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Fruit extracts of Momordica charantia potentiate glucose uptake and up-regulate Glut-4, PPARγ and PI3K
Journal of Ethnopharmacology 126 (2009) 533–537
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Fruit extracts of Momordica charantia potentiate glucose uptake and up-regulate Glut-4, PPAR␥ and PI3K Ramadhar Kumar, S. Balaji, T.S. Uma, P.K. Sehgal ∗ Bio-products Laboratory, Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600 020, Tamil Nadu, India
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Article history: Received 21 December 2008 Received in revised form 17 July 2009 Accepted 31 August 2009 Available online 8 September 2009 Keywords: Momordica charantia (MC) Fruit extracts Glucose transporter (Glut-4) Peroxisome proliferator activator receptor gamma (PPAR␥) Phosphotidylinositol-3 kinase (PI3K) L6 myotubes
a b s t r a c t Ethnopharmacological relevance: Momordica charantia fruit is a widely used traditional medicinal herb as, anti-diabetic, anti-HIV, anti-ulcer, anti-inflammatory, anti-leukemic, anti-microbial, and anti-tumor. Aims of study: The present study is undertaken to investigate the possible mode of action of fruit extracts derived from Momordica charantia (MC) and study its pharmacological effects for controlling diabetic mellitus. Effects of aqueous and chloroform extracts of Momordica charantia fruit on glucose uptake and up-regulation of glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPAR␥) and phosphatidylinositol-3 kinase (PI3K), were investigated to show its efficacy as a hypoglycaemic agent. Materials and methods: Dose dependent glucose uptake assay was performed on L6 myotubes using 2deoxy-d-[1-3 H] glucose. Up-regulatory effects of the extracts on the mRNA expression level of Glut-4, PPAR␥ and PI3K have been studied. Results: The association of Momordica charantia with the aqueous and chloroform extracts of Momordica charantia fruit at 6 g/ml has shown significant up-regulatory effect, respectively, by 3.6-, 2.8- and 3.8fold on the battery of targets Glut-4, PPAR␥ and PI3K involved in glucose transport. The up-regulation of glucose uptake was comparable with insulin and rosiglitazone which was approximately 2-fold over the control. Moreover, the inhibitory effect of the cyclohexamide on Momordica charantia fruit extract mediated glucose uptake suggested the requirement of new protein synthesis for the enhanced glucose uptake. Conclusion: This study demonstrated the significance of Glut-4, PPAR␥ and PI3K up-regulation by Momordica charantia in augmenting the glucose uptake and homeostasis. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Momordica charantia Linn. (bitter melon, bitter gourd or karela in Hindi), a plant native to the semi-tropical climate of China, India, Asia, and Africa, bears fruits which are traditionally used medicinal herbs as, anti-HIV, anti-ulcer, anti-inflammatory, antileukemic, anti-microbial, anti-diabetic, and anti-tumor, to name a few (Taylor, 2002; Grover and Yadav, 2004) and is one of the most promising alternative medicines for the disease. Herbal drugs have potential therapeutic applications because of their effectiveness, less side effects and relatively low cost (Venkatesh et al., 2003). Patients may prefer herbal drugs formulations if doctors readily make it available to them for common diseases. Therefore, investigation on such agents from traditional medicinal plants has become more important (Suba et al., 2004). Mukherjee et al. (2006) provided comprehensive information on various plant species from Indian biosphere and their constituents,
∗ Corresponding author. Tel.: +91 044 24420709; fax: +91 044 2491158. E-mail address: sehgal [email protected] (P.K. Sehgal). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.048
which have been shown to display potent hypoglycemic activity. In his review, he has stated that hypoglycaemic herbs are major Avenue in Indian perspectives particularly for treating diabetes, it required effectively to explore the literatures available on these aspects. A few active components, polypeptide-p (Khanna et al., 1981), flavonoids and charatin, a mixture of two steroid glycosides (Lotlikar and Rajarama, 1966) have been identified from the fruit and its seeds. These components, along with pure juice, methanol, chloroform and ethanol extracts from the fruit, have shown hypoglycaemic activity via in vitro, animal and human studies (Khanna et al., 1981; Welihinda and Karunanayake, 1986; Sitasawad et al., 2000; Rathi et al., 2002; Virdi et al., 2003). Mukherjee et al. (2006) described the chemistry, activity and usage of the constituents isolated from Indian hypoglycemic plants for the treatment of diabetes. Glucose transport is the rate-limiting step in glucose utilization, especially in insulin targeted skeletal muscle and mediated by major glucose transporter (Glut) proteins, Glut-4 and Glut-1 (Ziel et al., 1988). Insulin resistance in type 2 diabetes is manifested by decreased insulin-stimulated glucose transport and
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impaired metabolism in adipocytes and skeletal muscle, resulting in down-regulation of the major insulin-responsive Glut, the Glut-4 (Kellerer et al., 1999). PI3K is a key molecular switch, which mediates the metabolic effects of insulin, glucose transport and Glut-4 translocation (Okada et al., 1994; Tsakiridis et al., 1995). PPAR␥, a transcription factor belonging to the nuclear receptor super family (Desvergne and Wahli, 1999), essential for adipocyte differentiation (Ntambi and Young Cheul, 2000) and directly enhances insulin signalling and glucose uptake in muscle on binding with the PPAR␥ agonists (Ciaraldi et al., 1995). Although, Momordica charantia has been studied at cellular level and has shown the glucose uptake activity and up-regulation of PI3K as well as Glut-4 (Singh et al., 2004; Ben et al., 2007), but further mechanisms with clear evidences are yet to be found out. In the present work we have selected the L6 rat myoblasts as a model to see the expression level of mRNA of the battery of target Glut-4, PPAR␥ and PI3K to find out the possible mode of action for glucose uptake and homeostasis by fruit extract of Momordica charantia and we are also reporting the effect on mRNA expression level of PPAR␥.
2.5. Culture of L6 cells Monolayer of L6 muscle cell (NCCS, Pune, India) culture were maintained in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal calf serum (FCS) supplemented with penicillin (120 units/ml), streptomycin (75 mg/ml), gentamycin (160 mg/ml) and amphotericin B (3 mg/ml) at 37 ◦ C humidified with 5% CO2 . After 4–6 days confluence, L6 myoblast were transferred to DMEM with 2% FCS for differentiation. The extent of differentiation was established by observing multinucleation of cells and ∼90% fusions of myoblasts into myotubes. The differentiated myotubes were then used to investigate the effects of Momordica charantia fruit juice extracts. Differentiated myotubes were incubated for 30 min with insulin and 24 h with rosiglitazone, wherever indicated. 2.6. Cytotoxicity assay
2. Materials and methods
The effect of the extracts on cell viability of L6 was determined by using a colorimetric technique, which is 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosman, 1983). The cells were grown in 24-well plates (Corning, NY) and the differentiated cells were treated with both extracts at 5, 10, 15 and 20 g/ml.
2.1. Chemicals and reagents
2.7. Measurement of 2-deoxy-d-[1-3 H] glucose
All cell culture supplements were purchased from Life Technologies, USA. All chemicals, Primers and RT-PCR kits were obtained from Sigma–Aldrich, St. Louis, USA. All other chemicals and organic solvents used were of the highest analytical grade.
L6 cells grown in a 12-well plate (Corning, NY) were subjected to glucose uptake as reported (Yonemitsu et al., 2001). Fully differentiated myotubes serum starved for 5 h were incubated with the different concentration of fruit extracts for 12, 24, 36 or 48 h and control experiments in the absence of any stimulant were also performed for the same incubation periods. There were no significant difference in glucose uptake was observed on the basis of different incubation period; hence experimental incubation was optimised and reported for 24 h for all further studies. After incubation, cells were rinsed once with N-2-hydroxy ethyl piperazine-N -2ethane sulphonic acid (HEPES)-buffered Krebs Ringer phosphate solution (118 mM NaCl, 5 mM KCl, 1.3 mM CaCl2 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 and 30 mM HEPES ∼ pH 7.4) and further incubated for 15 min in HEPES-buffered solution containing 0.5 Ci/ml 2-deoxy-d-[1-3 H] glucose. The media were aspirated to terminate the uptake. Then the cells were washed thrice with ice cold HEPESbuffered solution and lysed in 0.1% SDS. An aliquot was used to measure the radioactivity. Glucose uptake values were corrected for non-specific uptake in presence of 10 mM cytochalasin B. All the assays were performed in triplicate.
2.2. Plant material The fruits of Momordica charantia Linn. were collected from local vegetable market, Chennai, Tamilnadu, India, and authenticated by a Pharmacognosy expert, Head, Department of Botany, Central Leather Research Institute, Chennai, India, before subjecting it to extraction and phytochemical characterization. The voucher specimens (MC1BPT) were identified and stored.
2.3. Preparation of fruit extract The 0.5 kg of fresh fruits were cut to remove the seeds and chopped into small pieces and then homogenised with water (1:2) in a commercial blender. The fresh juice was then centrifuged at 5000 rpm and the supernatant was lyophilized. The chloroform extract was prepared by extracting 0.5 kg of shade dried chopped pieces of fruits using chloroform in ratio of 1:10. The extraction was carried out exactly as described above and supernatant was dried under reduced pressure at low temperature. Both extracts were employed at different concentration.
2.4. Qualitative preliminary phytochemical group test The qualitative preliminary phytochemical group test for alkaloids, glucosides, saponins, phytosterols, tannin and flavonoids of aqueous and chloroform extracts were performed by the standard methods (Brain and Turner, 1975; Evans, 1996). HPLC analysis of the extracts with charantin (0.5 mg/ml) as a reference substance was performed with C-18 Inertsil ODS-3 column (5 m particle, 4.6 mm × 250 mm I.D.). The mobile phase was 100:2 (v/v) methanol–water and was delivered at a flow rate of 1 ml/min. The UV detection wavelength was 204 nm and the sample injection volume was 200 l.
2.8. Reverse transcriptase-polymerase chain reaction (RT-PCR) RT-PCR was carried out according to Kit supplier (Sigma, USA). After incubation, cells were lysed in Trizol, proteins were extracted with chloroform, and total RNA was precipitated with isopropanol. The RNA precipitate was washed with 70% ethanol and resuspended in 50 l of DEPC-treated water (Hall et al., 1998). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been taken as an internal control. The primers used were as follows. GAPDH: sense, 5 -CCA CCC ATG GCA AAT TCC ATG GCA-3 ; antisense, 5 -TCT AGA CGG CAG GTC AGG TCC ACC-3 (588-bp, Sahni et al., 1999); Glut-4: sense, 5 -CGG GAC GTG GAG CTG GCC GAG GAG3 ; anti-sense, 5 -CCC CCT CAG CAG CGA GTG A-3 (318-bp, Buhl et al., 2001); PI3K: sense, 5 -TGA CGC TTT CAA ACG CTA TC-3 ; antisense, 5 -CAG AGA GTA CTC TTG CAT TC-3 (248 bp, Laville et al., 1996); and PPAR␥: sense, 5 -GGA TTC ATG ACC AGG GAG TTC CTC3 ; anti-sense, 5 -GCG GTC TCC ACT GAG AAT AAT GAC-3 (155-bp, Yonemitsu et al., 2001). For PCR reaction, 1 l of cDNA mixture was added to a PCR reaction mix containing 10× PCR buffer, 2 mM dNTP,
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Fig. 1. Dose dependent analysis of 2-deoxyglucose uptake by aqueous extract. The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
10 pM of paired primers, 2 units of Taq polymerase. Semi-quantified PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed to analysis the transcripts using scanning densitometry. Signals of transcript in the agarose gel were quantified arbitrarily. 2.9. Statistical analysis Results are represented as mean ± S.D. of three independent experiments and statistical significance was determined via Student’s t-test with p < 0.05 considered to be significant. 3. Results
Fig. 2. Representative agarose gel photograph of (A) GAPDH (588 bp) and (B) Glut4 (318-bp) transcript. (C) Semi-quantification analysis of Glut-4 transcripts using scanning densitometry. Signals of Glut-4 in agarose gel were quantified arbitrarily. Lanes—1: control; 2: insulin; 3: rosiglitazone; 4: aqueous extract of Momordica charantia (MCA); 5: chloroform extract of Momordica charantia (MCC); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
In association with glucose uptake, extracts significantly increased the PPAR␥ expression (Fig. 3A) by 2.8-fold over control and this was comparable with rosiglitazone (2.4-fold) as revealed by densitometry scanning (Fig. 3C) while insulin-treated cells did not alter the PPAR␥ expression significantly. Densitometry scanning (Fig. 4C) showed approximately 3.8-fold increased PI3K expression (Fig. 4A) by Momordica charantia fruit extracts comparable with insulin (4.2-fold) over untreated control
3.1. Fruit extracts and phytochemical groups The yield of aqueous and chloroform extract was 2.6 w/w% and 4 w/w%, respectively. The preliminary qualitative phytochemical group tests showed the presence of alkaloids, glycosides, tannin and flavonoids in both extracts but saponins and steroids were detected in aqueous and chloroform extract respectively. The HPLC analysis of the extracts showed the presence of charantin molecules along with some other constituents. 3.2. Glucose uptake analysis Differentiated L6 myotubes after experimental incubation were analysed for glucose uptake analysis. Among the different doses, both extracts showed a better uptake of approximately 2-fold increases over control at 6 g/ml dose levels. Doses level greater than 6 g/ml have shown decreased uptake (Fig. 1). 3.3. Cytotoxicity assay The MTT analysis for all doses level shown 100% cell viability (data not shown) and did not have any cytotoxic effects on L6 cells. 3.4. Effect on Glut-4, PPAR and PI3K at transcription level Our observations reveal almost same elevated expression of Glut-4, PPAR␥ and PI3K transcripts by both chloroform and aqueous extracts of Momordica charantia fruit. The relative densitometry scanning (Fig. 2C) revealed significantly increase in Glut-4 transcript (Fig. 2B) approximately 3.6-fold by Momordica charantia fruit extracts over untreated control cells and equivalent to insulin (3.8fold) and rosiglitazone (3.2-fold).
Fig. 3. Representative agarose gel photograph of (A) PPAR␥ (155 bp) and (B) GAPDH (588 bp) transcript (C) Semi-quantification analysis of PPAR␥ transcripts using scanning densitometry. Signals of PPAR␥ in agarose gel were quantified arbitrarily. Lanes—1: control; 2: insulin; 3: rosiglitazone; 4: chloroform extract of Momordica charantia (MCC); 5: aqueous extract of Momordica charantia (MCA); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
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Fig. 4. Representative agarose gel photograph of (A) PI3K (248 bp), and (B) GAPDH transcript. (C) Semi-quantification analysis of PI3K transcripts using scanning densitometry. Signals of PI3K in agarose gel were quantified arbitrarily. Lanes—1: control; 2: rosiglitazone; 3: insulin; 4: aqueous extract of Momordica charantia (MCA); 5: chloroform extract of Momordica charantia (MCC); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
Fig. 5. Effect of cycloheximide on glucose uptake. MCA: aqueous extracts of Momordica charantia; MCC: chloroform extracts of Momordica charantia; and CHI: cycloheximide. The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
cells. Rosiglitazone-treated cells did not enhance the PI3K expression significantly. 3.5. Effect of cycloheximide on 2-deoxy-d-[1-3 H] glucose uptakes Incubation of Momordica charantia fruit extracts in the presence of cycloheximide (1 mg/ml) demonstrated complete inhibition of glucose transport (Fig. 5) indicating that new protein synthesis is pivotal for increased Glut-4 translocation. 4. Discussion In the present work we have studied the effects of Momordica charantia fruit extracts with maximum glucose uptake activities and probable underlying mechanisms. Phytochemical compounds such as charantin, steroid, glycosides, flavonoid and their derivatives have been implicated in hypoglycenic activity (Lotlikar and Rajarama, 1966; Anandharajan et al., 2005) and these compounds
which have been found in fruit extracts of Momordica charantia may in part have been responsible for the observed up-regulatory activities of glucose uptake and mRNA expression of Glut4, PI3K and PPAR␥ but the responsible particular component(s) or combination of more than one component are yet to be found out by individual and combinational screening of the components. L6 muscle cell line, as suitable in vitro model (Koivisto et al., 1991) has been selected for the current work and the earlier reports (Okada et al., 1994; Yonemitsu et al., 2001) demonstrated the maximum glucose uptake activity by troglitazone (10 M) and rosiglitazone (100 M) and further Yonemitsu et al. (2001) authenticated the role of increased Glut-4 level for elevated glucose uptake in L6 cells. Similarly our current findings evaluated the concomitant increase of Glut-4 levels parallel with glucose uptake, reinforced the enhanced glucose transport by Momordica charantia fruit extracts. Glucose uptake was dose dependent and 6 g/ml was found to be optimum, other doses after 6 g/ml have shown decreased uptake which may be due to down-regulation of Glut-4 translocation or down-regulation of the concerned battery of the genes. There were no significant difference between chloroform and aqueous extract on glucose uptake which reveals the efficacious role of the common phytochemicals present in two extracts. Interestingly, in the current observations, we report the effect of Momordica charantia fruit extracts on PPAR␥ up-regulation and confirm the increased glucose uptake and Glut-4 transcription are indeed due to the activation of PPAR␥ by PPAR␥ agonists/insulin sensitizers (Shimaya et al., 1998). Several studies have demonstrated that a vegetable bitter gourd (Momordica charantia) exhibited anti-diabetic effects, and that it contained anti-diabetic substances such as charantin, vicine, polypeptide-p, 5-,19-epoxy-3-,25-dihydroxycucurbita-6,23(E)-diene and 3,7-,25-trihydroxycucurbita-5,23(E)-dien-19-al. Chuang et al. (2006) have further found that a compound of Momordica charantia, 9c, 11t, 13t-conjugated linolenic acid, was the active compound in wild bitter gourd and acted as a PPAR␥ agonist. Although, Yasui et al. (2005) suggested that bitter gourd seed fatty acid rich in 9c, 11t, 13t-conjugated linolenic acid may induce apoptosis in Caco-2 cells through up-regulation of GADD45, p53 and PPAR␥ but we did not find any cytotoxic effect of the used extracts at mentioned doses. Cycloheximide, a protein synthesis inhibitor completely blocked the glucose uptake mediated by Momordica charantia fruit extracts, clearly justified the need for synthesis of new protein relevant to glucose transport. The classical pathway of insulin-mediated glucose transport involves the activation of PI3K (Laville et al., 1996; Bandyopadhyay et al., 1997) and the same was reported for troglitazone (Petersen et al., 2000; Kausch et al., 2001). Our current findings made on Glut4, PPAR␥ and PI3K expression, hypothesise Momordica charantia fruit extracts act as insulin sensitizer and activate the glucose transport in a PI3K dependent fashion and the results have significant implication to understand the mechanism leading to the glucose transport and glucose homeostasis. In conclusion the present study demonstrated the integrative approach of medicinal substitute and in vitro screening assays, which ensured the validation of a battery of targets on glucose transport. Also this study demonstrated the significance of Glut4, PPAR␥ and PI3K up-regulation by Momordica charantia fruit extracts in augmenting the glucose uptake and homeostasis. Purification of the above plant extracts towards the isolation of novel lead molecule is worth pursuing and the same is in progress. Acknowledgments We are grateful to Dr. A.B. Mandal, Director, CLRI, Chennai, for his kind permission to publish this work. The authors gratefully acknowledge CSIR & NMITLI for the financial support.
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Fruit extracts of Momordica charantia potentiate glucose uptake and up-regulate Glut-4, PPAR␥ and PI3K Ramadhar Kumar, S. Balaji, T.S. Uma, P.K. Sehgal ∗ Bio-products Laboratory, Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600 020, Tamil Nadu, India
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Article history: Received 21 December 2008 Received in revised form 17 July 2009 Accepted 31 August 2009 Available online 8 September 2009 Keywords: Momordica charantia (MC) Fruit extracts Glucose transporter (Glut-4) Peroxisome proliferator activator receptor gamma (PPAR␥) Phosphotidylinositol-3 kinase (PI3K) L6 myotubes
a b s t r a c t Ethnopharmacological relevance: Momordica charantia fruit is a widely used traditional medicinal herb as, anti-diabetic, anti-HIV, anti-ulcer, anti-inflammatory, anti-leukemic, anti-microbial, and anti-tumor. Aims of study: The present study is undertaken to investigate the possible mode of action of fruit extracts derived from Momordica charantia (MC) and study its pharmacological effects for controlling diabetic mellitus. Effects of aqueous and chloroform extracts of Momordica charantia fruit on glucose uptake and up-regulation of glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPAR␥) and phosphatidylinositol-3 kinase (PI3K), were investigated to show its efficacy as a hypoglycaemic agent. Materials and methods: Dose dependent glucose uptake assay was performed on L6 myotubes using 2deoxy-d-[1-3 H] glucose. Up-regulatory effects of the extracts on the mRNA expression level of Glut-4, PPAR␥ and PI3K have been studied. Results: The association of Momordica charantia with the aqueous and chloroform extracts of Momordica charantia fruit at 6 g/ml has shown significant up-regulatory effect, respectively, by 3.6-, 2.8- and 3.8fold on the battery of targets Glut-4, PPAR␥ and PI3K involved in glucose transport. The up-regulation of glucose uptake was comparable with insulin and rosiglitazone which was approximately 2-fold over the control. Moreover, the inhibitory effect of the cyclohexamide on Momordica charantia fruit extract mediated glucose uptake suggested the requirement of new protein synthesis for the enhanced glucose uptake. Conclusion: This study demonstrated the significance of Glut-4, PPAR␥ and PI3K up-regulation by Momordica charantia in augmenting the glucose uptake and homeostasis. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Momordica charantia Linn. (bitter melon, bitter gourd or karela in Hindi), a plant native to the semi-tropical climate of China, India, Asia, and Africa, bears fruits which are traditionally used medicinal herbs as, anti-HIV, anti-ulcer, anti-inflammatory, antileukemic, anti-microbial, anti-diabetic, and anti-tumor, to name a few (Taylor, 2002; Grover and Yadav, 2004) and is one of the most promising alternative medicines for the disease. Herbal drugs have potential therapeutic applications because of their effectiveness, less side effects and relatively low cost (Venkatesh et al., 2003). Patients may prefer herbal drugs formulations if doctors readily make it available to them for common diseases. Therefore, investigation on such agents from traditional medicinal plants has become more important (Suba et al., 2004). Mukherjee et al. (2006) provided comprehensive information on various plant species from Indian biosphere and their constituents,
∗ Corresponding author. Tel.: +91 044 24420709; fax: +91 044 2491158. E-mail address: sehgal [email protected] (P.K. Sehgal). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.048
which have been shown to display potent hypoglycemic activity. In his review, he has stated that hypoglycaemic herbs are major Avenue in Indian perspectives particularly for treating diabetes, it required effectively to explore the literatures available on these aspects. A few active components, polypeptide-p (Khanna et al., 1981), flavonoids and charatin, a mixture of two steroid glycosides (Lotlikar and Rajarama, 1966) have been identified from the fruit and its seeds. These components, along with pure juice, methanol, chloroform and ethanol extracts from the fruit, have shown hypoglycaemic activity via in vitro, animal and human studies (Khanna et al., 1981; Welihinda and Karunanayake, 1986; Sitasawad et al., 2000; Rathi et al., 2002; Virdi et al., 2003). Mukherjee et al. (2006) described the chemistry, activity and usage of the constituents isolated from Indian hypoglycemic plants for the treatment of diabetes. Glucose transport is the rate-limiting step in glucose utilization, especially in insulin targeted skeletal muscle and mediated by major glucose transporter (Glut) proteins, Glut-4 and Glut-1 (Ziel et al., 1988). Insulin resistance in type 2 diabetes is manifested by decreased insulin-stimulated glucose transport and
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impaired metabolism in adipocytes and skeletal muscle, resulting in down-regulation of the major insulin-responsive Glut, the Glut-4 (Kellerer et al., 1999). PI3K is a key molecular switch, which mediates the metabolic effects of insulin, glucose transport and Glut-4 translocation (Okada et al., 1994; Tsakiridis et al., 1995). PPAR␥, a transcription factor belonging to the nuclear receptor super family (Desvergne and Wahli, 1999), essential for adipocyte differentiation (Ntambi and Young Cheul, 2000) and directly enhances insulin signalling and glucose uptake in muscle on binding with the PPAR␥ agonists (Ciaraldi et al., 1995). Although, Momordica charantia has been studied at cellular level and has shown the glucose uptake activity and up-regulation of PI3K as well as Glut-4 (Singh et al., 2004; Ben et al., 2007), but further mechanisms with clear evidences are yet to be found out. In the present work we have selected the L6 rat myoblasts as a model to see the expression level of mRNA of the battery of target Glut-4, PPAR␥ and PI3K to find out the possible mode of action for glucose uptake and homeostasis by fruit extract of Momordica charantia and we are also reporting the effect on mRNA expression level of PPAR␥.
2.5. Culture of L6 cells Monolayer of L6 muscle cell (NCCS, Pune, India) culture were maintained in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal calf serum (FCS) supplemented with penicillin (120 units/ml), streptomycin (75 mg/ml), gentamycin (160 mg/ml) and amphotericin B (3 mg/ml) at 37 ◦ C humidified with 5% CO2 . After 4–6 days confluence, L6 myoblast were transferred to DMEM with 2% FCS for differentiation. The extent of differentiation was established by observing multinucleation of cells and ∼90% fusions of myoblasts into myotubes. The differentiated myotubes were then used to investigate the effects of Momordica charantia fruit juice extracts. Differentiated myotubes were incubated for 30 min with insulin and 24 h with rosiglitazone, wherever indicated. 2.6. Cytotoxicity assay
2. Materials and methods
The effect of the extracts on cell viability of L6 was determined by using a colorimetric technique, which is 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosman, 1983). The cells were grown in 24-well plates (Corning, NY) and the differentiated cells were treated with both extracts at 5, 10, 15 and 20 g/ml.
2.1. Chemicals and reagents
2.7. Measurement of 2-deoxy-d-[1-3 H] glucose
All cell culture supplements were purchased from Life Technologies, USA. All chemicals, Primers and RT-PCR kits were obtained from Sigma–Aldrich, St. Louis, USA. All other chemicals and organic solvents used were of the highest analytical grade.
L6 cells grown in a 12-well plate (Corning, NY) were subjected to glucose uptake as reported (Yonemitsu et al., 2001). Fully differentiated myotubes serum starved for 5 h were incubated with the different concentration of fruit extracts for 12, 24, 36 or 48 h and control experiments in the absence of any stimulant were also performed for the same incubation periods. There were no significant difference in glucose uptake was observed on the basis of different incubation period; hence experimental incubation was optimised and reported for 24 h for all further studies. After incubation, cells were rinsed once with N-2-hydroxy ethyl piperazine-N -2ethane sulphonic acid (HEPES)-buffered Krebs Ringer phosphate solution (118 mM NaCl, 5 mM KCl, 1.3 mM CaCl2 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 and 30 mM HEPES ∼ pH 7.4) and further incubated for 15 min in HEPES-buffered solution containing 0.5 Ci/ml 2-deoxy-d-[1-3 H] glucose. The media were aspirated to terminate the uptake. Then the cells were washed thrice with ice cold HEPESbuffered solution and lysed in 0.1% SDS. An aliquot was used to measure the radioactivity. Glucose uptake values were corrected for non-specific uptake in presence of 10 mM cytochalasin B. All the assays were performed in triplicate.
2.2. Plant material The fruits of Momordica charantia Linn. were collected from local vegetable market, Chennai, Tamilnadu, India, and authenticated by a Pharmacognosy expert, Head, Department of Botany, Central Leather Research Institute, Chennai, India, before subjecting it to extraction and phytochemical characterization. The voucher specimens (MC1BPT) were identified and stored.
2.3. Preparation of fruit extract The 0.5 kg of fresh fruits were cut to remove the seeds and chopped into small pieces and then homogenised with water (1:2) in a commercial blender. The fresh juice was then centrifuged at 5000 rpm and the supernatant was lyophilized. The chloroform extract was prepared by extracting 0.5 kg of shade dried chopped pieces of fruits using chloroform in ratio of 1:10. The extraction was carried out exactly as described above and supernatant was dried under reduced pressure at low temperature. Both extracts were employed at different concentration.
2.4. Qualitative preliminary phytochemical group test The qualitative preliminary phytochemical group test for alkaloids, glucosides, saponins, phytosterols, tannin and flavonoids of aqueous and chloroform extracts were performed by the standard methods (Brain and Turner, 1975; Evans, 1996). HPLC analysis of the extracts with charantin (0.5 mg/ml) as a reference substance was performed with C-18 Inertsil ODS-3 column (5 m particle, 4.6 mm × 250 mm I.D.). The mobile phase was 100:2 (v/v) methanol–water and was delivered at a flow rate of 1 ml/min. The UV detection wavelength was 204 nm and the sample injection volume was 200 l.
2.8. Reverse transcriptase-polymerase chain reaction (RT-PCR) RT-PCR was carried out according to Kit supplier (Sigma, USA). After incubation, cells were lysed in Trizol, proteins were extracted with chloroform, and total RNA was precipitated with isopropanol. The RNA precipitate was washed with 70% ethanol and resuspended in 50 l of DEPC-treated water (Hall et al., 1998). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been taken as an internal control. The primers used were as follows. GAPDH: sense, 5 -CCA CCC ATG GCA AAT TCC ATG GCA-3 ; antisense, 5 -TCT AGA CGG CAG GTC AGG TCC ACC-3 (588-bp, Sahni et al., 1999); Glut-4: sense, 5 -CGG GAC GTG GAG CTG GCC GAG GAG3 ; anti-sense, 5 -CCC CCT CAG CAG CGA GTG A-3 (318-bp, Buhl et al., 2001); PI3K: sense, 5 -TGA CGC TTT CAA ACG CTA TC-3 ; antisense, 5 -CAG AGA GTA CTC TTG CAT TC-3 (248 bp, Laville et al., 1996); and PPAR␥: sense, 5 -GGA TTC ATG ACC AGG GAG TTC CTC3 ; anti-sense, 5 -GCG GTC TCC ACT GAG AAT AAT GAC-3 (155-bp, Yonemitsu et al., 2001). For PCR reaction, 1 l of cDNA mixture was added to a PCR reaction mix containing 10× PCR buffer, 2 mM dNTP,
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Fig. 1. Dose dependent analysis of 2-deoxyglucose uptake by aqueous extract. The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
10 pM of paired primers, 2 units of Taq polymerase. Semi-quantified PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed to analysis the transcripts using scanning densitometry. Signals of transcript in the agarose gel were quantified arbitrarily. 2.9. Statistical analysis Results are represented as mean ± S.D. of three independent experiments and statistical significance was determined via Student’s t-test with p < 0.05 considered to be significant. 3. Results
Fig. 2. Representative agarose gel photograph of (A) GAPDH (588 bp) and (B) Glut4 (318-bp) transcript. (C) Semi-quantification analysis of Glut-4 transcripts using scanning densitometry. Signals of Glut-4 in agarose gel were quantified arbitrarily. Lanes—1: control; 2: insulin; 3: rosiglitazone; 4: aqueous extract of Momordica charantia (MCA); 5: chloroform extract of Momordica charantia (MCC); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
In association with glucose uptake, extracts significantly increased the PPAR␥ expression (Fig. 3A) by 2.8-fold over control and this was comparable with rosiglitazone (2.4-fold) as revealed by densitometry scanning (Fig. 3C) while insulin-treated cells did not alter the PPAR␥ expression significantly. Densitometry scanning (Fig. 4C) showed approximately 3.8-fold increased PI3K expression (Fig. 4A) by Momordica charantia fruit extracts comparable with insulin (4.2-fold) over untreated control
3.1. Fruit extracts and phytochemical groups The yield of aqueous and chloroform extract was 2.6 w/w% and 4 w/w%, respectively. The preliminary qualitative phytochemical group tests showed the presence of alkaloids, glycosides, tannin and flavonoids in both extracts but saponins and steroids were detected in aqueous and chloroform extract respectively. The HPLC analysis of the extracts showed the presence of charantin molecules along with some other constituents. 3.2. Glucose uptake analysis Differentiated L6 myotubes after experimental incubation were analysed for glucose uptake analysis. Among the different doses, both extracts showed a better uptake of approximately 2-fold increases over control at 6 g/ml dose levels. Doses level greater than 6 g/ml have shown decreased uptake (Fig. 1). 3.3. Cytotoxicity assay The MTT analysis for all doses level shown 100% cell viability (data not shown) and did not have any cytotoxic effects on L6 cells. 3.4. Effect on Glut-4, PPAR and PI3K at transcription level Our observations reveal almost same elevated expression of Glut-4, PPAR␥ and PI3K transcripts by both chloroform and aqueous extracts of Momordica charantia fruit. The relative densitometry scanning (Fig. 2C) revealed significantly increase in Glut-4 transcript (Fig. 2B) approximately 3.6-fold by Momordica charantia fruit extracts over untreated control cells and equivalent to insulin (3.8fold) and rosiglitazone (3.2-fold).
Fig. 3. Representative agarose gel photograph of (A) PPAR␥ (155 bp) and (B) GAPDH (588 bp) transcript (C) Semi-quantification analysis of PPAR␥ transcripts using scanning densitometry. Signals of PPAR␥ in agarose gel were quantified arbitrarily. Lanes—1: control; 2: insulin; 3: rosiglitazone; 4: chloroform extract of Momordica charantia (MCC); 5: aqueous extract of Momordica charantia (MCA); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
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Fig. 4. Representative agarose gel photograph of (A) PI3K (248 bp), and (B) GAPDH transcript. (C) Semi-quantification analysis of PI3K transcripts using scanning densitometry. Signals of PI3K in agarose gel were quantified arbitrarily. Lanes—1: control; 2: rosiglitazone; 3: insulin; 4: aqueous extract of Momordica charantia (MCA); 5: chloroform extract of Momordica charantia (MCC); 6: negative control and M: marker (100 bp). The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
Fig. 5. Effect of cycloheximide on glucose uptake. MCA: aqueous extracts of Momordica charantia; MCC: chloroform extracts of Momordica charantia; and CHI: cycloheximide. The values are means ± S.E.M. of three independent experiments. *P < 0.05, as compared with untreated control group.
cells. Rosiglitazone-treated cells did not enhance the PI3K expression significantly. 3.5. Effect of cycloheximide on 2-deoxy-d-[1-3 H] glucose uptakes Incubation of Momordica charantia fruit extracts in the presence of cycloheximide (1 mg/ml) demonstrated complete inhibition of glucose transport (Fig. 5) indicating that new protein synthesis is pivotal for increased Glut-4 translocation. 4. Discussion In the present work we have studied the effects of Momordica charantia fruit extracts with maximum glucose uptake activities and probable underlying mechanisms. Phytochemical compounds such as charantin, steroid, glycosides, flavonoid and their derivatives have been implicated in hypoglycenic activity (Lotlikar and Rajarama, 1966; Anandharajan et al., 2005) and these compounds
which have been found in fruit extracts of Momordica charantia may in part have been responsible for the observed up-regulatory activities of glucose uptake and mRNA expression of Glut4, PI3K and PPAR␥ but the responsible particular component(s) or combination of more than one component are yet to be found out by individual and combinational screening of the components. L6 muscle cell line, as suitable in vitro model (Koivisto et al., 1991) has been selected for the current work and the earlier reports (Okada et al., 1994; Yonemitsu et al., 2001) demonstrated the maximum glucose uptake activity by troglitazone (10 M) and rosiglitazone (100 M) and further Yonemitsu et al. (2001) authenticated the role of increased Glut-4 level for elevated glucose uptake in L6 cells. Similarly our current findings evaluated the concomitant increase of Glut-4 levels parallel with glucose uptake, reinforced the enhanced glucose transport by Momordica charantia fruit extracts. Glucose uptake was dose dependent and 6 g/ml was found to be optimum, other doses after 6 g/ml have shown decreased uptake which may be due to down-regulation of Glut-4 translocation or down-regulation of the concerned battery of the genes. There were no significant difference between chloroform and aqueous extract on glucose uptake which reveals the efficacious role of the common phytochemicals present in two extracts. Interestingly, in the current observations, we report the effect of Momordica charantia fruit extracts on PPAR␥ up-regulation and confirm the increased glucose uptake and Glut-4 transcription are indeed due to the activation of PPAR␥ by PPAR␥ agonists/insulin sensitizers (Shimaya et al., 1998). Several studies have demonstrated that a vegetable bitter gourd (Momordica charantia) exhibited anti-diabetic effects, and that it contained anti-diabetic substances such as charantin, vicine, polypeptide-p, 5-,19-epoxy-3-,25-dihydroxycucurbita-6,23(E)-diene and 3,7-,25-trihydroxycucurbita-5,23(E)-dien-19-al. Chuang et al. (2006) have further found that a compound of Momordica charantia, 9c, 11t, 13t-conjugated linolenic acid, was the active compound in wild bitter gourd and acted as a PPAR␥ agonist. Although, Yasui et al. (2005) suggested that bitter gourd seed fatty acid rich in 9c, 11t, 13t-conjugated linolenic acid may induce apoptosis in Caco-2 cells through up-regulation of GADD45, p53 and PPAR␥ but we did not find any cytotoxic effect of the used extracts at mentioned doses. Cycloheximide, a protein synthesis inhibitor completely blocked the glucose uptake mediated by Momordica charantia fruit extracts, clearly justified the need for synthesis of new protein relevant to glucose transport. The classical pathway of insulin-mediated glucose transport involves the activation of PI3K (Laville et al., 1996; Bandyopadhyay et al., 1997) and the same was reported for troglitazone (Petersen et al., 2000; Kausch et al., 2001). Our current findings made on Glut4, PPAR␥ and PI3K expression, hypothesise Momordica charantia fruit extracts act as insulin sensitizer and activate the glucose transport in a PI3K dependent fashion and the results have significant implication to understand the mechanism leading to the glucose transport and glucose homeostasis. In conclusion the present study demonstrated the integrative approach of medicinal substitute and in vitro screening assays, which ensured the validation of a battery of targets on glucose transport. Also this study demonstrated the significance of Glut4, PPAR␥ and PI3K up-regulation by Momordica charantia fruit extracts in augmenting the glucose uptake and homeostasis. Purification of the above plant extracts towards the isolation of novel lead molecule is worth pursuing and the same is in progress. Acknowledgments We are grateful to Dr. A.B. Mandal, Director, CLRI, Chennai, for his kind permission to publish this work. The authors gratefully acknowledge CSIR & NMITLI for the financial support.
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