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Author's personal copy Biochimica et Biophysica Acta 1800 (2010) 359–366
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n
3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes Kadapakkam Nandabalan Sangeetha a, Sundaresan Sujatha a, Velusamy Shanmuganathan Muthusamy a, Singaravel Anand a, Nirmal Nithya c, Devadasan Velmurugan c, Arun Balakrishnan b, Baddireddi Subhadra Lakshmi a,⁎ a b c
Centre for Biotechnology, Anna University, India Department of Pharmacology, Nicholas Piramal Research Centre, India Centre of Advanced study in Crystallography and Biophysics, University of Madras, India
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 5 October 2009 Accepted 3 December 2009 Available online 21 December 2009 Keywords: 3β-Taraxerol Mangifera indica Glycogen synthesis Glucose uptake 3T3-L1 adipocytes GSK3β PI3K
a b s t r a c t Background: The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage. Methods: Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process. Results: The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β. General significance: This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Type 2 diabetes is a multifactorial metabolic disorder that has affected an estimated 2.8% of the world's population as of the year 2000 and the figure is expected to rise to 4.4% by the year 2030 [1]. The distinctive feature of the disease is insulin resistance involving
Abbreviations: EAE, Ethyl acetate extract; IRβ, insulin receptor β; IRS, insulin receptor substrate; IRTK, insulin receptor tyrosine kinase; PI3K, phosphotidyl inositol 3kinase; PKB, protein kinase B; GLUT4, glucose transporter; GSK3 β, Glycogen synthase kinase 3β; NBT, Nitro blue tetrazolium chloride; BCIP, 5-Bromo-4-chloro-3-indolylphosphate; MTT-3-(4,5-Dimethylthiazol-2-yl)-2,5, diphenyltetrazolium bromide; DMEM, Dulbecco's Modified Eagles medium; PTP1B, protein tyrosine phosphatase 1B; GS, glycogen synthesis; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; NIDDM, Non insulin dependent diabetes mellitus; RT-PCR, reverse transcription polymerase chain reaction; LDH, lactate dehydrogenase; pNPP, p-nitrophenyl phosphate; 2-DOG, 2deoxy-D-3[1-H]-glucose uptake; WT, wortmannin; Gen, genistein; NMR, nuclear magnetic resonance spectroscopy ⁎ Corresponding author. Tel.: + 91 44 22350772; fax: + 91 44 22350299. E-mail address:
[email protected] (B.S. Lakshmi). 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.12.002
impaired glucose transport and synthesis of glycogen leading to hyperglycemia. Glucose transport is the process of efficient clearance of glucose from the blood through the activation of glucose transporter (GLUTs) that helps in its uptake into the cells. The glucose entering the cell is either metabolized or converted into glycogen (a storage form) via glycogen synthesis. Hence, enhancement of these two metabolic processes of glucose uptake and glycogen synthesis can reduce insulin resistance and are therefore considered as the most advantageous treatment method for diabetes [2–4]. Numerous antihyperglycemic drugs like TZD, sulphonylureas and biguanides are in use to balance the glycemic level and to prevent the long term complications associated with Type 2 diabetes. However these agents induce potential deleterious effects that include hypoglycemic episodes, gastrointestinal disturbances, lactic acidosis, edema, weight gain, elevation of LDL cholesterol level, etc. [5]. Therefore, it is desirable to identify bioactive molecules with limited or no adverse effects along with exploring these phytochemicals for their diverse pharmacological and biochemical properties which could be of potential pharmaceutical interest. The present study attempts to identify a bioactive compound from plants and unravel the mechanism involved in triggering the anti-
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diabetic effect via glucose transport and glycogen synthesis. Mangifera indica is a well-known plant in traditional medicine. It belongs to the Anacardaceae family and has been reported to have in vivo antihyperglycemic [6], antioxidant [7] and in vitro anti-inflammatory activities [8]. The constituents isolated and reported from the plant are flavonols, xanthone glycosides [9], triterpenes [10] etc. However, the in vitro anti-diabetic effect of Mangifera indica has not been explored so far. Hence this study evaluated the anti-diabetic potential of Mangifera indica and identified the active principle(s) in a bioactivity (in terms of its glucose uptake potential) guided fashion. The active component was a triterpenoid isolated from the ethyl acetate extract (EAE) and was characterized as 3β-taraxerol, which has been isolated from a variety of plants including Bridella micranthus [11] and has a wide spectrum of activities such as anti-microbial [12], anti-ulcer and anti-tumor activity [13]. This work reports for the first time in vitro studies evaluating the anti-diabetic potential of 3βtaraxerol from Mangifera indica. In addition to these, the study also attempts to unravel the mechanism of action of 3β-taraxerol and EAE of M. indica. 2. Materials and methods 2.1. Chemicals and reagents DMEM and FCS for cell culture were purchased from GIBCO BRL (U.S. A). MMLV Reverse Transcriptase, dNTPs, Taq polymerase, primers for PCR were purchased from GIBCO BRL (U.S.A). Radiolabelled 2-deoxyD-3[1-H]-glucose and [14C]-UDP-glucose were from Amersham Pharmacia Biotech (U.K). Trizol reagent, insulin, genistein were from Sigma (U.K). Rosiglitazone was a gift from Dr. Reddy's Laboratories, India. MTT kit was from Promega (U.S.A). Antibodies IRβ, PKB, PI3K, phospho GSK3β were from Calbiochem (Germany). Leaves of Mangifera indica were collected from Chennai, India. The collected materials were authenticated by Department of Life sciences, Bharathidasan University, Trichy, India and deposited in the University herbarium. Chemicals and solvents were obtained from Merck, USA. 2.2. Cell culture 3T3-L1 cells were cultured in DMEM supplemented with 2 mM glutamine, antibiotics (penicillin 120 U/mL, streptomycin 75 µg/mL, gentamycin 160 µg/mL, amphotericin B 3 µg/mL) and 10% FCS. The cell cultures were maintained at 37 °C in a humidified incubator with 5% CO2. 2.2.1. Measurement of 2-deoxy-D-3[1-H]-glucose uptake Preadipocytes were seeded in 24 well plates and differentiated as described [14]. Differentiated adipocytes were treated with the solvent extracts, fractions or the compound for 24 h. Glucose uptake experiments were performed as described earlier [15] with slight modifications. Cells were stimulated with insulin (10 nM) for 15 min followed by the addition of 0.5 µCi/well of 2-deoxy-D-3[1-H]glucose in KRPH buffer (118 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 30 mM HEPES-pH 7.4) for 45 min at 37 °C. Cells were then washed with KRPH and then lysed with 0.1% SDS. The lysates were quantitated using scintillation counter and the results expressed as % glucose uptake. The uptake effects were compared with the positive control rosiglitazone (50 μM). 2.2.2. Plant extraction The leaves of Mangifera indica were air dried, powdered and used for the study. A 100 g sample of the dried powder was sequentially extracted by cold maceration using hexane, dichloromethane, ethyl acetate and methanol for three days. The extracts were concentrated under reduced pressure to make them completely devoid of solvents.
They were re-dissolved in methanol and serially diluted to the desired concentrations (10 μg, 1 μg, 100 ng, 10 ng, and 1 ng per mL). The final volume of solvent used for the assay was 10 μL. The extracts were then tested for their glucose uptake activity. 2.2.3. Bioactivity guided column fractionation and purification The active extract of Mangifera indica was used for bioactivity guided purification using column chromatography. A column of length 60 cm was used as the first stage column. It was packed with silica gel of mesh size 60–120 μm as stationary phase and hexane as the mobile phase. A slurry containing 2 g of the extract was loaded onto the packed column. The fractions were eluted using a combination of hexane and ethyl acetate, the percentage of the latter solvent being increased by increments of 5. Fractions were then subjected to TLC analysis and those with similar profile were pooled. The pooled fractions were dried for total solvent removal, made up to the desired concentrations (10 μg, 1 μg, 100 ng, 10 ng, and 1 ng per mL) using methanol and tested for their glucose uptake activity. The active fraction was then subjected to purification on a second stage column using silica gel of mesh size 120–240 μm and assayed for their glucose uptake activity. The third and final purification step involved using silica gel of size 240–400 μm and a pure compound showing maximum activity was isolated after elution with hexane and ethyl acetate. 2.2.4. Structure elucidation The purified active compound was subjected to various spectroscopic studies such as 1H, 13C, NMR and mass spectroscopy for elucidating the possible structure. 2.2.5. Cytotoxicity assay using MTT Dose response studies were performed for the EAE and 3βtaraxerol to assess their cytotoxicity. The LDH released was measured at 492 nm as described [16]. 2.2.6. Measurement of GLUT-4 mRNA by RT-PCR 3T3-L1 adipocytes were treated with 1 µg/mL of EAE or 100 ng/ mL of 3β-taraxerol for 6, 12, 18 and 24 h. The positive controls for the experiment were insulin (100 nM) and rosiglitazone (50 μM) treated for 15 min and 24 h respectively. Following these incubation periods, the cells were stimulated with insulin (10 nM) for 15 min and RT-PCR was carried out as described previously [17]. PCR was performed with specific primers for GLUT4 Forward 5′-GCACAGCCAGGACATTGTTG and Reverse 5′-CCCCCTCAGCAGCGAGTA. The PCR products were analyzed by 1.4% agarose gel electrophoresis. Normalization was done using GAPDH as control. 2.2.7. Western blotting 3T3-L1 adipocytes were treated for 24 h with optimum doses of EAE or 3β-taraxerol following which the cells were stimulated with 10 nM insulin. The cells were then lysed with extraction buffer (20 nM HEPES, 150 nM NaCl, 1% Triton X-100, 1 nM EDTA, 1 nM PMSF, 10 µg/mL leupeptin and 10 µg/mL aprotinin) and centrifuged at 12,000 rpm at 4 °C for 30 min. Equal concentrations of total protein lysates were resolved in 10% SDS-polyacrylamide gel electrophoresis and immunoblotted as described before [18] and probed with specific antibodies of IRβ, PI3K, GLUT4, PKB and phosphoGSK3β. Colorimetric detection was carried out using alkaline phosphatase conjugated secondary antibody and NBT/BCIP. Normalization of protein expression was carried out using β-actin as control. 2.2.8. Subcellular membrane fractionation Subcellular membrane fractions were obtained using density gradient centrifugation method as described earlier [19]. Briefly, 3T3L1 adipocytes after treatment with EAE or 3β-taraxerol were washed and resuspended in buffer I (250 mM/L sucrose, 5 mM NaN3, 20 mM
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HEPES, 200 μM/L PMSF, 1 μM/L pepstatin, 1 μM/L aprotinin and 2 mM/ L EGTA). Cell lysates were homogenized using 20 strokes of a Dounce homogenizer (0.5 cycles, 10 pulses; 2 min each and lag time of 1 min for each pulse). Lysates were then centrifuged at 750 g for 5 min at 4 °C to remove cell debris. The plasma membrane (PM) fraction was obtained by centrifugation of the resulting supernatant at 30,000 g for 40 min at 4 °C. The resultant pellet was resuspended in buffer I and this constitutes the PM fraction. Supernatant was removed and centrifuged at 100,000 g for 75 min at 4 °C to isolate the cytosol fraction. The light microsome (LM) pellet was resuspended in buffer I and assayed for soluble protein content (Bradford). Fractions were subjected to electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with anti-GLUT4 antibody.
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2.2.9. Inhibitor studies Inhibitor studies using genistein and wortmannin was performed as described [20,21]. Cells were pre-incubated with or without 50 µM of genistein, 100 µM of wortmannin for 30 min, followed by incubation with EAE or 3β-taraxerol for 24 h and stimulated with 10 nM insulin for 15 min. Analysis for glucose uptake and glycogen synthesis was performed as mentioned earlier and results were expressed as percentage inhibition of glucose uptake and glycogen synthesis. 2.2.10. Glycogen synthesis assay Differentiated 3T3-L1 adipocytes were treated with different concentrations of the EAE or 3β-taraxerol for 24 h. Glycogen synthesis experiments were performed as mentioned earlier [22] with minor
Fig. 1. a and b): Dose response study for insulin-stimulated 2-deoxy-D-3[1-H]-glucose uptake of Mangifera indica (a) Extracts (b) Fractions of Ethyl acetate extract (EAE) on 3T3-L1 adipocytes. Cells were pretreated with or without the extracts/fractions for 24 h and stimulated with insulin (10 nM) for 15 min and then assayed for 2-DOG uptake. All the experiments were performed twice in triplicates and expressed as mean ± S.E. c): Chemical structure of 3β-taraxerol (C30H50O). d): Comparative dose response analysis of EAE and 3β-taraxerol on insulin-stimulated 2-deoxy-D-3[1-H]-glucose uptake (2-DOG). Cells were pretreated with or without the EAE and 3β-taraxerol for 24 h stimulated with insulin (10 nM) for 15 min and then assayed for 2-DOG uptake. All the experiments were performed twice in triplicates and expressed as mean ± S.E. e): Cytotoxic assessment of EAE and 3β-taraxerol on 3T3-L1 cells at 24 h.
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modifications. Cells were stimulated with or without insulin and pulsed with 1 µCi/mL [14C]-UDP-glucose in GS buffer (2.5 mM glucose, 0.1% BSA, 25 mM HEPES at pH 7.4) for 1 h. Following incubation, the cells were lysed in 30% KOH and carrier glycogen at a concentration of 20 mg/mL was added and heated for 30 min at 70 °C. The glycogen synthesized was then precipitated using ice cold ethanol at 20 °C for 24 h and centrifuged at 2000 g for 10 min. The pellet was dissolved in water, dried and read using the Liquid scintillation counter. Results were expressed as % [14C]-UDP-glucose incorporated into glycogen. Insulin (100 nM) was used as positive control for the experiment. 2.2.11. Statistical analysis All data are expressed as mean ± S.E. The statistical significance between means of the independent groups was analyzed using one way ANOVA and p value of less than 0.05 was considered to be statistically significant. 2.2.12. Densitometric analysis Densitometric analysis was performed for all the experiments and the integral density values (IDV) of untreated controls were assigned an arbitrary value of 1. The arbitrary values of treated samples were represented in comparison to the untreated values. 3. Results and discussion Aderibigbe et al. have reported the in vivo anti-hyperglycemic activity of Mangifera indica [6]. The anti-diabetic potential of this plant has been further investigated using in vitro studies in the current work. The principle finding of our study is that 3β-taraxerol isolated from the active ethyl acetate extract of Mangifera indica exhibited its anti-diabetic potential by enhancing glucose uptake and glycogen synthesis. Mangifera indica leaves were subjected to sequential solvent extraction and the extracts were screened for glucose uptake activity to identify the active extract. Results from the radiolabelled glucose uptake assay indicated that, among the four extracts of Mangifera indica , the ethyl acetate extract (EAE) showed an increased glucose uptake at a concentration of 1 µg/mL, comparable with the positive control rosiglitazone (50 μM) (Fig. 1a), suggesting that the effective components with glucose transport inducing activity in Mangifera indica extract are moderately polar. Hence the EAE was chosen for bioactivity guided fractionation to identify active compound. Column purification of the EAE resulted in eight pooled fractions which were subsequently tested for their glucose uptake effect. Among the eight fractions (data shown only for fractions II,III and IV in Fig. 1b), fraction II eluting at 70:30 ratio of hexane and ethyl acetate showed maximum glucose uptake effect and was subjected to the second and third stages of purification, following which a single compound exhibiting maximum activity was isolated. The bioactive compound isolated from the active EAE of Mangifera indica leaves was characterized using spectroscopic techniques. Amorphous powder (MeOH), 1H-NMR (300 MHz, CDCl3) δ: 5.49 (1H, dd, 3.6 Hz, H-15), 3.18 (1H, dd, H-3), 1.1 (3H, s, CH3), 1.0 (3H, s, CH3), 0.94 (6H, s, 2×CH3), 0.92 (3H, s, CH3), 0.91 (3H, s, CH3), 0.81 (3H, s, CH3), 0.80 (3H, s, CH3). 13C-NMR (100 MHz, CDCl3) δ: 157.2 (C-14), 116.1 (C-15), 79.1 (C-3), 54.9 (C-5), 48.2 (C-18), 48.1 (C-9), 41.4 (C-19), 38.8 (C-4), 38.6 (C-8), 37.9 (C-17), 37.8 (C-1), 37.8 (C-13), 37.3 (C-10), 36.2 (C-16), 35.5 (C-12), 34.9 (C-7), 33.9 (C-21), 33.6 (C-29), 32.9 (C-22), 30.0 (C-28), 29.9 (C-26), 29.6 (C-20), 28.0 (C-23), 26.9 (C-2), 25.9 (C-27), 21.3 (C-30), 18.4 (C-6), 17.2 (C-11), 15.1 (C-24), 15.0 (C-25). Mass data EIMS m/z: 426 [M]+, 408 (20), 306 (8), 148 (80). Characterization studies elucidated the compound as 3β-taraxerol (Fig. 1c) which was compared and verified with existing reports [23]. The molecular formula of the compound is C30H50O. 3β-taraxerol is a triterpenoid that has been isolated and reported from Mangifera indica [24,25].
In this work, it is hypothesized that EAE and 3β-taraxerol, activates the key components in the insulin signaling cascade. The hypothesis is supported by the significant effects observed in terms of glucose transport and its storage. Comparison of the effect of concentration dependence of EAE and 3β-taraxerol on glucose uptake exhibited a dose dependent increase in glucose uptake which was maximum at 1 μg/mL with a decrease in the activity at 10 μg/mL for EAE, whereas for 3β-taraxerol maximum glucose uptake was observed at 100 ng/ mL after which a decline was observed (Fig. 1d). Liu et al. have reported a similar profile for Lagestroma [26], and we have observed a similar profile for Chichorium intybus [27]. The cytotoxicity studies for EAE and 3β-taraxerol (Fig. 1e) showed a maximum toxicity of
