30 and 60 mg/kg/day) for 14 days reduced the
hepatic triglyceride content but the amount of visceral fat was unchanged (Shimoda et al., 2006). However, in this study, chlorogenic
acid significantly lowered the body weight gain and perirenal adipose weight compared to the high-fat control group and caffeic
acid group. Thus, chlorogenic acid suppressed body weight gain
and lowered visceral fat mass more efficiently than caffeic acid in
high-fat diet fed mice.
Visceral adipose tissue releases a large amount of free fatty
acids (FFA) and hormones/cytokines in the portal vein that can
be delivered to the liver and interact with hepatocytes and various
immune cells (Lafontan and Girard, 2008). It was also observed
that both the body weight and epididymal adipose weight were
positively correlated with plasma leptin and insulin levels, respectively. The plasma leptin and insulin levels were significantly
942
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
Fig. 5. Effect of caffeic acid and chlorogenic acid supplementation on the expression
of PPAR a in high-fat diet-induced-obese mice.
higher in the high-fat group than in the normal diet group; however, these hormone levels were significantly lowered by caffeic
acid and chlorogenic acid supplementation. These results showed
a significant positive correlation between leptin and insulin levels
in the plasma (r = 0.594, p < 0.01).
During the development of leptin resistance, there is an increased release of FFA from adipose tissue to other tissues, mainly
to the liver (Torre-Villalvazo et al., 2008). As a result, hepatic esterification of FFA to triglycerides leads to the formation of fatty liver
that is accelerated by an increased lipogenesis as a consequence of
hyperinsulinemia and decreased FFA oxidation (Torre-Villalvazo
et al., 2008). In this study, it was confirmed that high-fat feeding
for 8 weeks to mice induced a (Mathieu et al., 2008) decrease of
fatty acid oxidation in the liver. Several studies have pointed out
that the most common form of insulin resistance is associated with
an increased accumulation visceral fat (Slawik and Vidal-Puig,
2006). Prevention of hyperinsulinemia may ameliorate metabolic
abnormalities that occur in the liver as a consequence of obesity
(Torre-Villalvazo et al., 2008). In particular, visceral fat depot was
associated with metabolic abnormalities contributing to the development of cardiovascular disease (Mathieu et al., 2008). The lipotoxic theory states that the accumulation of lipid in target organs
such as skeletal muscle, kidney, pancreas and heart, may contribute to the development of obesity-related disorders (Slawik and Vidal-Puig, 2006). Recently, it has been reported that chlorogenic
acid (5 mg/kg body weight, intravenously) improved glucose tolerance and decreased plasma and hepatic lipids without changing
triglyceride concentration in the adipose tissue of (fa/fa) Zucker
rats, which is an animal model for obese type 2 diabetes (de Sotillo
and Hadley, 2002). However, in this study, caffeic acid and chlorogenic acid significantly lowered the plasma FFA, triglyceride and
total cholesterol concentrations as well as lipid contents of tissues
such as liver, epididymal adipose tissue and heart.
This study showed that caffeic acid and chlorogenic acid increased the expression of PPAR a with a simultaneous increase in
fatty acid b-oxidation in the liver compared to the high-fat control
group. In particular, chlorogenic acid normalized the activity of hepatic fatty acid b-oxidation in diet-induced-obese mice to similar
to values of a normal diet fed group. In some preclinical studies,
more potent and selective PPAR a agonists have been shown to
prevent the development of diabetes/insulin resistance in rodent
models of diabetes by reducing adiposity, improving peripheral
insulin action, and exerting beneficial effects on pancreatic b-cells
(Guerre-Millo et al., 2000; Kim et al., 2003; Bergeron et al., 2006).
Previous research suggested that adiponectin increased fatty acid
combustion and energy consumption via PPAR a activation at least
in part, which led to decreased triglyceride content in the liver and
skeletal muscle and thus coordinately increased in vivo insulin sensitivity (Kadowaki and Yamauchi, 2005). Plasma adiponectin level
has been reported to be significantly reduced in obese/diabetic
mice and humans (Yamauchi et al., 2001; Arita et al., 1999) and
to be decreased in patients with cardiovascular disease (Kumada
et al., 2003), hypertension (Ouchi et al., 2003), or metabolic syndrome (Trujillo and Scherer, 2005). Although both chlorogenic acid
and caffeic acid improved adiponectin to leptin ratio in diet-induced-obese mice, only chlorogenic acid significantly elevated
plasma adiponectin concentration compared to the high-fat control group and caffeic acid group. Moreover, chlorogenic acid
seems to be a more potential stimulator of hepatic fatty acid b-oxidation than caffeic acid. Furthermore, we showed that caffeic acid
and chlorogenic acid also efficiently inhibited fatty acid and cholesterol biosynthesis as evidenced by suppressing the activities of
fatty acid synthase, HMG-CoA reductase and ACAT in the liver.
In conclusion, chlorogenic acid and caffeic acid exhibited a potential anti-obesity effect in high-fat diet-induced mice, which
may be mediated by altering plasma adipokine level and body fat
distribution and down-regulating fatty acid and cholesterol biosynthesis, whereas up-regulating fatty acid oxidation and PPAR a
expression in the liver. Chlorogenic acid seemed to be more potent
and efficacious anti-obesity agent in diet-induced-obese mice than
caffeic acid. Although, more studies are needed to support this
promising mechanism, this research might provide implication in
human anti-obesity effect.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This work was supported by the Science Research Center (SRC)
program of Korea Science and Engineering Foundation (KOSEF, No.
2009-0063409).
References
American Institute of Nutrition, 1977. Report of the American institute of nutrition
ad hoc committee on standards for nutritional studies. J. Nutr. 107, 1340–1348.
American Institute of Nutrition, 1980. Report of ad hoc committee on standards for
nutritional studies. J. Nutr. 110, 1717–1726.
Arita, Y., Kihara, S., Ouchi, N., Takahashi, M., Maeda, K., Miyagawa, J., Hotta, K.,
Shimomura, I., Nakamura, T., Miyaoka, K., Kuriyama, H., Nishida, M., Yamashita,
S., Okubo, K., Matsubara, K., Muraguchi, M., Ohmoto, Y., Funahashi, T.,
Matsuzawa, Y., 1999. Paradoxical decrease of an adipose-specific protein
adiponectin, in obesity. Biochem. Biophys. Res. Commun. 257, 79–83.
Azuma, K., Ippoushi, K., Nakayama, M., Ito, H., Higashio, H., Terao, J., 2000.
Absorption of chlorogenic acid and caffeic acid in rats after oral administration.
J. Agric. Food Chem. 48, 5496–5500.
Bergeron, R., Yao, J., Woods, J.W., Zycband, E.I., Liu, C., Li, Z., Adams, A., Berger, J.P.,
Zhang, B.B., Moller, D.E., Doebber, T.W., 2006. Peroxisome proliferatorsactivated receptor (PPAR)-alpha agonism prevents the onset of type 2
diabetes in Zucker diabetic fatty rats: a comparison with PPAR gamma
agonism. Endocrinology 147, 4252–4262.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein–dye binding.
Anal. Biochem. 72, 248–254.
Chen, X., Matthews, J., Zhou, L., Pelton, P., Liang, Y., Xu, J., Yang, M., Cryan, E.,
Rybczynski, P., Demarest, K., 2008. Improvement of dyslipidemia, insulin
sensitivity, and energy balance by a peroxisome proliferators-activated
receptor a agonist. Metab. Clin. Exp. 57, 1516–1525.
Clifford, M.N., 1999. Chlorogenic acids and other cinnamates-nature, occurrence
and dietary burden. J. Sci. Food Agric. 320, 362–372.
de Sotillo, D.V.R., Hadley, M., 2002. Chlorogenic acid modifies plasma and liver
concentrations of cholesterol, triacylglycerol, and minerals in (fa/fa) Zucker rats.
J. Nutr. Biochem. 13, 717–726.
Erickson, S.K., Schrewsbery, M.A., Brooks, C., Meyer, D.J., 1980. Rat liver acylcoenzyme A:cholesterol acyltransferase: its regulation in vivo and some of
properties in vitro. J. Lipid Res. 21, 930–941.
Folch, J., Lees, M., Sloan-Stanley, G.H., 1957. A simple method for isolation and
purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509.
Gillies, P.J., Rathgeb, K.A., Perri, M.A., Robinson, C.S., 1986. Regulation of acylCoA:cholesterol acyltransferase activity in normal control and atherosclerotic
rabbit aortas: role of a cholesterol substrate pool. Exp. Mol. Pathol. 44, 320–339.
Gonthier, M.P., Remesy, C., Scalbert, A., Cheynier, V., Souquet, J.M., Poutanen, K.,
Aura, A.M., 2006. Microbial metabolism of caffeic acid and its esters chlorogenic
A.-S. Cho et al. / Food and Chemical Toxicology 48 (2010) 937–943
and caftaric acids by human faecal microbiota in vitro. Biomed. Pharmacother.
60, 536–540.
Guerre-Millo, M., Gervois, P., Raspé, E., Madsen, L., Poulain, P., Derudas, B., Herbert,
J.M., Winegar, D.A., Willson, T.M., Fruchart, J.C., Berge, R.K., Staels, B., 2000.
Peroxisome proliferators-activated receptor a activators improve insulin
sensitivity and reduce adiposity. J. Biol. Chem. 275, 16638–16642.
Hemmerle, H., Berger, H.J., Below, P., Schubert, G., Rippel, R., Schindler, P.W., Paulus,
E., Herling, A.W., 1997. Chlorogenic acid and synthetic chlorogenic acid
derivatives: novel inhibitors of hepatic glucose-6-translocase. J. Med. Chem.
40, 137–145.
Hertog, M.G., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F.,
Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., 1995. Flavonoid intake and
long-term risk of coronary heart disease and cancer in the seven countries
study. Arch. Intern. Med. 155, 381–386.
Hsu, C.L., Hung, S.L., Yen, G.C., 2006. Inhibitiory effect of phenolic acids on the
proliferation of 3T3-L1 preadipocytes in relation to their antioxidant activity. J.
Agric. Food Chem. 54, 4191–4197.
Hulcher, F.H., Oleson, W.H., 1973. Simplified spectrophotometric assay for
microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of
coenzyme A. J. Lipid Res. 14, 625–631.
Jung, U.J., Lee, M.K., Park, Y.B., Jeon, S.M., Choi, M.S., 2006. Antihyperglycemic and
antioxidant properties of caffeic acid in db/db mice. J. Pharmacol. Exp. Ther.
318, 476–483.
Kadowaki, T., Yamauchi, T., 2005. Adiponectin and adiponectin receptors. Endocr.
Rev. 26, 439–451.
Kim, H., Haluzik, M., Asghar, Z., Yau, D., Joseph, J.W., Fernandez, A.M., Reitman, M.L.,
Yakar, S., Stannard, B., Heron-Milhavet, L., Wheeler, M.B., LeRoith, D., 2003.
Peroxisome proliferators-activated reverses the lipotoxic state and improves
glucose homeostasis. Diabetes 52, 1770–1778.
Kim, H.K., Jeong, T.S., Lee, M.K., Park, Y.B., Choi, M.S., 2003. Lipid-lowering efficacy of
hespertin metabolites in high-cholesterol fed rats. Clin. Chim. Acta 327, 129–
137.
Kumada, M., Kihara, S., Sumitsuji, S., Kawamoto, T., Matsumoto, S., Ouchi, N., Arita,
Y., Okamoto, Y., Shimomura, I., Hiraok, H., Nakamura, T., Funahashi, T.,
Matsuzawa, Y.Yuji Matsuzawa for the Osaka CAD Study Group, 2003.
Association of hypoadiponectinemia with coronary artery disease in men.
Arterioscler. Thromb. Vasc. Biol. 23, 85–89.
Lafontan, M., Girard, J., 2008. Impact of visceral adipose tissue on liver metabolism.
Part I: heterogeneity of adipose tissue and functional properties of visceral
adipose tissue. Diabetes Metab. 34, 317–327.
Lazarow, P.B., 1981. Assay of peroxisomal b-oxidation of fatty acids. Methods
Enzymol. 72, 315–319.
Lee, M.K., Park, E.M., Bok, S.H., Jung, U.J., Kim, J.Y., Park, Y.B., Huh, T.L., Kwon, O.S.,
Choi, M.S., 2003. Two cinnamate derivatives produce similar alteration in mRNA
expression and activity of antioxidant enzymes in rats. J. Biochem. Mol. Toxicol.
17, 255–262.
Mathieu, P., Pibarot, P., Larose, E., Poirier, P., Marette, A., Despres, J.P., 2008. Visceral
obesity and the heart. Biochem. Cell Biol. 40, 821–836.
943
Mori, H., Tanaka, T., Shima, H., Kunniyasu, T., Takahashi, M., 1986. Inhibitory effect
of chlorogenic acid on methlazoxymethanol acetate-induced carcinogenesis in
large intestine and liver of hamsters. Cancer Lett. 30, 49–50.
Nardini, M., Aquino, M.D., Tomassi, G., Gentili, V., Di-Felice, M., Scaccini, C., 1995.
Inhibition of human low-density lipoprotein oxidation by caffeic acid and other
hydroxycinnamic acid derivatives. Free Radical Biomed. Med. 19, 542–552.
Nepokroeff, C.M., Lakshmanan, M.R., Poter, J.W., 1975. Fatty acid synthase from rat
liver. Methods Enzymol. 35, 37–44.
Ouchi, N., Ohishi, M., Kihara, S., Funahashi, T., Nakamura, T., Nagaretani, H., Kumada,
M., Ohashi, K., Okamoto, Y., Nishizawa, H., Kishida, K., Maeda, N., Nagasawa, A.,
Kobayashi, H., Hiraoka, H., Komai, N., Kaibe, M., Rakugi, H., Ogihara, T.,
Matsuzawa, Y., 2003. Association of hypoadiponectinemia with impaired
vasoreactivity. Hypertension 42, 231–234.
Radtke, J., Linseisen, J., Wolfram, G., 1998. Phenolic acid intake of adults in a
Bavarian subgroup of the national food consumption survey. Z. Ernahrungswiss.
37, 190–197.
Scalbert, A., Williamson, G., 2000. Dietary intake and bioavailability of polyphenols.
J. Nutr. 130, 20735–20855.
Shapiro, D.J., Nordstrom, J.L., Mitschelen, J.J., Rodwell, V.W., Schimke, R.T., 1974.
Micro assay for 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver and in Lcell fibroblasts. Biochim. Biophys. 370, 369–377.
Shimoda, H., Seki, E., Aitani, M., 2006. Inhibitory effect of green coffee bean extract
on fat accumulation and body weight gain mice. BMC Complement Altern. Med.
6, 9–17.
Slawik, M., Vidal-Puig, A.J., 2006. Lipotoxicity, overnutrition and energy metabolism
in aging. Ageing Res. Rev. 5, 144–164.
Tanaka, T., Kojima, T., Kawamori, T., Wang, A., Suzui, M., Okamoto, K., Mori, H., 1993.
Inhibition of 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis by
the naturally occurring plant phenolics caffeic, ellagic, chlorogenic and ferulic
acids. Carcinogenesis 14, 1321–1325.
Torre-Villalvazo, I., Tovar, A.R., Ramos-Barragán, V.E., Cerbón-Cervantes, M.A.,
Torres, N., 2008. Soy protein ameliorates metabolic abnormalities in liver and
adipose tissue of rats fed a high fat diet. J. Nutr. 138, 462–468.
Trujillo, M.E., Scherer, P.E., 2005. Adiponectin: journey from an adipocyte secretory
protein to biomarker of the metabolic syndrome. J. Intern. Med. 257, 167–175.
Tsuchiya, T., Suzuki, O., Igarashi, K., 1996. Protective effects of chlorogenic acid on
paraquat-induced oxidative stress in rats. Biosci. Biotechnol. Biochem. 60, 765–
768.
Willson, T.M., Wahli, W., 1997. Peroxisome proliferators-activated receptor
agonists. Curr. Opin. Chem. Biol. 1, 235–241.
Yamauchi, T., Kamon, J., Waki, H., Terauchi, Y., Kubota, N., Hara, K., Mori, Y., Ide, T.,
Murakami, K., Tsuboyama-Kasaoka, N., Ezaki, O., Akanuma, Y., Gavrilova, O.,
Vinson, C., Reitman, M.L., Kagechika, H., Shudo, K., Yoda, M., Nakano, Y., Tobe, K.,
Nagai, R., Kimura, S., Tomita, M., Froguel, P., Kadowaki, T., 2001. The fat-derived
hormone adiponectin reverse insulin resistance associated with both
lipoatrophy and obesity. Nat. Med. 7, 941–946.