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The Korean traditional medicine Gyeongshingangjeehwan inhibits obesity through the regulation of leptin and PPARα action in OLETF rats
Journal of Ethnopharmacology 119 (2008) 245–251
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The Korean traditional medicine Gyeongshingangjeehwan inhibits obesity through the regulation of leptin and PPAR␣ action in OLETF rats Sunhyo Jeong a , Kyungsil Chae a , Yang Sam Jung b , Young Ho Rho b , Jinmi Lee a , Juran Ha a , Ki Hyeon Yoon b , Gyeong Cheol Kim c , Ki Sook Oh d , Soon Shik Shin b,∗∗ , Michung Yoon a,∗ a
Department of Life Sciences, Mokwon University, Taejon 302-729, Republic of Korea Department of Formula Sciences, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea Department of Diagnostics, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea d Department of Physiology and Biophysics, USC School of Medicine, Los Angeles, CA 90089-9142, USA b c
a r t i c l e
i n f o
Article history: Received 30 January 2008 Received in revised form 16 April 2008 Accepted 27 June 2008 Available online 15 July 2008 Keywords: Gyeongshingangjeehwan Obesity Hypertriglyceridemia Leptin Appetite PPAR␣ OLETF rat
a b s t r a c t Gyeongshingangjeehwan (GGEx), which comprises Liriope platyphylla F.T. Wang & T. Tang (Liliaceae), Platycodon grandiflorum A. DC. (Campanulaceae), Schisandra chinensis K. Koch (Magnoliaceae), and Ephedra sinica Stapf (Ephedraceae), has traditionally been used as an anti-obesity drug in Korean local clinics, although there is no evidence concerning the scientific analyses of its effects and mechanism(s) of action. Thus, we investigated the effects of GGEx on obesity, as well as the mechanism by which GGEx functions, in Otsuka Long-Evans Tokushima Fatty (OLETF) male rats. Compared with obese OLETF control rats, administration of GGEx for 8 weeks significantly decreased food intake and plasma leptin levels as well as body weight gain and abdominal fat in OLETF rats. GGEx treatment not only decreased circulating triglycerides, but also inhibited lipid accumulation in the liver. GGEx increased the hepatic mRNA levels of PPAR␣ target genes responsible for fatty acid -oxidation. Consistent with the in vivo data, GGEx elevated PPAR␣ reporter gene expression in NMu2Li liver cells. These results suggest that GGEx may effectively prevent obesity and hypertriglyceridemia in part through the inhibition of feeding and the activation of hepatic PPAR␣. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Obesity is the result of an energy imbalance caused by an increased ratio of caloric intake to energy expenditure. In conjunction with obesity, such related metabolic disorders of dyslipidemia, atherosclerosis, and type 2 diabetes have become global health problems. Over the past decade, there has been a great increase in the use of alternative treatments, such as herbal remedies, for these diseases (Das and Maulik, 2006; Sharpe et al., 2007; ValchevaKuzmanova et al., 2007). Synthetic drugs, including sibutramine and orlistat, approved by the FDA are currently used for the treatment of obesity. However, there remains an urgent need for safer and more effective alternative therapies to the long-term use of these drugs. Gyeongshingangjeehwan (GGEx), an herbal drug composed of four medicinal plants Liriope platyphylla, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica, has widely been used as
∗ Corresponding author. Tel.: +82 42 829 7585; fax: +82 42 829 7580. ∗∗ Corresponding author. Tel.: +82 51 850 7414; fax: +82 51 853 4036. E-mail addresses: [email protected] (S.S. Shin), [email protected] (M. Yoon). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.06.037
one of the most valuable medicines in Korea. GGEx is also known to exhibit many beneficial pharmacological effects on the endocrine, cardiovascular, and immune systems in Korean medicine (Lee, 1993; Kim, 1997; Eun et al., 2006). In particular, GGEx has already been used as an anti-obesity drug in local clinics of Korea, although the mechanism of its action remains unknown. Of the constituents of GGEx, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica are reported to have lipid-lowering, anti-obesity, and antidiabetes effects (Park et al., 2007; Zheng et al., 2007; Yao et al., 2008), supporting that GGEx may regulate efficiently obesity and lipid disorders. Several candidate molecules involved in energy expenditure have been implicated in a number of prevalent metabolic disorders, such as obesity, atherosclerosis, and type 2 diabetes. The family of peroxisome proliferator-activated receptors (PPARs) is thought to be involved in the control of energy homeostasis. Among the three PPAR isoforms, fibrate-activated PPAR␣ appears to be important in obesity and lipid metabolism due to its ability to restore overall energy balance. Since the PPAR␣ ligand fibrate increases fatty acid oxidation and thus decreases the levels of plasma triglycerides responsible for adipose cell hypertrophy and hyperplasia (Bourgeois et al., 1983; Costet et al., 1998; Chaput et al., 2000), it
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may also inhibit body weight gain, suggesting that PPAR␣ may be important in obesity. Furthermore, recent studies have suggested that fibrates can modulate body weight and adiposity in experimental animal models (Guerre-Millo et al., 2000; Mancini et al., 2001; Yoon et al., 2002; Jeong et al., 2004a). Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of obesity and type 2 diabetes, are known to have a congenital defect in the expression of the cholecystokinin A (CCK-A) receptor gene. Since CCK plays an important role in the inhibition of food intake through its interaction with CCK-A receptor, the lack of CCK-A receptors results in a satiety deficit which leads to overall hyperphagia and obesity (Moran et al., 1998; Hajnal et al., 2005). It is also known that fatty acid oxidative PPAR␣ target genes as well as PPAR␣ expression are suppressed (Liang and Tall, 2001; Muoio and Lynis Dohm, 2002) and leptin is overexpressed, which results in leptin resistance (Maffei et al., 1995a; Zimmet et al., 1999; Hidaka et al., 2001), in obese animals, suggesting that obesity and lipid disorders in OLETF rats may be due, at least in part, to a reduction of PPAR␣ activity and an elevation of leptin. Thus, we hypothesized that obesity and hypertriglyceridemia in OLETF rats may be improved by GGEx-mediated suppression of appetite and activation of hepatic PPAR␣. The aim of our present study is to examine whether the Korean herbal medicine GGEx can regulate food intake, body weight gain, adiposity, and dyslipidemia in OLETF rats, and to investigate the mechanism by which GGEx modulates these effects.
OLETF-C), two groups of obese OLETF rats given GGEx [n = 4; 60.85 mg/(kg day) or 121.7 mg/(kg day) for OLETF-G (X5) or OLETFG (X10), respectively], and an obese OLETF group given sibutramine (n = 4; 2.5 mg/(kg day), OLETF-R). GGEx or sibutramine was given orally by gavage once a day for 8 weeks. In all experiments, body weights were measured daily using a top-loading balance and the person measuring the body weights was blinded to each treatment group. Food intake was determined by estimating the amount of food consumed by the rats throughout the treatment period. Cages were inspected for food spillage, but only a little spillage was noticed; it was collected and measured to determine food intake amount. At the end of the 8-week period, the animals were sacrificed under diethyl ether anesthesia and their tissues were harvested, weighed, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use. Blood was collected from the saphenous vein after a 12-h fast and the plasma was separated and stored at −80 ◦ C until analysis. All animal experiments were approved by the Institutional Animal Care and Use Committees of Dongeui University, and followed National Research Council Guidelines. 2.3. Blood analysis
2. Materials and methods
Plasma concentrations of triglycerides and total cholesterol were measured using an automatic blood chemical analyzer (CIBA Corning, Oberlin, OH, USA). Plasma concentrations of leptin were measured using a rat leptin radioimmunoassay kit (Linco, St. Charles, MO, USA).
2.1. Preparation of GGEx
2.4. RT-PCR
GGEx was prepared from food-grade aqueous extracts of the four herbs Liriope platyphylla, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica (Hwalim, Busan, Korea), and the composition of GGEx is shown in Table 1. The proportions used in this study are same as the proportions that are used to treat patients. Boucher specimens for Liriope platyphylla (FOS-05-01), Platycodon grandiflorum (FOS-05-02), Schisandra chinensis (FOS-05-03), and Ephedra sinica (FOS-05-04) were deposited at the Department of Formula Sciences, Dongeui University. Briefly, dried herbs were extracted with distilled water for 22 h at 95 ◦ C after powdering and mixing according to their proportions. Then, the extracts were freeze-dried under vacuum and combined for the production of GGEx.
Total cellular RNA was prepared using the Trizol reagent (Gibco-BRL, Grand Island, NY, USA). After 2 g total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and an antisense primer, cDNA was generated. Briefly, RNA was denatured for 5 min at 72 ◦ C and then immediately placed on ice for 5 min. Denatured RNA was mixed with MMLV-RT, MMLV-RT buffer, and the dNTP mixture, and incubated for 1 h at 42 ◦ C. Synthesized cDNA fragments were amplified by a PCR process in an MJ Research Thermocycler (Waltham, MA, USA). PCR primers used to assess the mRNA expression of each gene are shown in Table 2. Briefly, cDNA was mixed with the PCR primers, Taq DNA polymerase (Solgent, Taejon, Korea), and the dNTP mixture. The PCR procedure consisted of 30 cycles of denaturation for 1 min at 94 ◦ C, annealing for 1 min at 58 ◦ C, and elongation for 1 min at 72 ◦ C. The PCR products were analyzed by electrophoresis on a 1% agarose gel. Relative expression levels were presented as a ratio of target gene cDNA vs. -actin cDNA. The results were determined using an image analyzer (Syngene, Cambridge, UK).
2.2. Animal treatments Sixteen male OLETF and four Long-Evans Tokushima Otsuka (LETO) rats were obtained as a generous gift of the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). Tenweek-old animals were housed at the Dongeui University under pathogen-free conditions with a standard 12-h light/dark cycle. Prior to administration of special treatments, the rats were fed with standard rodent chow and water ad libitum. Rats were divided into five groups: a non-obese LETO control group given water (n = 4; LETO-C), an obese OLETF control group given water (n = 4;
2.5. Histological analysis The right lobe of the liver was fixed in 10% phosphate-buffered formalin for 1 day and processed in a routine manner for paraffin section. Five-micrometer thick sections were stained with hematoxylin and eosin (HE) for microscopic examination.
Table 1 Composition of GGEx Scientific name
Common name
Family name
Part used
Content (%)
Liriope platyphylla F.T. Wang & T. Tang Platycodon grandiflorum A. DC. Schisandra chinensis K. Koch Ephedra sinica Stapf
Big blue lily turf Chinese bellflower Chinese magnolia vine Chinese ephedra
Liliaceae Campanulaceae Magnoliaceae Ephedraceae
Root Root Fruit Stem
42.86 28.57 14.29 14.28
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Table 2 Sequences of primers used for the RT-PCR assays Genes
Gene bank
Primer sequences
ACOX
J02752
Forward: 5 -actatatttggccaattttgtg-3 Reverse: 5 -tgtggcagtggtttccaagcc-3
195
VLCAD
AF017176
Forward: 5 -cgtcagaggtgtactttgatgg-3 Reverse: 5 -catggactcagtcacatactgc-3
263
MCAD
NM007382
Forward: 5 -gacatttggaaagctgctagtg-3 Reverse: 5 -tcacgagctatgatcagcctctg-3
321
CPT1
L07736
Forward: 5 -tatgtgaggatgctgcttcc-3 Reverse: 5 -ctcggagagctaagcttgtc-3
-Acitn
J00691
Product size (bp)
587
Forward: 5 -tggaatcctgtggcatccatgaaa-3 Reverse: 5 -taaaacgcagctcagtaacagtcc-3
350
2.6. Measurement of visceral fat levels All animals underwent computed tomography (CT, HiSpeed NX/I, General Electric Medical System, Piscataway, USA) to measure cross-sectional abdominal subcutaneous and visceral fat. The animals were examined in the dorsal position and CT scans were performed (120 kV, 150 mA, 1-mm thickness). The cross-sectional areas were determined by the automatic calculation of the scans with a CT number of −160 to −20 Hounsfield units. 2.7. Statistics All values are expressed as the mean ± standard deviation (S.D.). Statistical analysis was performed by ANOVA followed by either post hoc Turkey’s multiple comparison test or Dunnett’s test. Statistical significance was defined as a value of p < 0.05. 3. Results
Fig. 1. Regulation of body weight and food intake by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Body weight gain (A) and food intake (B) were measured daily and all values are expressed as the mean ± S.D. for n = 4 rats. Statistical significance was analyzed at the end of the treatment period. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
3.1. GGEx reduces body weight gain and food intake in genetically obese OLETF rats Compared with OLETF-C rats, OLETF rats given GGEx significantly decreased body weight gain by 14.1% and 29.9% for OLETF-G (X5) and OLETF-G (X10) groups, respectively (Fig. 1A). GGEx treatment also significantly decreased food intake compared with OLETF-C rats. Food intake was reduced by 7.4% and 22.0% in OLETFG (X5) and OLETF-G (X10) groups, respectively (Fig. 1B). The effects of GGEx (X10) on reductions in body weight gain and food intake were comparable to those of sibutramine, an appetite-lowering, anti-obesity drug, in obese OLETF controls (Fig. 1A and 1B). No dose of GGEx used in the present study had any observed toxic effects. 3.2. GGEx reduces abdominal fat areas in genetically obese OLETF rats At the end of the study, the OLETF-C group had abdominal total fat areas of 105.73 ± 3.8 mm2 , whereas the OLETF-G (X5) and OLETF-G (X10) groups had fat areas of 92.95 ± 5.38 and 60.51 ± 1.04 mm2 , respectively (Fig. 2A). Compared with OLETF-C animals, feeding rats with a chow diet supplemented with GGEx significantly decreased total abdominal fat areas by 12.1% in OLETFG (X5) and by 42.8% in OLETF-G (X10) groups. Both visceral and subcutaneous fat areas in the abdomen were significantly reduced by GGEx treatment. In addition, reductions of fat areas were higher in the OLETF-G (X10) than in the OLETF-R group (decreases of 28.2% in total fat areas). Representative CT images are shown in Fig. 2B.
3.3. GGEx lowers the elevation in plasma leptin levels in genetically obese OLETF rats Leptin is a long-term satiety signal that often reflects changes in body weight and adipose tissue mass (Maffei et al., 1995b; Considine et al., 1996). Marked elevations in leptin are thought to reflect leptin resistance and a diminution of satiety responses (Ahima and Flier, 2000). Compared with the LETO-C group, plasma leptin levels were 172% higher in the OLETF-C group (Fig. 3A). In contrast, GGEx (X10) treatment significantly decreased leptin plasma levels by 23.5% in OLETF-C rats with similar levels to those in sibutramine-treated obese rats. To evaluate whether leptin expression is disproportionate to body weight, body weight vs. leptin values were examined. Body weights positively correlated with leptin levels following GGEx treatment (Fig. 3B). 3.4. GGEx improves lipid profiles in genetically obese OLETF rats To determine whether GGEx has beneficial effects on circulating lipid profiles, its effects on plasma triglycerides and total cholesterol were examined. After 8 weeks, OLETF-C rats had significantly higher plasma levels of triglycerides and total cholesterol than did the LETO-C rats. However, a reduction of plasma triglycerides by 28.7% was seen in the OLETF-G (X10) group, although total cholesterol levels were unaffected by GGEx compared with obese control rats (Fig. 4).
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Fig. 2. Modulation of abdominal fat by GGEx in genetically obese OLETF rats. (A) Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Rats underwent CT to measure cross-sectional abdominal subcutaneous and visceral fat areas. All values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. (B) Representative CT images are shown. Gray and white arrows indicate subcutaneous and visceral fat, respectively.
3.5. Inhibition of hepatic lipid accumulation by GGEx in genetically obese OLETF rats Obese OLETF controls showed considerable hepatic lipid accumulation compared with lean LETO controls. However, we found that hepatic lipid accumulation was markedly lower in GGExtreated OLETF rats than in obese OLETF controls (Fig. 5A). Moreover, when OLETF rats were treated with GGEx (X10), there was a complete inhibition of hepatic lipid accumulation, as indicated by the decreased hepatic lesion scores (Fig. 5B). 3.6. Regulation of hepatic PPAR˛ target gene expression by GGEx in genetically obese OLETF rats To evaluate whether GGEx causes alterations in PPAR␣ actions, we measured the mRNA levels of PPAR␣ target enzymes involved in fatty acid -oxidation in the liver. As expected, OLETF control rats had lower mRNA levels of hepatic PPAR␣ target genes, such as carnitine palmitoyl transferase-1 (CPT-1), very long chain acyl-CoA dehydrogenase (VLCAD), medium chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA oxidase (ACOX) by 18%, 2%, 5%, and 9%, respectively, than LETO control rats (Fig. 6A–D). However, GGEx (X10) administration up-regulated the mRNA levels of liver CPT-1, VLCAD, MCAD, and ACOX by 18%, 16%, 21%, and 19%, respectively, in OLETF rats compared with vehicle-treated controls.
Fig. 3. Correlation between plasma leptin levels and body weights after GGEx treatment in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. (A) GGEx significantly decreased leptin plasma levels that were elevated in obese OLETF control rats. All values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. (B) Plasma leptin levels positively correlated with body weights after GGEx treatment.
3.7. Modulation of PPAR˛ reporter gene expression by GGEx To examine the mechanism by which GGEx increases the mRNA expression of PPAR␣ target genes, NMu2Li cells were cotransfected with PPAR␣ and RXR␣ expression constructs as well as the luciferase reporter construct PPRE3 -tk-luc, which contains three copies of the PPRE from the rat ACOX gene. The transfected cells were treated with GGEx at doses that did not induce cytotoxic effects as measured by trypan blue exclusion. Treatment of the transfected cells with GGEx increased luciferase activities in a dose-dependent manner (Fig. 7). 4. Discussion The present study was undertaken to investigate the antiobesity and hypolipidemic effects of GGEx in obese and type 2 diabetic OLETF rats. Since GGEx has been known to modulate human obesity in traditional Korean medicine, it is important to demonstrate the effects of GGEx on obesity experimentally and to verify its mechanism of action. Our results clearly demonstrate that GGEx significantly reduces body weight gain and inhibits hyperphagia. As expected, obese OLETF control rats had much higher food intake compared with lean LETO controls. However, the administration of GGEx (X10)
S. Jeong et al. / Journal of Ethnopharmacology 119 (2008) 245–251
Fig. 4. Changes in circulating triglycerides and total cholesterol by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Plasma concentrations of triglycerides (A) and total cholesterol (B) were measured and all values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
decreased food intake in obese OLETF controls. The magnitude of this inhibition by GGEx was similar to the effects of sibutramine that is widely used as an anti-obesity drug via a feeding-inhibitory action (Barkeling et al., 2003). Body weight gain was also significantly decreased following GGEx treatment in OLETF control rats. Body weight gain began to decrease after 18 days of treatment and became significantly different by day 43. In parallel with the effects of GGEx on body weight gain, abdominal fat areas were also decreased by GGEx. GGEx treatment significantly reduced both subcutaneous and visceral fat areas in obese OLETF rats. In particular, the reduction after GGEx (X10) treatment was greater than that induced by sibutramine. Our results show that GGEx effectively decreases body weight gain and adipose tissue areas, and that these effects of GGEx may be due to its feeding-inhibitory actions. Obesity is associated with elevated leptin production in the blood (Considine et al., 1996). Leptin is produced mainly in adipocytes and is present in the serum in direct proportion to the amount of adipose tissue (Maffei et al., 1995b; Considine et al., 1996). Leptin exerts negative feedback effects on energy intake, but loses its ability both to inhibit energy intake and stimulate energy expenditure in conditions of obesity. This apparent leptin resistance is explained by the findings that neither endogenous hyperleptinemia nor exogenous leptin treatment is able to prevent weight gain in obese humans and rodents (Considine et al., 1996; Flier, 2004). Similarly, our results showed that obese OLETF controls had 2.7 times
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Fig. 5. Inhibition of hepatic lipid accumulation by GGEx in genetically obese OLETF rats. (A) Representative hematoxylin- and eosin-stained sections of livers are shown (original magnification 200×). Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Arrows indicate lipid droplets in hepatocytes. (B) Histological analyses of hepatic lipid accumulation. Pathological scores of hepatic lipid accumulation are as follows: 0, no lesion; 1, mild; 2, moderate; 3, severe; 4, very severe. All values are expressed as the mean ± S.D. of four independent experiments. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
higher levels of plasma leptin compared with lean LETO controls. In contrast, plasma leptin levels were significantly decreased by GGEx (X10). In fact, increased leptin levels did not induce decreased food intake in OLETF rats and a decrease in its levels after GGEx treatment did not result in increased food intake. Regression analysis also revealed that plasma leptin levels positively correlated with body weight gain. Thus, it is plausible that GGEx prevents hyperphagia, body weight gain, and abdominal fat increases in OLETF rats in part through a decrease in leptin levels contributing to the amelioration of leptin resistance. OLETF rats are known to exhibit hypertriglyceridemia, which is largely responsible for obesity and cardiovascular disease (Sattar et al., 1998; Nakaya et al., 2002; Mori et al., 2005). We observed that OLETF controls had 4.3 times higher plasma triglyceride levels than LETO controls. However, plasma triglycerides were significantly decreased by GGEx (X10) in these rats, although plasma total cholesterol levels were not altered after GGEx treatment, indicating that GGEx efficiently prevented hypertriglyceridemia in obese animals. In order to investigate whether GGEx inhibits hepatic lipid accumulation, HE staining of triglycerides was performed in liver. Compared with LETO control rats, lipid droplets were found to be
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Fig. 6. Modulation of liver PPAR␣ target gene expression by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. RNA was extracted from liver, and relative mRNA levels of CPT-1 (A), VLCAD (B), MCAD (C), and ACOX (D) were measured as described in Section 2. All values are expressed as the mean ± S.D. of R.D.U. (relative density units) using -actin as a reference. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. ACOX, acyl-CoA oxidase; CPT-1, carnitine palmitoyl transferase-1; VLCAD and MCAD, very long chain and medium chain acyl-CoA dehydrogenases, respectively.
substantially increased in the livers of obese OLETF rats. Consistent with the effects of GGEx on circulating triglycerides, GGEx inhibited hepatic lipid accumulation in OLETF control rats. These results demonstrate that GGEx stimulates fat catabolism in liver. Ligand-activated PPAR␣ has been shown to regulate the expression of a number of genes critical for fatty acid -oxidation and lipid catabolism. Our previous studies also showed that PPAR␣ effectively improved high fat diet-induced obesity and hypertriglyceridemia (Yoon et al., 2003; Jeong et al., 2004b). To evaluate whether the effects of GGEx on obesity and lipid profiles were mediated by alterations in PPAR␣ target gene expression in liver, we measured the mRNA levels of various PPAR␣ targets, including CPT-1, VLCAD, MCAD, and ACOX. Obese OLETF control rats had
Fig. 7. Modulation of PPAR␣ reporter gene expression by GGEx. All values are expressed as mean ± S.D. of relative luciferase units/-galactosidase activity. The experiments were performed at least three times. *, p < 0.05 vs. vehicle.
lower PPAR␣ target mRNA levels than lean LETO controls. However, OLETF rats fed GGEx were found to have significantly higher CPT-1, VLCAD, MCAD, and ACOX mRNA levels than OLETF control rats. These results are in accordance with our data on the substantial reduction of lipid droplets by GGEx in liver. While GGEx administration substantially increased hepatic PPAR␣ functions in rats, our results can overestimate the pharmacological effects of GGEx on PPAR␣ actions in humans since humans have much less PPAR␣ expression and activity compared with rodents (Holden and Tugwood, 1999; Wilson et al., 2000). Therefore, GGEx-induced PPAR␣ effects obtained in rodents are thought to be less effective in humans. To determine the mechanism by which GGEx increases PPAR␣ target gene expression and to examine whether GGEx-induced PPAR␣ activation regulates obesity and lipid metabolism in an appetite-independent manner, we examined the effects of GGEx on PPAR␣ transactivation using NMu2Li cells, which do not express leptin. Consistent with the in vivo data, GGEx induced the expression of a PPRE-luciferase reporter construct in a transactivation assay. GGEx treatment increased luciferase activity in a dosedependent manner compared with reporter gene activation of cells treated with vehicle, suggesting that GGEx can positively regulate the action of PPAR␣. Although, we cannot exclude the possibility that GGEx stimulates PPAR␣ function through leptin-dependent actions. According to Suzuki et al. (2007), leptin stimulates fatty acid oxidation through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as PPAR␣. In fact, decreased leptin levels and food intake can lead to not only decreases in plasma lipid levels and hepatic lipid accumulation, but also increases in PPAR␣ target gene expression. Thus, it is necessary to investigate whether GGEx-activated PPAR␣ functions are mediated by leptin-AMPK pathways. In addition to the effects of GGEx on obesity, GGEx use in elderly seems to slow aging and prevent age-associated metabolic
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disorders by compensating the age-associated decrease in PPAR␣ expression (Ye et al., 2005; Erol, 2007). Recently, it is reported that PPAR␣ activators work for longevity and may be good candidates as anti-aging agents (Erol, 2005, 2007). Based on these results, GGEx, one of the potential PPAR␣ agonists, may have therapeutic applications in age-related diseases. 5. Conclusion Overall, the results demonstrate that abnormal food intake and obesity, as well as the obesity-related disorder, dyslipidemia, in OLETF rats are effectively inhibited by GGEx. GGEx appears to act via important mechanisms by which a marked elevation in leptin levels is decreased and PPAR␣ is activated in liver. Thus, the use of GGEx may be very useful in obese patients with hypertriglyceridemia. Acknowledgement This research was supported by the Korea Research Foundation (KRF-2006-531-00052), Korea. References Ahima, R.S., Flier, J.S., 2000. Leptin. Annual Review of Physiology 62, 413–437. Barkeling, B., Elfhag, K., Rooth, P., Rössner, S., 2003. Short-term effects of sibutramine (Reductil) on appetite and eating behaviour and the long-term therapeutic outcome. International Journal of Obesity and Related Metabolic Disorders 27, 693–700. Bourgeois, F., Alexiu, A., Lemonnier, D., 1983. Dietary-induced obesity: effect of dietary fats on adipose tissue cellularity in mice. The British Journal of Nutrition 49, 17–26. Considine, R.V., Sinha, M.K., Heiman, M.L., Kriauciunas, A., Stephens, T.W., Nyce, M.R., Ohannesian, J.P., Marco, C.C., McKee, L.J., Bauer, T.L., et al., 1996. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. New England Journal of Medicine 334, 292–295. Chaput, E., Saladin, R., Silvestre, M., Edgar, A.D., 2000. Fenofibrate and rosiglitazone lower serum triglycerides with opposing effects on body weight. Biochemical and Biophysical Research Communications 271, 445–450. Costet, P., Legendre, C., More, J., Edgar, A., Galtier, P., Pineau, T., 1998. Peroxisome proliferator-activated receptor alpha-isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis. The Journal of Biological Chemistry 273, 29577–29585. Das, D.K., Maulik, N., 2006. Resveratrol in cardioprotection: a therapeutic promise of alternative medicine. Molecular Interventions 6, 36–47. Erol, A., 2005. PPARalpha activators may be good candidates as antiaging agents. Medical Hypotheses 65, 35–38. Erol, A., 2007. The functions of PPARs in aging and longevity. PPAR Research 2007, 1–10. Eun, J.S., Jeon, Y.K., Lee, K.A., Leem, J.Y., Kwon, J., Jang, I.S., Song, J.M., 2006. Effect of Bopaewon-tang on Immune Reaction. Journal of Orient Physiology and Pathology 20, 125–130. Flier, J.S., 2004. Obesity wars: molecular progress confronts an expanding epidemic. Cell 116, 337–350. Guerre-Millo, M., Gervois, P., Raspe, 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 proliferator-activated receptor alpha activators improve insulin sensitivity and reduce adiposity. The Journal of Biological Chemistry 275, 16638–16642. Hajnal, A., Covasa, M., Bello, N.T., 2005. Altered taste sensitivity in obese, prediabetic OLETF rats lacking CCK-1 receptors. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 289, R1675–R1686. Hidaka, S., Ogawa, Y., Ebihara, K., Shintani, M., Abe, M., Miyanaga, F., Nakao, K., 2001. Pathology and significance of leptin resistance in obesity Nippon Rinsho. Japanese Journal Clinical Medicine 59, 472–480. Holden, P.R., Tugwood, J.D., 1999. Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences. Journal of Molecular Endocrinology 22, 1–8. Jeong, S., Han, M., Lee, H., Kim, M., Nicol, C.J., Kim, B.H., Choi, J.H., Oh, G.T., Yoon, M., 2004a. Effects of fenofibrate on high fat diet-induced body weight gain and adiposity in female C57BL/6J mice. Metabolism 53, 1284–1289.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
The Korean traditional medicine Gyeongshingangjeehwan inhibits obesity through the regulation of leptin and PPAR␣ action in OLETF rats Sunhyo Jeong a , Kyungsil Chae a , Yang Sam Jung b , Young Ho Rho b , Jinmi Lee a , Juran Ha a , Ki Hyeon Yoon b , Gyeong Cheol Kim c , Ki Sook Oh d , Soon Shik Shin b,∗∗ , Michung Yoon a,∗ a
Department of Life Sciences, Mokwon University, Taejon 302-729, Republic of Korea Department of Formula Sciences, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea Department of Diagnostics, College of Oriental Medicine, Dongeui University, Busan 614-052, Republic of Korea d Department of Physiology and Biophysics, USC School of Medicine, Los Angeles, CA 90089-9142, USA b c
a r t i c l e
i n f o
Article history: Received 30 January 2008 Received in revised form 16 April 2008 Accepted 27 June 2008 Available online 15 July 2008 Keywords: Gyeongshingangjeehwan Obesity Hypertriglyceridemia Leptin Appetite PPAR␣ OLETF rat
a b s t r a c t Gyeongshingangjeehwan (GGEx), which comprises Liriope platyphylla F.T. Wang & T. Tang (Liliaceae), Platycodon grandiflorum A. DC. (Campanulaceae), Schisandra chinensis K. Koch (Magnoliaceae), and Ephedra sinica Stapf (Ephedraceae), has traditionally been used as an anti-obesity drug in Korean local clinics, although there is no evidence concerning the scientific analyses of its effects and mechanism(s) of action. Thus, we investigated the effects of GGEx on obesity, as well as the mechanism by which GGEx functions, in Otsuka Long-Evans Tokushima Fatty (OLETF) male rats. Compared with obese OLETF control rats, administration of GGEx for 8 weeks significantly decreased food intake and plasma leptin levels as well as body weight gain and abdominal fat in OLETF rats. GGEx treatment not only decreased circulating triglycerides, but also inhibited lipid accumulation in the liver. GGEx increased the hepatic mRNA levels of PPAR␣ target genes responsible for fatty acid -oxidation. Consistent with the in vivo data, GGEx elevated PPAR␣ reporter gene expression in NMu2Li liver cells. These results suggest that GGEx may effectively prevent obesity and hypertriglyceridemia in part through the inhibition of feeding and the activation of hepatic PPAR␣. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Obesity is the result of an energy imbalance caused by an increased ratio of caloric intake to energy expenditure. In conjunction with obesity, such related metabolic disorders of dyslipidemia, atherosclerosis, and type 2 diabetes have become global health problems. Over the past decade, there has been a great increase in the use of alternative treatments, such as herbal remedies, for these diseases (Das and Maulik, 2006; Sharpe et al., 2007; ValchevaKuzmanova et al., 2007). Synthetic drugs, including sibutramine and orlistat, approved by the FDA are currently used for the treatment of obesity. However, there remains an urgent need for safer and more effective alternative therapies to the long-term use of these drugs. Gyeongshingangjeehwan (GGEx), an herbal drug composed of four medicinal plants Liriope platyphylla, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica, has widely been used as
∗ Corresponding author. Tel.: +82 42 829 7585; fax: +82 42 829 7580. ∗∗ Corresponding author. Tel.: +82 51 850 7414; fax: +82 51 853 4036. E-mail addresses: [email protected] (S.S. Shin), [email protected] (M. Yoon). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.06.037
one of the most valuable medicines in Korea. GGEx is also known to exhibit many beneficial pharmacological effects on the endocrine, cardiovascular, and immune systems in Korean medicine (Lee, 1993; Kim, 1997; Eun et al., 2006). In particular, GGEx has already been used as an anti-obesity drug in local clinics of Korea, although the mechanism of its action remains unknown. Of the constituents of GGEx, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica are reported to have lipid-lowering, anti-obesity, and antidiabetes effects (Park et al., 2007; Zheng et al., 2007; Yao et al., 2008), supporting that GGEx may regulate efficiently obesity and lipid disorders. Several candidate molecules involved in energy expenditure have been implicated in a number of prevalent metabolic disorders, such as obesity, atherosclerosis, and type 2 diabetes. The family of peroxisome proliferator-activated receptors (PPARs) is thought to be involved in the control of energy homeostasis. Among the three PPAR isoforms, fibrate-activated PPAR␣ appears to be important in obesity and lipid metabolism due to its ability to restore overall energy balance. Since the PPAR␣ ligand fibrate increases fatty acid oxidation and thus decreases the levels of plasma triglycerides responsible for adipose cell hypertrophy and hyperplasia (Bourgeois et al., 1983; Costet et al., 1998; Chaput et al., 2000), it
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may also inhibit body weight gain, suggesting that PPAR␣ may be important in obesity. Furthermore, recent studies have suggested that fibrates can modulate body weight and adiposity in experimental animal models (Guerre-Millo et al., 2000; Mancini et al., 2001; Yoon et al., 2002; Jeong et al., 2004a). Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of obesity and type 2 diabetes, are known to have a congenital defect in the expression of the cholecystokinin A (CCK-A) receptor gene. Since CCK plays an important role in the inhibition of food intake through its interaction with CCK-A receptor, the lack of CCK-A receptors results in a satiety deficit which leads to overall hyperphagia and obesity (Moran et al., 1998; Hajnal et al., 2005). It is also known that fatty acid oxidative PPAR␣ target genes as well as PPAR␣ expression are suppressed (Liang and Tall, 2001; Muoio and Lynis Dohm, 2002) and leptin is overexpressed, which results in leptin resistance (Maffei et al., 1995a; Zimmet et al., 1999; Hidaka et al., 2001), in obese animals, suggesting that obesity and lipid disorders in OLETF rats may be due, at least in part, to a reduction of PPAR␣ activity and an elevation of leptin. Thus, we hypothesized that obesity and hypertriglyceridemia in OLETF rats may be improved by GGEx-mediated suppression of appetite and activation of hepatic PPAR␣. The aim of our present study is to examine whether the Korean herbal medicine GGEx can regulate food intake, body weight gain, adiposity, and dyslipidemia in OLETF rats, and to investigate the mechanism by which GGEx modulates these effects.
OLETF-C), two groups of obese OLETF rats given GGEx [n = 4; 60.85 mg/(kg day) or 121.7 mg/(kg day) for OLETF-G (X5) or OLETFG (X10), respectively], and an obese OLETF group given sibutramine (n = 4; 2.5 mg/(kg day), OLETF-R). GGEx or sibutramine was given orally by gavage once a day for 8 weeks. In all experiments, body weights were measured daily using a top-loading balance and the person measuring the body weights was blinded to each treatment group. Food intake was determined by estimating the amount of food consumed by the rats throughout the treatment period. Cages were inspected for food spillage, but only a little spillage was noticed; it was collected and measured to determine food intake amount. At the end of the 8-week period, the animals were sacrificed under diethyl ether anesthesia and their tissues were harvested, weighed, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use. Blood was collected from the saphenous vein after a 12-h fast and the plasma was separated and stored at −80 ◦ C until analysis. All animal experiments were approved by the Institutional Animal Care and Use Committees of Dongeui University, and followed National Research Council Guidelines. 2.3. Blood analysis
2. Materials and methods
Plasma concentrations of triglycerides and total cholesterol were measured using an automatic blood chemical analyzer (CIBA Corning, Oberlin, OH, USA). Plasma concentrations of leptin were measured using a rat leptin radioimmunoassay kit (Linco, St. Charles, MO, USA).
2.1. Preparation of GGEx
2.4. RT-PCR
GGEx was prepared from food-grade aqueous extracts of the four herbs Liriope platyphylla, Platycodon grandiflorum, Schisandra chinensis, and Ephedra sinica (Hwalim, Busan, Korea), and the composition of GGEx is shown in Table 1. The proportions used in this study are same as the proportions that are used to treat patients. Boucher specimens for Liriope platyphylla (FOS-05-01), Platycodon grandiflorum (FOS-05-02), Schisandra chinensis (FOS-05-03), and Ephedra sinica (FOS-05-04) were deposited at the Department of Formula Sciences, Dongeui University. Briefly, dried herbs were extracted with distilled water for 22 h at 95 ◦ C after powdering and mixing according to their proportions. Then, the extracts were freeze-dried under vacuum and combined for the production of GGEx.
Total cellular RNA was prepared using the Trizol reagent (Gibco-BRL, Grand Island, NY, USA). After 2 g total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and an antisense primer, cDNA was generated. Briefly, RNA was denatured for 5 min at 72 ◦ C and then immediately placed on ice for 5 min. Denatured RNA was mixed with MMLV-RT, MMLV-RT buffer, and the dNTP mixture, and incubated for 1 h at 42 ◦ C. Synthesized cDNA fragments were amplified by a PCR process in an MJ Research Thermocycler (Waltham, MA, USA). PCR primers used to assess the mRNA expression of each gene are shown in Table 2. Briefly, cDNA was mixed with the PCR primers, Taq DNA polymerase (Solgent, Taejon, Korea), and the dNTP mixture. The PCR procedure consisted of 30 cycles of denaturation for 1 min at 94 ◦ C, annealing for 1 min at 58 ◦ C, and elongation for 1 min at 72 ◦ C. The PCR products were analyzed by electrophoresis on a 1% agarose gel. Relative expression levels were presented as a ratio of target gene cDNA vs. -actin cDNA. The results were determined using an image analyzer (Syngene, Cambridge, UK).
2.2. Animal treatments Sixteen male OLETF and four Long-Evans Tokushima Otsuka (LETO) rats were obtained as a generous gift of the Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). Tenweek-old animals were housed at the Dongeui University under pathogen-free conditions with a standard 12-h light/dark cycle. Prior to administration of special treatments, the rats were fed with standard rodent chow and water ad libitum. Rats were divided into five groups: a non-obese LETO control group given water (n = 4; LETO-C), an obese OLETF control group given water (n = 4;
2.5. Histological analysis The right lobe of the liver was fixed in 10% phosphate-buffered formalin for 1 day and processed in a routine manner for paraffin section. Five-micrometer thick sections were stained with hematoxylin and eosin (HE) for microscopic examination.
Table 1 Composition of GGEx Scientific name
Common name
Family name
Part used
Content (%)
Liriope platyphylla F.T. Wang & T. Tang Platycodon grandiflorum A. DC. Schisandra chinensis K. Koch Ephedra sinica Stapf
Big blue lily turf Chinese bellflower Chinese magnolia vine Chinese ephedra
Liliaceae Campanulaceae Magnoliaceae Ephedraceae
Root Root Fruit Stem
42.86 28.57 14.29 14.28
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Table 2 Sequences of primers used for the RT-PCR assays Genes
Gene bank
Primer sequences
ACOX
J02752
Forward: 5 -actatatttggccaattttgtg-3 Reverse: 5 -tgtggcagtggtttccaagcc-3
195
VLCAD
AF017176
Forward: 5 -cgtcagaggtgtactttgatgg-3 Reverse: 5 -catggactcagtcacatactgc-3
263
MCAD
NM007382
Forward: 5 -gacatttggaaagctgctagtg-3 Reverse: 5 -tcacgagctatgatcagcctctg-3
321
CPT1
L07736
Forward: 5 -tatgtgaggatgctgcttcc-3 Reverse: 5 -ctcggagagctaagcttgtc-3
-Acitn
J00691
Product size (bp)
587
Forward: 5 -tggaatcctgtggcatccatgaaa-3 Reverse: 5 -taaaacgcagctcagtaacagtcc-3
350
2.6. Measurement of visceral fat levels All animals underwent computed tomography (CT, HiSpeed NX/I, General Electric Medical System, Piscataway, USA) to measure cross-sectional abdominal subcutaneous and visceral fat. The animals were examined in the dorsal position and CT scans were performed (120 kV, 150 mA, 1-mm thickness). The cross-sectional areas were determined by the automatic calculation of the scans with a CT number of −160 to −20 Hounsfield units. 2.7. Statistics All values are expressed as the mean ± standard deviation (S.D.). Statistical analysis was performed by ANOVA followed by either post hoc Turkey’s multiple comparison test or Dunnett’s test. Statistical significance was defined as a value of p < 0.05. 3. Results
Fig. 1. Regulation of body weight and food intake by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Body weight gain (A) and food intake (B) were measured daily and all values are expressed as the mean ± S.D. for n = 4 rats. Statistical significance was analyzed at the end of the treatment period. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
3.1. GGEx reduces body weight gain and food intake in genetically obese OLETF rats Compared with OLETF-C rats, OLETF rats given GGEx significantly decreased body weight gain by 14.1% and 29.9% for OLETF-G (X5) and OLETF-G (X10) groups, respectively (Fig. 1A). GGEx treatment also significantly decreased food intake compared with OLETF-C rats. Food intake was reduced by 7.4% and 22.0% in OLETFG (X5) and OLETF-G (X10) groups, respectively (Fig. 1B). The effects of GGEx (X10) on reductions in body weight gain and food intake were comparable to those of sibutramine, an appetite-lowering, anti-obesity drug, in obese OLETF controls (Fig. 1A and 1B). No dose of GGEx used in the present study had any observed toxic effects. 3.2. GGEx reduces abdominal fat areas in genetically obese OLETF rats At the end of the study, the OLETF-C group had abdominal total fat areas of 105.73 ± 3.8 mm2 , whereas the OLETF-G (X5) and OLETF-G (X10) groups had fat areas of 92.95 ± 5.38 and 60.51 ± 1.04 mm2 , respectively (Fig. 2A). Compared with OLETF-C animals, feeding rats with a chow diet supplemented with GGEx significantly decreased total abdominal fat areas by 12.1% in OLETFG (X5) and by 42.8% in OLETF-G (X10) groups. Both visceral and subcutaneous fat areas in the abdomen were significantly reduced by GGEx treatment. In addition, reductions of fat areas were higher in the OLETF-G (X10) than in the OLETF-R group (decreases of 28.2% in total fat areas). Representative CT images are shown in Fig. 2B.
3.3. GGEx lowers the elevation in plasma leptin levels in genetically obese OLETF rats Leptin is a long-term satiety signal that often reflects changes in body weight and adipose tissue mass (Maffei et al., 1995b; Considine et al., 1996). Marked elevations in leptin are thought to reflect leptin resistance and a diminution of satiety responses (Ahima and Flier, 2000). Compared with the LETO-C group, plasma leptin levels were 172% higher in the OLETF-C group (Fig. 3A). In contrast, GGEx (X10) treatment significantly decreased leptin plasma levels by 23.5% in OLETF-C rats with similar levels to those in sibutramine-treated obese rats. To evaluate whether leptin expression is disproportionate to body weight, body weight vs. leptin values were examined. Body weights positively correlated with leptin levels following GGEx treatment (Fig. 3B). 3.4. GGEx improves lipid profiles in genetically obese OLETF rats To determine whether GGEx has beneficial effects on circulating lipid profiles, its effects on plasma triglycerides and total cholesterol were examined. After 8 weeks, OLETF-C rats had significantly higher plasma levels of triglycerides and total cholesterol than did the LETO-C rats. However, a reduction of plasma triglycerides by 28.7% was seen in the OLETF-G (X10) group, although total cholesterol levels were unaffected by GGEx compared with obese control rats (Fig. 4).
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Fig. 2. Modulation of abdominal fat by GGEx in genetically obese OLETF rats. (A) Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Rats underwent CT to measure cross-sectional abdominal subcutaneous and visceral fat areas. All values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. (B) Representative CT images are shown. Gray and white arrows indicate subcutaneous and visceral fat, respectively.
3.5. Inhibition of hepatic lipid accumulation by GGEx in genetically obese OLETF rats Obese OLETF controls showed considerable hepatic lipid accumulation compared with lean LETO controls. However, we found that hepatic lipid accumulation was markedly lower in GGExtreated OLETF rats than in obese OLETF controls (Fig. 5A). Moreover, when OLETF rats were treated with GGEx (X10), there was a complete inhibition of hepatic lipid accumulation, as indicated by the decreased hepatic lesion scores (Fig. 5B). 3.6. Regulation of hepatic PPAR˛ target gene expression by GGEx in genetically obese OLETF rats To evaluate whether GGEx causes alterations in PPAR␣ actions, we measured the mRNA levels of PPAR␣ target enzymes involved in fatty acid -oxidation in the liver. As expected, OLETF control rats had lower mRNA levels of hepatic PPAR␣ target genes, such as carnitine palmitoyl transferase-1 (CPT-1), very long chain acyl-CoA dehydrogenase (VLCAD), medium chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA oxidase (ACOX) by 18%, 2%, 5%, and 9%, respectively, than LETO control rats (Fig. 6A–D). However, GGEx (X10) administration up-regulated the mRNA levels of liver CPT-1, VLCAD, MCAD, and ACOX by 18%, 16%, 21%, and 19%, respectively, in OLETF rats compared with vehicle-treated controls.
Fig. 3. Correlation between plasma leptin levels and body weights after GGEx treatment in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. (A) GGEx significantly decreased leptin plasma levels that were elevated in obese OLETF control rats. All values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. (B) Plasma leptin levels positively correlated with body weights after GGEx treatment.
3.7. Modulation of PPAR˛ reporter gene expression by GGEx To examine the mechanism by which GGEx increases the mRNA expression of PPAR␣ target genes, NMu2Li cells were cotransfected with PPAR␣ and RXR␣ expression constructs as well as the luciferase reporter construct PPRE3 -tk-luc, which contains three copies of the PPRE from the rat ACOX gene. The transfected cells were treated with GGEx at doses that did not induce cytotoxic effects as measured by trypan blue exclusion. Treatment of the transfected cells with GGEx increased luciferase activities in a dose-dependent manner (Fig. 7). 4. Discussion The present study was undertaken to investigate the antiobesity and hypolipidemic effects of GGEx in obese and type 2 diabetic OLETF rats. Since GGEx has been known to modulate human obesity in traditional Korean medicine, it is important to demonstrate the effects of GGEx on obesity experimentally and to verify its mechanism of action. Our results clearly demonstrate that GGEx significantly reduces body weight gain and inhibits hyperphagia. As expected, obese OLETF control rats had much higher food intake compared with lean LETO controls. However, the administration of GGEx (X10)
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Fig. 4. Changes in circulating triglycerides and total cholesterol by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Plasma concentrations of triglycerides (A) and total cholesterol (B) were measured and all values are expressed as the mean ± S.D. for n = 4 rats. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
decreased food intake in obese OLETF controls. The magnitude of this inhibition by GGEx was similar to the effects of sibutramine that is widely used as an anti-obesity drug via a feeding-inhibitory action (Barkeling et al., 2003). Body weight gain was also significantly decreased following GGEx treatment in OLETF control rats. Body weight gain began to decrease after 18 days of treatment and became significantly different by day 43. In parallel with the effects of GGEx on body weight gain, abdominal fat areas were also decreased by GGEx. GGEx treatment significantly reduced both subcutaneous and visceral fat areas in obese OLETF rats. In particular, the reduction after GGEx (X10) treatment was greater than that induced by sibutramine. Our results show that GGEx effectively decreases body weight gain and adipose tissue areas, and that these effects of GGEx may be due to its feeding-inhibitory actions. Obesity is associated with elevated leptin production in the blood (Considine et al., 1996). Leptin is produced mainly in adipocytes and is present in the serum in direct proportion to the amount of adipose tissue (Maffei et al., 1995b; Considine et al., 1996). Leptin exerts negative feedback effects on energy intake, but loses its ability both to inhibit energy intake and stimulate energy expenditure in conditions of obesity. This apparent leptin resistance is explained by the findings that neither endogenous hyperleptinemia nor exogenous leptin treatment is able to prevent weight gain in obese humans and rodents (Considine et al., 1996; Flier, 2004). Similarly, our results showed that obese OLETF controls had 2.7 times
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Fig. 5. Inhibition of hepatic lipid accumulation by GGEx in genetically obese OLETF rats. (A) Representative hematoxylin- and eosin-stained sections of livers are shown (original magnification 200×). Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. Arrows indicate lipid droplets in hepatocytes. (B) Histological analyses of hepatic lipid accumulation. Pathological scores of hepatic lipid accumulation are as follows: 0, no lesion; 1, mild; 2, moderate; 3, severe; 4, very severe. All values are expressed as the mean ± S.D. of four independent experiments. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats.
higher levels of plasma leptin compared with lean LETO controls. In contrast, plasma leptin levels were significantly decreased by GGEx (X10). In fact, increased leptin levels did not induce decreased food intake in OLETF rats and a decrease in its levels after GGEx treatment did not result in increased food intake. Regression analysis also revealed that plasma leptin levels positively correlated with body weight gain. Thus, it is plausible that GGEx prevents hyperphagia, body weight gain, and abdominal fat increases in OLETF rats in part through a decrease in leptin levels contributing to the amelioration of leptin resistance. OLETF rats are known to exhibit hypertriglyceridemia, which is largely responsible for obesity and cardiovascular disease (Sattar et al., 1998; Nakaya et al., 2002; Mori et al., 2005). We observed that OLETF controls had 4.3 times higher plasma triglyceride levels than LETO controls. However, plasma triglycerides were significantly decreased by GGEx (X10) in these rats, although plasma total cholesterol levels were not altered after GGEx treatment, indicating that GGEx efficiently prevented hypertriglyceridemia in obese animals. In order to investigate whether GGEx inhibits hepatic lipid accumulation, HE staining of triglycerides was performed in liver. Compared with LETO control rats, lipid droplets were found to be
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Fig. 6. Modulation of liver PPAR␣ target gene expression by GGEx in genetically obese OLETF rats. Adult male LETO and OLETF rats were treated with water, GGEx, or sibutramine for 8 weeks. RNA was extracted from liver, and relative mRNA levels of CPT-1 (A), VLCAD (B), MCAD (C), and ACOX (D) were measured as described in Section 2. All values are expressed as the mean ± S.D. of R.D.U. (relative density units) using -actin as a reference. *, p < 0.05 vs. lean LETO control rats; **, p < 0.05 vs. obese OLETF control rats. ACOX, acyl-CoA oxidase; CPT-1, carnitine palmitoyl transferase-1; VLCAD and MCAD, very long chain and medium chain acyl-CoA dehydrogenases, respectively.
substantially increased in the livers of obese OLETF rats. Consistent with the effects of GGEx on circulating triglycerides, GGEx inhibited hepatic lipid accumulation in OLETF control rats. These results demonstrate that GGEx stimulates fat catabolism in liver. Ligand-activated PPAR␣ has been shown to regulate the expression of a number of genes critical for fatty acid -oxidation and lipid catabolism. Our previous studies also showed that PPAR␣ effectively improved high fat diet-induced obesity and hypertriglyceridemia (Yoon et al., 2003; Jeong et al., 2004b). To evaluate whether the effects of GGEx on obesity and lipid profiles were mediated by alterations in PPAR␣ target gene expression in liver, we measured the mRNA levels of various PPAR␣ targets, including CPT-1, VLCAD, MCAD, and ACOX. Obese OLETF control rats had
Fig. 7. Modulation of PPAR␣ reporter gene expression by GGEx. All values are expressed as mean ± S.D. of relative luciferase units/-galactosidase activity. The experiments were performed at least three times. *, p < 0.05 vs. vehicle.
lower PPAR␣ target mRNA levels than lean LETO controls. However, OLETF rats fed GGEx were found to have significantly higher CPT-1, VLCAD, MCAD, and ACOX mRNA levels than OLETF control rats. These results are in accordance with our data on the substantial reduction of lipid droplets by GGEx in liver. While GGEx administration substantially increased hepatic PPAR␣ functions in rats, our results can overestimate the pharmacological effects of GGEx on PPAR␣ actions in humans since humans have much less PPAR␣ expression and activity compared with rodents (Holden and Tugwood, 1999; Wilson et al., 2000). Therefore, GGEx-induced PPAR␣ effects obtained in rodents are thought to be less effective in humans. To determine the mechanism by which GGEx increases PPAR␣ target gene expression and to examine whether GGEx-induced PPAR␣ activation regulates obesity and lipid metabolism in an appetite-independent manner, we examined the effects of GGEx on PPAR␣ transactivation using NMu2Li cells, which do not express leptin. Consistent with the in vivo data, GGEx induced the expression of a PPRE-luciferase reporter construct in a transactivation assay. GGEx treatment increased luciferase activity in a dosedependent manner compared with reporter gene activation of cells treated with vehicle, suggesting that GGEx can positively regulate the action of PPAR␣. Although, we cannot exclude the possibility that GGEx stimulates PPAR␣ function through leptin-dependent actions. According to Suzuki et al. (2007), leptin stimulates fatty acid oxidation through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as PPAR␣. In fact, decreased leptin levels and food intake can lead to not only decreases in plasma lipid levels and hepatic lipid accumulation, but also increases in PPAR␣ target gene expression. Thus, it is necessary to investigate whether GGEx-activated PPAR␣ functions are mediated by leptin-AMPK pathways. In addition to the effects of GGEx on obesity, GGEx use in elderly seems to slow aging and prevent age-associated metabolic
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