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In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPAR[gamma] in L6 myotubes.

Abstract

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPAR[gamma]) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are antidiabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100 ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPAR[gamma] and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Aegles marmelos; Syzygium cumini; Glucose transport; PPAR[gamma]; PI3 kinase; L6 myotubes

Introduction

Medicinal plants have formed the basis for Indian traditional medicine systems. A wide array of plant-derived active principles, demonstrated for their possible use in the treatment of Type 2 diabetes mellitus has been reported (Bailey and Day, 1989). Leaves, fruits and barks of Aegles marmelos (Family--Rutaceae) and Syzygium cumini (Family--Myrtaceae) have been used in the Ayurvedic Medicine for centuries for their hypoglycemic activity. Earlier studies reported that aqueous leaf extracts of A. marmelos (Ponnachan et al., 1993) and S. cumini (Teixeira et al., 1997) has significantly lowered the blood glucose level of diabetic rats.

Glucose transport is the rate-limiting step in glucose utilization, especially in insulin targeted skeletal muscle, mediated by major glucose transporter (Glut) proteins, Glut-4 and Glut-1 (Ziel et al., 1988). Glut-4 and Glut-1 were shown to be expressed in human skeletal muscle by cloning and characterisation of insulin-responsive Gluts (Fukumoto et al., 1989). Insulin resistance in type 2 diabetes is manifested by decreased insulin-stimulated glucose transport and impaired metabolism in adipocytes and skeletal muscle, resulting in down-regulation of the major insulin-responsive Glut, Glut-4 (Kellerer and Lammers, 1999).

Impaired Glut-4 translocation and reduced expression of phosphatidylinositol 3' kinase (PI3 kinase) and peroxisome proliferator activator receptor gamma (PPAR[gamma]) have been studied in detail under diabetic condition. Studies explaining the role of PI3 kinase and PPAR[gamma] in insulin signalling enumerate the up-regulation of insulin-dependent glucose transport and Glut-4 translocation in cultured L6 myotubes (Tsakiridis et al., 1995; Ciaraldi et al., 1995).

PI3 kinase is a key molecular switch, which mediates the metabolic effects of insulin, glucose transport and Glut-4 translocation (Okada et al., 1994). PPAR[gamma], a transcription factor belonging to the nuclear receptor super family (Desvergne and Wahli, 1999) essential for adipocyte differentiation (Ntambi and Young-Cheul, 2000) directly enhances insulin signalling and glucose uptake in muscle on binding with the PPAR[gamma] agonists (Ciaraldi et al., 1995).

Both A. marmelos and S. cumini were studied in detail in in vivo model for its pancreatic effect. Very little is known of its hypoglycaemic effect at cellular and molecular level. So the current study was aimed to elucidate the molecular mechanistic action of methanolic extracts of A. marmelos and S. cumini at cellular level. Therefore the effect of A. marmelos and S. cumini on glucose transport, PI3 kinase and PPAR[gamma] expression were analysed using L6 myotubes as an in vitro model.

Materials and methods

Chemicals and reagents

All cell culture supplements were purchased from Life Technologies, USA. All fine chemicals were obtained from Sigma-Aldrich, St. Louis, USA. 2-deoxy-D-[1-[.sup.3.H]] glucose was obtained from Amersham Pharmacia Biotech, UK. Trizol reagent was procured from Gibco BRL, USA. Avian Moloney leukemic virus (AMLV) reverse transcriptase, dNTP, Taq polymerase were obtained from New England Biolabs, UK. A taxonomist authenticated both A. marmelos and S. cumini, procured from a reliable source and the voucher specimen of these plants are preserved in our laboratory. Thin layer chromatography (TLC) plates (60 [F.sub.254] grade) were from Merck, Germany. Rosiglitazone was a kind gift from Dr. Reddy's Laboratories, India. All other chemicals and organic solvents used were of the highest analytical grade. Primers were procured from Gibco BRL, USA.

Plant extraction

The dried powder (100 g) of A. marmelos and S. cumini was extracted sequentially using different organic solvents in increasing order of polarity (hexane, dichloromethane, ethylacetate and methanol) at room temperature. Extracts were concentrated (Rotavap--Ika Instruments, Germany) under reduced pressure. One milligram of dried extract was reconstituted to 1 ml with respective solvent and diluted to attain the final concentrations 10 [micro]g/ml, 1 [micro]g/ml, 100 ng/ml, 20 ng/ml and 1 ng/ml, for the glucose uptake studies.

Culture of L6 cells

L6 cells (ATCC, USA) were maintained in Dulbecco's modified Eagles medium (DMEM) with 10% fetal calf serum (FCS) supplemented with penicillin (120 units/ml), streptomycin (75 [micro]g/ml), gentamycin (160 [micro]g/ml) and amphotericin B (3 [micro]g/ml) at 37[degrees]C humidified with 5% C[O.sub.2]. For differentiation, L6 cells were transferred to DMEM with 2% FCS, 4-6 days post-confluence. The extent of differentiation was established by observing multinucleation of cells and ~90% fusion of myoblasts into myotubes were considered for our study. Differentiated myotubes were incubated with insulin and rosiglitazone for 30 min and 24 h, respectively, wherever indicated.

Measurement of 2-deoxy-D-[1-[.sup.3.H]] glucose

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 5h were incubated with plant extracts. After incubation, cells were rinsed once with N-2-hydroxy ethyl piperazine-N'-2-ethane sulphonic acid (HEPES)-buffered Krebs Ringer phosphate solution (118 mM NaCl, 5 mM KCl, 1.3 mM Ca[Cl.sub.2], 1.2 mM MgS[O.sub.4], 1.2 mM K[H.sub.2]P[O.sub.4] and 30 mM HEPES--pH 7.4) and further incubated for 15 min in HEPES-buffered solution containing 0.5 [micro]Ci/ml 2-deoxy-D-[1-[.sup.3.H]] glucose. The uptake was terminated by aspiration of media. Then the cells were washed thrice with ice-cold HEPES-buffered 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 [micro]M cytochalasin B, (~5-10% of total uptake). All the assays were performed in triplicate.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

RT-PCR was carried out as described previously (Hall et al., 1998). 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 [micro]l of DEPC-treated water. Reverse transcription was carried out using 200 units of avian reverse transcriptase and 200 ng/[micro]l oligo d[T]18. The primers used were as follows. Glut-4: sense, 5'-CGG GAC GTG GAG CTG GCC GAG GAG-3'; anti-sense, 5'-CCC CCT CAG CAG CGA GTG A-3' (318-bp, Buhl et al., 2001); PPAR[gamma]: sense, 5'-GGA TTC ATG ACC AGG GAG TTC CTC-3'; anti-sense, 5'-GCG GTC TCC ACT GAG AAT AAT GAC-3' (155-bp, Yonemitsu et al., 2001); PI 3kinase: sense, 5'-TGA CGC TTT CAA ACG CTA TC-3'; anti-sense, 5'-CAG AGA GTA CTC TTG CAT TC-3' (248-bp, Laville et al., 1996); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; anti-sense, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (588-bp, Sahni et al., 1999). For PCR reaction, 1 [micro]l of cDNA mixture was added to a PCR reaction mix containing 10X PCR buffer, 2 mM dNTP, 10 pM of paired primers, 2 units of Taq polymerase. PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed.

Statistical analysis

Analysis of variance was conducted and the effect of mean between the independent groups was found be significant, p < 0.05. The Tukey's HSD procedure revealed that all pair-wise differences among means were significant, p < 0.05. All data are expressed as mean [+ or -] SE.

Results

Extraction

Hexane and dichloromethane extracts of A. marmelos and S. cumini yielded 100-150 mg while the yield from ethyl acetate and methanol was ~2g each. All the extracts were subjected to glucose uptake analysis at the concentrations indicated.

Glucose uptake analysis

Fully differentiated L6 myotubes demonstrated high insulin-sensitive glucose uptake. In order to determine the extract with maximum glucose uptake activity, in vitro bioassay was performed. Differentiated myotubes were incubated with plant extracts. Among the different solvent extracts, methanolic extract of A. marmelos (Fig. 1A) and S. cumini (Fig. 1B) showed a better uptake of approximately 2 fold increase over control at 100 ng/ml dose (data not shown for other solvent extracts). No significant difference in uptake was observed between 24 and 48 h; hence experimental incubation was optimised to 24 h for all further studies (48 h data not shown).

Comparison of A. marmelos and S. cumini with insulin and rosiglitazone based on 2-deoxy-D-[1-[.sup.3.H]] glucose

To determine the efficacy of A. marmelos and S. cumini, we compared their effect with insulin and rosiglitazone. Insulin caused a dose-dependent (1-100 nM) stimulation of glucose uptake leading to ~2 fold increase from basal at a maximal concentration of 100 nM (Fig. 5) (data not shown for other concentrations). Also a similar dose-response study to rosiglitazone was carried out at 0.1, 1, 10, 50 and 100 [micro]M. Rosiglitazone caused dose-dependent stimulation of glucose uptake reaching a maximum at 50 [micro]M (Fig. 5). So, insulin was used at 100 nM and rosiglitazone at 50 [micro]M for all of our studies (data not shown for different concentration of rosiglitazone). Our findings on glucose uptake demonstrated ~2 fold increase by A. marmelos and S. cumini over untreated control cells (Fig. 5) and highly comparable with insulin and rosiglitazone (2.1 fold).

Effect of A. marmelos and S. cumini on Glut-4, PPAR[gamma] and PI3 kinase at transcript level

We then determined the effect of A. marmelos and S. cumini on Glut-4, PPAR[gamma] and PI3 kinase mRNA expression by semi-quantitative RT-PCR. A. marmelos and S. cumini demonstrated elevated Glut-4 transcripts comparable with insulin and rosiglitazone. A representative agarose gel is shown in Fig. 2A. The relative densitometry scanning (Fig. 2C) revealed an increase in Glut-4 transcript ~3.1 fold and 2.8 fold by A. marmelos and S. cumini over untreated control cells equivalent to insulin (3.4 fold) and rosiglitazone (3.2 fold).

As a next step, we analysed the role of PPAR[gamma] in glucose transport mediated by A. marmelos and S. cumini. Up-regulation of PPAR[gamma] transcript by A. marmelos and S. cumini (Fig. 3A) equivalent to rosiglitazone sheds light for the involvement of PPAR[gamma] in glucose transport. In association with glucose uptake, A. marmelos and S. cumini increased the PPAR[gamma] expression ~2.2 fold and 2.3 fold, respectively, over control cells. It was comparable with rosiglitazone (2.3 fold) as revealed by densitometry scanning (Fig. 3C), while insulin-treated cells did not alter the PPAR[gamma] expression (1.1 fold).

[FIGURE 1 OMITTED]

PI3 kinase plays a key role in insulin signalling. So the effect of A. marmelos and S. cumini on PI3 kinase in glucose transport was studied. Transcript analysis (Fig. 4A) showed the elevation of PI3 kinase expression by A. marmelos and S. cumini similar to insulin. Densitometry scanning (Fig. 4C) showed ~3.0 fold increased PI3 kinase expression by A. marmelos and S. cumini comparable with insulin (3.8 fold) over untreated control cells. Rosiglitazone-treated cells did not enhance the PI3 kinase expression (1.2 fold).

Effect of cycloheximide on 2-deoxy-D-[1-[.sup.3.H]] glucose uptake

Observations made on Glut-4, PPAR[gamma] and PI3 kinase transcripts confirmed the elevated glucose uptake by A. marmelos and S. cumini. To find out whether new protein synthesis is required for Glut-4 translocation, we decided to study the effect of cycloheximide on glucose transport. Incubation of A. marmelos and S. cumini in the presence of cycloheximide (1 [micro]g/ml) demonstrated complete inhibition of glucose transport (Fig. 5), indicating that new protein synthesis is pivotal for increased Glut-4 translocation.

Discussion

Altered glucose transport associated with defective Glut-4 translocation and impaired insulin signalling cascade was evidenced as one among the major defects in insulin resistance, type 2 diabetes. Acute elevation of Glut-4 and PI3 kinase mRNA by insulin in Euglycemic Hyperinsulinemic Clamp Study enlightened the role of PI3 kinase and Glut-4 in insulin-mediated glucose transport (Laville et al., 1996). PPAR[gamma] agonists have been shown to facilitate the glucose transport in type 2 diabetes (Petersen et al., 2000). In this present study we tried to identify the plant extracts with maximum glucose uptake. To the best of our knowledge, our report is the first to investigate the effect of medicinal plants on a series of targets in insulin signalling cascade (Anandharajan et al., 2005) using L6 myotubes as an in vitro model. Therefore the present study demonstrates the mechanism of glucose transport mediated by A. marmelos and S. cumini.

[FIGURE 2 OMITTED]

L6 muscle cell line a suitable in vitro model (Koivisto et al., 1991) used to study the glucose transport activity since skeletal muscle is the major site for primary glucose disposal and glucose utilization. Earlier reports on L6 myotubes (Ciaraldi et al., 1995; Yonemitsu et al., 2001) demonstrated the maximum glucose uptake activity by troglitazone and rosiglitazone at 10 and 100 [micro]M, respectively. Further, Yonemitsu et al. (2001) authenticated the elevated glucose uptake in L6 cells was due to increased Glut-4 level. Similarly our current findings evaluated the concomitant increase of Glut-4 levels parallel with glucose uptake, reinforced the enhanced glucose transport by A. marmelos and S. cumini. Cycloheximide, a protein synthesis inhibitor completely blocked the glucose uptake mediated by A. marmelos and S. cumini clearly justified the need for synthesis of new protein relevant to glucose transport. Taken together the above observations A. marmelos and S. cumini confirmed the glucose transport and Glut-4 translocation, which supports the fact that there is reduced expression of Glut-4 and encoding mRNA in type 2 diabetes patients (Garvey et al., 1993).

PPAR[gamma] agonists (insulin sensitisers) are currently being used in the treatment of insulin resistance associated with type 2 diabetes (Nolan et al., 1994). Interestingly, elevated levels of PPAR[gamma] transcript by A. marmelos and S. cumini correlated with enhanced Glut-4 transcription and glucose uptake. So the current observations confirms the increased glucose uptake and Glut-4 transcription are indeed due to the activation of PPAR[gamma] by PPAR[gamma] agonists (Shimaya et al., 1998).

The classical pathway of insulin-mediated glucose transport involves the activation of PI3 kinase. An interesting study on human muscle culture hinted the up-regulation of PI3 kinase and Glut-4 mRNA expression by insulin (Laville et al., 1996). Another study using L6 myotubes evidenced the active participation of PI3 kinase in insulin-mediated glucose transport (Bandyopadhyay et al., 1997). A recent report on satellite human muscle culture demonstrated the role of troglitazone in elevation of glucose transport associated with PI3 kinase (Kausch et al., 2001). Considering the observations made on elevated PI3 kinase transcript by A. marmelos and S. cumini, we tentatively concluded the activation of glucose transport is by a PI3 kinase-dependent fashion. However it is necessary to confirm the PI3 kinase expression at protein level and also at phosphorylation level by kinase assays.

[FIGURE 3 OMITTED]

Current results have significant implication to understand the mechanism leading to the activation of glucose transport. Collectively based on our findings made on Glut-4, PPAR[gamma] and PI3 kinase, we hypothesise A. marmelos and S. cumini extracts activate the glucose transport in a PI3 kinase-dependent fashion. Simulation of the above study in adipocytic cell-line 3T3-L1 could help us to better understand the mechanistic action of A. marmelos and S. cumini and the same is in progress.

Conclusion

In the present study we have demonstrated the integrative approach of medicinal chemistry and in vitro screening assays, which ensured the validation of a battery of targets on glucose transport. Also this study demonstrated the significance of Glut-4, PPAR[gamma] and PI3 kinase up-regulation by A. marmelos and S. cumini in augmenting the glucose transport. Purification of the above plant extracts towards the isolation of novel lead molecule is worth pursuing and the same is in progress.

Acknowledgements

The authors gratefully acknowledge Dr. Sandip Basu, Director, National Institute of Immunology, New Delhi for his valuable support. The authors would like to thank Dr. P. Giridharan, Lecturer, Anna University for his superb technical assistance. Financial assistance from Department of Biotechnology, New Delhi, is also acknowledged.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

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R. Anandharajan (a), S. Jaiganesh (a), N.P. Shankernarayanan (b), R.A. Viswakarma (c), A. Balakrishnan (d,*)

(a) Centre For Biotechnology, Anna University, Chennai 600025, India

(b) V.H.S Leprosy project, Shakthi Nagar, Erode 638 315, India

(c) Bio-organic Laboratory, National Institute of Immunology, New Delhi 110041, India

(d) Screening and Biotechnology, Department of Pharmacology, Nicholas Piramal Research Centre, I Nirlon Complex, Mumbai 40063, India

Received 15 October 2004; accepted 24 March 2005

Abbreviations: Glut, glucose transporter; PI3 kinase, phosphatidylinositol 3' kinase; PPAR[gamma], peroxisome proliferator activator receptor gamma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagles medium; FCS, fetal calf serum; HEPES, N-2-hydroxy ethyl piperazine-N'-2-ethane sulphonic acid; TLC, thin layer chromatography; RT-PCR, reverse transcriptase-polymerase chain reaction; AMLV, avian Moloney leukemic virus

*Corresponding author. Tel.: +91 44 30818000.

E-mail address: abalakrishnan@nicholaspiramal.co.in (A. Balakrishnan).
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Author:Anandharajan, R.; Jaiganesh, S.; Shankernarayanan, N.P.; Viswakarma, R.A.; Balakrishnan, A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Clinical report
Date:Jun 1, 2006
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