Journal of Ethnopharmacology 135 (2011) 156161

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Wild bitter gourd extract up-regulates mRNA expression of PPAR, PPAR and their target genes in C57BL/6J mice
Che-Yi Chao a, , Mei-Chin Yin a,b , Ching-jang Huang c
a b c

Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan Department of Nutrition, China Medical University and Hospital, Taichung, Taiwan Department of Biochemical Science and Technology and Institute of Micobiology and Biochemistry, National Taiwan University, Taipei, Taiwan

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) was commonly used as a medicinal herb in Asia, Africa, and South America because of its anti-diabetic, antibacterial, anti-viral, and chemopreventive functions. Materials and methods: C57BL/6J mice were orally administered with 250, 500 or 1000 mg/kg BW of WBGE in 0.2 mL/mouse of olive oil daily for 2 weeks. Results: Compared to control (vehicle treated) mice, mice receiving WBGE showed signicantly higher PPAR, ACO (acyl-CoA oxidase) and L-FABP (liver-fatty acid binding protein) mRNA expression, ACO activity and protein in the liver (P < 0.05), as clobrate-treated mice. WBGE treatment also resulted in signicantly higher PPAR and LPL (lipoprotein lipase) mRNA (P < 0.05) in the epididymal adipose tissue. Liver triglyceride and non-esteried fatty acid concentration in WBGE treated mice were signicantly lower than those of control mice (P < 0.05). Plasma adiponectin level was signicantly higher in mice receiving WBGE than in control mice (P < 0.05), as the rosiglitazone treated mice. Conclusion: Results of this study demonstrated that WBGE also activates PPAR and PPAR signaling pathway in vivo. 2011 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 21 June 2010 Received in revised form 2 January 2011 Accepted 2 March 2011 Available online 8 March 2011 Keywords: Wild bitter gourd PPAR PPAR Adiponectin

1. Introduction A wild species of Momordica charantia, wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.), is native to tropical areas of Asia. Wild bitter gourd (WBG) grows in the eastern and southern regions of Taiwan and is consumed not only as a vegetable but also as a folklore medicine for disease prevention by local residents (Lii et al., 2009). The most noticeable pharmacological property of WBG is the hypoglycemic activity which has not only been used widely in the herbal medicine but also been demonstrated in rodent models (Teoh et al., 2009; Uebanso et al., 2007) and type 2 diabetic patients (Leatherdale et al., 1981; Welihinda et al., 1986). However, the mechanism and well-controlled clinical trials are still lacking (Leung et al., 2009). WBG is considered to be more potent in disease prevention than bitter gourd; however, little is known about the biological and physiological characteristics of WBG.

Corresponding author at: Department of Health and Nutrition Biotechnology, Asia University, 500, Liufeng Rd., Wufeng, Taichung, Taiwan. Tel.: +886 4 23323456x1859; fax: +886 4 23013858. E-mail address: cychao@asia.edu.tw (C.-Y. Chao). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.03.001

Peroxisome proliferator activated receptors (PPARs) are ligandactivated transcriptional factors that belong to the steroid hormone nuclear receptor superfamily. These receptors regulate various metabolic processes by controlling the expression of specic cascades of genes (Desvergne and Wahli, 1999). Fibrates, a class of therapeutic agents that effectively lower serum triglycerides and raise HDL-cholesterol in human, are synthetic ligand of PPAR and the pharmacologic action was demonstrated to mediated by this receptor. Thiazolidinediones (TZDs), a new class of anti-diabetic drug that effectively lower blood glucose and insulin level and improving insulin sensitivity, are synthetic ligand of PPAR and sensitize insulin response through the activation of this receptor (Picard and Auwerx, 2002). Hence, PPARs are molecular targets for the development of drugs for treating obesity, hyperlipidemia, type 2 diabetes and cardiovascular disease (Plutzky, 2003). PPARs regulate genes for which a functional PPAR response element (PPRE) has been identied includes peroxisomal -oxidation enzymes, acyl-CoA oxidase (ACO); fatty acid carriers, adipocyte lipid-binding protein (aP2), liver fatty acid binding protein (L-FABP) and lipoprotein lipase (LPL), indicating the important role of PPARs in the regulation of lipid metabolism (Juge-Aubry et al., 1997). PPARs are regarded as important therapeutic targets for cardiovascular disease because the activation of these receptors can not

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only directly regulate genes of vascular and inammatory cells that involved in the development of atherosclerosis, but also indirectly improve glucose utilization and serum lipid proles (Plutzky, 2003). We have used a transactivation assay to screen a wide range of food materials and found that the organic solvent extract of WBG activated both PPAR and PPAR to an extent that is comparable to known potent agonists of PPAR (Wy-14643) and PPAR (rosiglitazone) in a previous report (Chao and Huang, 2003). This result implied that the hypoglycemic activity of WBG maybe through its activation on PPAR. This study was conducted to test the hypothesis that WBG extract also regulate the expression of PPAR and PPAR target genes in vivo. 2. Materials and methods 2.1. Preparation of test samples Whole fruit of wild bitter gourd was cut and pressed in an electric juicer. The residue was collected, freeze-dried, ground up and extracted with ethyl acetate (1:30 w/v) by stirring overnight at room temperature. The ethyl acetate solutions were ltered, and the ltrates were evaporated in a rotary evaporator (Buchi) at 40 C to remove the solvent. The extract (WBGE) was weighed and stored at 20 C. The preparation efcacy of freeze-dried bitter gourd residue was around 5%. The yield of WBGE in this extraction procedure was around 0.1% of the wild bitter gourd whole fruit. The WBGE was dissolved in olive oil for oral administration of mice. 2.2. Animals and grouping Six-week old male C57BL/6J mice weighing 1820 g (National Laboratory Animal Center, Taipei, Taiwan) were housed individually in stainless steel wire cages in a room maintained at 25 2 C, with a controlled 12 h lightdark cycle and free access to food and tap water. They were fed the chow diet and adapted for one week and then randomly assigned to seven groups (V, L, M, H, CF, RSG and BB) for treatments, with 8 mice in each of the V, L, M, H and the BB groups and 4 mice in each of the CF (Fluka) and the RSG (Avandia) groups. Body weight and food intake were recorded weekly and every 23 days, respectively. All animal experiments were carried out in accordance with Institutional Animal Care and Use Committee Guidelines of Asia University. Throughout the treatment period, V, L, M, H, CF, RSG groups of mice were daily gavaged with vehicle (olive oil, V group), low (250 mg/kg BW, L group), medium (500 mg/kg BW, M group) or high dose (1000 mg/kg BW, H group) of WBGE, clobrate (100 mg/kg BW, CF group) or rosiglitazone (10 mg/kg BW, RSG group), respectively. Treated substances were dissolved in olive oil (vehicle) and each mouse received 0.2 mL per day. These animals were fed with a basal diet which was modied from the AIN-93G puried diet and contained 39.8% cornstarch, 20% casein, 13.2% dextrinized cornstarch, 10% sucrose, 7% soybean oil, 5% ber, 3.5% AIN-93G mineral mix, 1% AIN-93 vitamin mix, 0.3% l-cysteine and 0.2% choline bitartrate. BB group of mice was fed with a diet containing 5% of freeze-dried wild bitter gourd juicing residue without gavage feeding. This diet was prepared by replacing 0.5% casein (Sigma, USA), 1% starch (Samyang genex, Korea), 1% sucrose (Taiwan Sugar Co., Taiwan) and 2.5% ber (JRS, Germany) of the basal diet with 5% freeze-dried wild bitter gourd juicing residue that was used for extracting WBGE. 2.3. Blood and tissue preparation After 14 days of treatment, mice were fasted overnight (12 h) and sacriced by CO2 asphyxiation. Blood was collected from supe-

rior vena cava in an EDTA-tube. Liver, epididymal fat pad, and retroperioneal fat pad were excised, weighed and a small portion was immediately frozen in liquid nitrogen and stored at 80 C for the analysis of mRNA expression. A second portion of liver was frozen at 20 C for the analysis of liver lipids. Remaining portions of liver were freshly homogenized for the preparation of post-nuclear supernatant (PNS) and microsome, respectively as described (Chao et al., 2001). Plasma samples were obtained by centrifugation of blood and stored at 80 C for the analysis. Plasma and liver lipids were measured by enzymatic methods using commercial kits (RANDOX, Amtrim, UK) for cholesterol, TG and nonesteried fatty acid (NEFA), these biochemical analysis as previously described (Chao et al., 2001). Plasma adiponectin concentration was measured by ELISA also using a commercial kit (Mouse/Rat Adiponectin ELISA kit, B-Bridge, USA). 2.4. Western blot analysis The peroxisomal ACO in PNS of liver were determined by the method of Small et al. (1985). Liver microsomal protein was subjected to 10% SDSPAGE, and then transferred to a polyvinylidene uoride (PVDF) membrane (NEN Life Science, Boston, MA). The blot was immunodetected with enhanced chemiluminescence Western blotting kit (Amersham International, Amersham, UK) in which goat anti-rat ACO antiserum was used as the primary antibody and goat anti-rabbit IgG-biotinylated species-specic whole antibody (Calbiochem) was used as the secondary antibody. 2.5. Northern blot and semi-quantitative RT-PCR Total RNA was extracted from liver and adipose tissue using trizol reagent (GIBCO/BRL). Total RNA was separated by electrophoresis in denaturing formaldehyde agarose gel, and then transferred to nylon membrane. The blots were prehybridized at 42 C for 6 h in the hybridization buffer containing salmon sperm DNA, then hybridized at 42 C for 1216 h with 32 P-labelled cDNA probes of ACO, L-FABP, aP2, LPL or 18S RNA sequentially after deprobing previous probe remained on membrane. All the cDNA probes were synthesized by RT-PCR to amplify encoding base pairs 74-2059 for ACO, 33-405 for L-FABP, 301-697 for aP2, 1005-1864 for LPL and 1-1297 for 18S RNA. To correct for possible difference in transfer and loading, 18S RNA was used as an internal control. After washing at the appropriate stringency, the blots were exposed to X-OMAT AR lm (Kodak). Signals were quantied using the microcomputer imaging device (MCID) image analysis system (Fuji, Tokyo, Japan). The content of PPAR mRNA in liver and PPAR mRNA in adipose tissue were measured by a semiquantitative RT-PCR as previously described (Chao et al., 2001). Primers used for PPAR was: 5 -CCTGTGAACACGATCTGAAAG3 and 5 -TCTGACTCGGTCTTCTTGATG-3 , that used for PPAR was 5 -ATGGGTGAAACTCTGGGAGAT-3 and 5ACCACCCATTGGGTCAGCTCT-3 . 2.6. Statistical analysis All values were expressed as mean SD. The signicant difference between the means of the control and the experimental animals was evaluated by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically signicant. Statistical analysis was performed using the SPSS13.0 statistic program (Chicago, IL, USA). 3. Results As shown in Table 1, the initial body weight of the seven groups of mice was not signicantly different. After 2 weeks of treat-

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Table 1 The initial body weight, nal body weight, body weight gain, food intake, relative tissue weight of mice orally gavaged with WBGE for 2 weeks. Groups V (n = 8) L (n = 8) M (n = 8) H (n = 8) BB (n = 8) CF (n = 4) RSG (n = 4) Initial BW 20.7 20.3 20.1 20.1 20.1 20.5 20.8 1.5 1.9 1.7 1.7 1.7 1.5 1.5 Final BW 22.9 22.8 23.1 23.9 21.3 21.9 24.0 1.2 0.8 0.8 1.2 1.1* 1.0* 1.4 BW gain 2.2 2.5 3.0 3.8 1.2 1.4 3.2 0.3 0.5 0.2 0.4* 0.5* 0.4* 0.2* Food intake (g/day) 4.94 5.01 4.78 4.91 5.04 4.85 4.81 0.84 0.94 0.68 0.99 0.62 0.46 0.67 Liver (g/100 g BW) 4.81 5.01 5.03 5.10 5.67 5.65 4.81 0.35 0.45 0.28 0.26 0.36* 0.18* 0.08 Epididymal fat pad (g/100 g BW) 1.88 1.73 1.94 1.93 1.73 1.74 1.99 0.42 0.45 0.47 0.51 0.23 0.25 0.36 Retroperitoneal fat pad (g/100 g BW) 0.60 0.44 0.46 0.51 0.44 0.44 0.60 0.15 0.12* 0.11* 0.13 0.10* 0.09* 0.18

Data are expressed as mean SD of each group. * Signicant difference compared to the vehicle control group (P < 0.05).

ment, mice orally gavaged with three doses of WBGE (L, M and H groups) or 10 mg/kg BW of Rosiglitazone or vehicle showed similar nal body weight, relative weight of liver and epididymal fat pad (P > 0.05). The H and RSG groups of mice had signicantly higher body weight gain than V, L and M groups (P < 0.05). However, mice dosed with 100 mg/kg of clobrate (CF group) and mice fed diets containing 5% wild bitter gourd freeze-dried juicing residue (BB) showed signicantly lower nal body weight, body weight gain and higher relative liver weight compared to the remaining 5 groups (P < 0.05). Moreover, the relative weight of the retroperitoneal fat pad of the L, M, BB and CF groups were signicantly lower than that of V, H and RSG groups (P < 0.05). The food intake was not signicantly different among the seven groups of mice. Liver acyl-CoA oxidase (ACO) is the target gene of PPAR. Oral administration of WBGE (L, M and H groups) dose-dependently increased liver acyl-CoA oxidase activity of mice. (P < 0.05) (Fig. 1A). The ACO activity of the H group was signicantly higher than that of V, L and M group, ACO activity of the M group was signicantly higher than that of V and L group, ACO activity of the L group was also signicantly higher than that of the V group (P < 0.05). The extent of the increase in the WBGE treated mice was not as high as that in the CF treated mice. The ACO activity of CF group was about 1.5 fold that of the H group (P < 0.05). Oral administration of WBGE also signicantly increased the expression of ACO protein (Fig. 1B) and mRNA (Fig. 2A) (P < 0.05), similar to the effect of CF. The ACO protein and mRNA expression of the BB and RSG groups were also signicantly higher than that the V group but lower than the CF groups. The mRNA expressions of liver L-FABP, other PPAR target genes, were also up-regulated by oral administration of WBGE (Fig. 2A). Mice dosed with the three doses of WBGE had signicantly higher mRNA expressions of liver L-FABP than mice of the V group, and there were a dos-dependent tendency. In the case of L-FABP mRNA, the H group had signicantly higher level than the remaining six groups of mice (P < 0.05). The mRNA expression of aP2 as well as LPL in the epididymal fat pad is shown in Fig. 2B. Oral administration of the three doses of WBGE did not change the aP2 mRNA (P > 0.05). Mice of the BB group had signicantly higher aP2 mRNA compared to mice of the V group. The LPL mRNA in the epididymal fat pad of mice receiving wild bitter gourd (L, M, H and BB groups) were signicantly higher than that of the V group (P < 0.05). The mRNA of PPAR in liver and PPAR in the adipose tissue were analyzed by a semi-quantitative PCR and the result is shown in Fig. 3. Oral administration of WBGE dose dependently increased the mRNA of PPAR in liver and PPAR in the adipose tissue (P < 0.05). The CF and BB groups of mice also showed signicantly higher level of liver PPAR mRNA, but not the RSG group. The RSG and BB groups of mice had signicantly higher level of PPAR mRNA in the adipose tissue, but not the CF groups. Serum adiponectin concentration, measured by an ELISA kit, was shown in Fig. 4. Mice of the H, BB and RSG groups had signicantly

higher level of adiponectin in the plasma than the V group (P < 0.05). The plasma adiponectin of the RSG group was further higher than the H and BB group. As shown in Table 2, the liver triglyceride concentration of the H group was signicantly lower than that of the V group, similar to the effect of CF treatment (P < 0.05). The liver NEFA concentration was signicantly lower in L, M, H and BB groups than in the V group (P < 0.05). The triglyceride, cholesterol and NEFA in

Fig. 1. The protein expression and enzyme activity of acyl-CoA oxidase (ACO) in the liver of mice orally gavaged with vehicle (V), 250 (L), 500 (M) or 1000 (H) mg/kg of WBGE for 2 weeks. The results are presented as mean SD of each group. *Signicant difference compared to the vehicle control group (P < 0.05).

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Fig. 2. Northern blot analysis for mRNA of PPAR target genes in liver and adipose tissue of mice orally gavaged with vehicle (V), 250 (L), 500 (M) or 1000 (H) mg/kg of WBGE for 2 weeks. Tissue total RNA was extracted, separated by electrophoresis, transferred to nylon membranes, and then hybridized with various cDNA probes. Signals were quantitated by image analysis. Values were normalized by 18S. The results are presented as mean SD of each group. *Signicant difference compared to the vehicle control group (P < 0.05).

serum of mice dosed with WBGE were not signicantly different from those of the V group (P > 0.05). 4. Discussions Oral administration of WBGE up-regulated the mRNA expression of liver ACO, L-FABP as well as adipose tissue LPL in the normal

mouse model (C57BL/6J on an AIN-93G standard diet) used in this study. These results demonstrated that WBGE activates the PPAR signaling pathway in vivo as well. As mRNA expression of PPAR and PPAR were also up-regulated, WBGE activated PPARs signaling pathway not only by providing agonists but also increased the expression of the receptor. As most of the type 2 diabetic patients also have dyslipidemia, attempts have been made to develop thera-

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Fig. 3. Semi-quantitative RT-PCR analysis for mRNA of PPAR in liver and PPAR in adipose tissue of mice orally gavaged with vehicle (V), 250 mg/kg (L), 500 mg/kg (M) or 1000 mg/kg (H) of WBGE for 2 weeks. Tissue total RNA was extracted and reverse transcribed by MMLV-RT. The RT products were used as templates for PCR (B). Signals were quantitated by image analysis and normalized by -actin (A). The results are presented as mean SD of each group. *Signicant difference compared to the vehicle control group (P < 0.05).

Table 2 Lipid concentration in serum and liver of mice orally gavaged with WBGE for 2 weeks. Groups Serum TG (mg/dL) V (n = 8) L (n = 8) M (n = 8) H (n = 8) BB (n = 8) CF (n = 4) RSG (n = 4) 141 124 119 106 84 65 61 27 26 27 29 19* 19* 21* Cholesterol (mg/dL) 157 149 143 139 124 160 155 11 13 5 15* 22* 10 18 NEFA (mmol/L) 0.88 0.85 0.74 0.69 0.79 0.52 0.65 0.25 0.21 0.14 0.08 0.06 0.02* 0.27 Liver TG (mg/g liver) 4.80 4.73 4.42 3.22 3.85 2.09 3.84 0.7 0.7 0.4 0.5* 0.5 0.4* 0.1 Cholesterol (mg/g liver) 2.01 1.79 1.60 1.50 1.73 1.53 1.91 0.2 0.3 0.6 0.6 0.2 0.1* 0.2 NEFA (mol/g liver) 59 42 31 33 32 48 52 6 7* 2* 7* 4* 7* 8

Data are expressed as mean SD of each group. * Signicant difference compared to the vehicle control group (P < 0.05).

50

40

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30

20

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0 V
vehicle

L
250 mg/kgBW

BB
5% (w/w)

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500 1000 mg/kgBW mg/kgBW

100 10 mg/kgBW mg/kgBW

Fig. 4. Serum adiponectin concentrations of mice orally gavaged with vehicle (V), 250 mg/kg (L), 500 mg/kg (M) or 1000 mg/kg (H) of WBGE for 2 weeks. The results are presented as mean SD of each group. *Signicant difference compared to the vehicle control group (P < 0.05).

peutic agents that are dual agonists for both PPAR and PPAR. For example, KRP297 (Murakami et al., 1998), GW2331 (Dana et al., 2001) and Ragaglitazar (Chakrabarti et al., 2003; Ye et al., 2003) were shown to be effective in lipid lowering and insulin sensitizing in rodents. Being able to activate both PPAR and PPAR, wild bitter gourd extract may have similar properties. Target genes of PPAR in liver include an array of enzymes responsible for the -oxidation of fatty acids. Activation of the PPAR signaling, therefore, leads to increased fatty acid catabolism (Desvergne and Wahli, 1999). This may contribute to signicantly lowered liver NEFA concentrations in mice treated with WBGE and clobrate. Although there was up-regulation of liver ACO mRNA, protein and activity, L-FABP and LPL mRNA in mice receiving WBGE, indicating an activation of PPAR, the serum triglycerides level was not lowered in these mice as in mice receiving clobrate. A number of attributive factors may be speculated. The liver ACO activity of mice receiving WBGE was signicantly lower than that of mice receiving clobrate. It is possible that the nal effective concentration of the active component in WBGE present in mouse liver is not as high as clobrate due to a low absorption or a faster bioconversion, i.e. the pharmacokinetic pattern of the active component in WBGE is different from that of clobrate. In addition, Caira et al. (1995) found that the

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fold of increase in the transcription rate was much lower than the fold of increase in the ACO mRNA and activity in rats treated with ciprobrate. The result led these authors to speculate that there may be a post-transcription regulation, such as the stabilization of mRNA. It is also possible that this type of post-transcription regulation may be different between PPAR activator of WBGE and clobrate. Furthermore, WBGE sample used may have high content of other lipid species which may interfere with the metabolic process induced by PPAR signaling or may even elevated serum triglycerides. It has been identied the active component in WBGE was to be 9cis, 11trans, 13trans-conjugated linolenic acid (9c, 11t, 13t-CLN). The isolated 9c, 11t, 13t-CLN rich fraction also signicantly induced acyl-CoA oxidase (ACO) activity in a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, implying that 9c, 11t, 13t-CLN was able to act on a natural PPAR signaling pathway as well (Chuang et al., 2006). Physiological condition, such as fasting and developmental condition, such as adipocyte differentiation, are known to increase the expression of PPAR in liver and PPAR in adipose tissue, respectively. However, inconsistent results were reported in literature regarding whether a PPAR agonist up-regulate the expression of PPAR. It appears that the differences in agonist used (Davies et al., 2002), species (Motojima et al., 1997) and diet condition (Pearson et al., 1996) may contribute to the discrepancy. In conclusion, oral administrations of wild bitter gourd extract up-regulated mRNA expression of PPAR, PPAR as well as their target genes in mice. Wild bitter gourd can potentially be developed to a diet supplement that exhibit hypolipidemic and insulinsensitizing activity similar to dual agonist of PPAR and PPAR. It would be of great value for the improvement of metabolic syndrome, a major health hazard that predict very high risk of cardiovascular disease, premature death and diabetes (Isomma, 2003; Meigs, 2003). Acknowledgement This study is supported in part by National Science Council, Taiwan (NSC 97-2320-B-468-001-MY3). References
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