Chlorogensäure

Titel: Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice
Orginaltitel: Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice
Erschienen in: Elsevier (2010) Untertitel: Food and Chemical Toxicology

Autoren: • Ae-Sim Cho, • Seon-Min Jeon, • Myung-Joo Kim, • Jiyoung Yeo, • Kwon-Il Seo, • Myung-Sook Choi, • Mi-Kyung Lee • Ibrahim Akyazie,

Abstract

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.

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-coumaricacid, 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 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 (Table1) 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.

2.2. Plasma leptin, insulin and adiponectin levels

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.

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

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 presenceof 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

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

3.1. Body weight, food intake, energy intake and visceral fat weight

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- 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. he 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) afterthe 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).

"3.5. Expression of hepatic PPAR a"

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).

Schlussfolgerung

Curcumin verringerte eine reihe von proinflammtorischer Parameter. AP-1, NF-?B und die Trypsin-Aktivität wurden gehemmt. Die Spiegel von TNF-a, IL-6 iNOS und NO. Es konnte eine Verringerung der Gewebeverletzung im Rahmen einer akuten Pankreatitis festgestellt werden. Allerdings konnte keine Korrelation zwischen des Einflusses auf die Zytokine und des schützenden Effektes auf das Gewebe festgestellt werden.

4. Discussion

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 by approximately 22% and 46%, respectively, compared to the highfat control group without changing daily food or energy intake. nder 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 roup 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 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).

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