AKT pathway in the hypoglycemic effects of saponins from Helicteres isora

Journal of Ethnopharmacology 126 (2009) 386–396

Contents lists available at ScienceDirect

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

Involvement of the PI3K/AKT pathway in the hypoglycemic effects of saponins from Helicteres isora Shefalee K. Bhavsar a,1 , Michael Föller c , Shuchen Gu c , Sanjay Vir d , Mamta B. Shah b , K.K. Bhutani d , Dev D. Santani a , Florian Lang c,∗ a

Department of Pharmacology, L.M. College of Pharmacy, Ahmedabad, India Department of Pharmacognosy, L.M. College of Pharmacy, Ahmedabad, India Department of Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany d Department of Natural Products, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, India b c

a r t i c l e

i n f o

Article history: Received 1 July 2009 Received in revised form 7 September 2009 Accepted 10 September 2009 Available online 23 September 2009 Keywords: AKT C2C12 myotubes Glut4 translocation GSK-3␣/␤ Helicteres isora PI3K

a b s t r a c t Aim of the study: Saponins from Helicteres isora have previously been shown to exert antidiabetic effects. The present study explored the underlying mechanisms in C2C12 skeletal muscle cells. Materials and methods: C2C12 cells were incubated with saponins and sapogenin followed by Western blotting and immunofluorescence analysis. Results: Western blotting revealed that incubation with saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) induced the phosphorylation of the phosphatidylinositol-3-kinase (PI3K) as well as of the downstream targets protein kinase B/Akt (at Ser473) and glycogen synthase kinase GSK-3␣/␤ (at Ser21/9) in a timedependent manner. In contrast, no phosphorylation of the AMP-sensitive kinase AMPK (at Thr172) was observed. Within 48 h saponins/sapogenin treatment further increased the protein abundance of the insulin-sensitive glucose transporter Glut4. Confocal microscopy confirmed that saponins/sapogenin treatment stimulated Akt phosphorylation and revealed that the treatment was followed by translocation of Glut4 into the cell membrane of C2C12 muscle cells. Conclusions: Saponins and sapogenin activate the PI3K/Akt pathway thus leading to phosphorylation and inactivation of GSK-3␣/␤ with subsequent stimulation of glycogen synthesis as well as increase of Glut4-dependent glucose transport across the cell membrane. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1.1. Background The hyperglycemia of diabetes mellitus results from impairment of insulin release or decreased insulin sensitivity (American Diabetes Association, 2008). As chronic increases in circulating glucose and lipid levels may compromise insulin secretion and action (Muoio and Newgard, 2008), the hyperglycemia and hyperlipidemia of diabetes may lead to a vicious circle. Besides the use of insulin and drugs inducing normoglycemia, the hyperglycemia of diabetes mellitus could be favourably influenced by compo-

∗ Corresponding author. Tel.: +49 7071 2972194; fax: +49 7071 295618. E-mail addresses: [email protected] (S.K. Bhavsar), [email protected] (M. Föller), [email protected] (S. Gu), [email protected] (S. Vir), mamta b [email protected] (M.B. Shah), [email protected] (K.K. Bhutani), fl[email protected] (F. Lang). 1 Present address: Department of Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany. 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.09.027

nents derived from plants (Aquino et al., 1995). Among those, Helicteres isora Linn. (Sterculiaceae) root juice has been used in the treatment of diabetes, emphysema and gastric disorders by several ethnic groups in different parts of India (Kirtikar et al., 1995). Ethanol extracts of the root have been reported to possess significant antidiabetic and hypolipidemic activity in C57BL/KsJdb/db mice and mildly hypertriglyceridemic swiss albino mice (Chakrabarti et al., 2002). Moreover, ethanol, ethyl acetate and butanol extracts of Helicteres isora root showed significant oral hypoglycemic activity in glucose-loaded rats (Venkatesh et al., 2004). Moreover, stem–bark aqueous extract produced significant antidiabetic and hypolipidaemic effects in streptozotocin-induced type 1 diabetic rats (Kumar et al., 2006a,b; Kumar and Murugesan, 2008). In addition, Helicteres isora administration caused significant changes in tissue fatty acid composition and erythrocyte membrane and antioxidant status in streptozotocin-induced type 1 diabetic rats (Kumar et al., 2009a,b). In a previous study, we evaluated the effects of Helicteres isora in the fat-fed, low dosestreptozotocin-treated rat model and characterized saponins as active constituents exerting insulin-sensitizing and antihyperlipidemic effects (Bhavsar et al., 2009). Furthermore, we described

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

Helicteres isora induced changes in the expression of glucose and lipid metabolism regulating genes (Bhavsar et al., 2009). 1.2. Aim of present work The mechanisms underlying the antidiabetic and antihyperlipidemic effects of saponins have, however, remained elusive thus far. The present investigation explored, whether the effect of saponins involves the phosphatidylinositol (PI) 3 kinase, the protein kinase B PKB/AKT, the AMP-activated protein kinase (AMPK), the glucose transporter Glut4 and/or the peroxisome proliferator-activated receptor gamma (PPAR␥). To this end, saponins were added to C2C12 skeletal muscle cells. The activation of the respective signaling molecules was studied by Western blotting and confocal immunohistochemistry. 2. Materials and methods 2.1. Collection, identification and authentication of Helicteres isora

387

20 mg sample (saponins) was dissolved in 1.0 ml water: methanol (1:1, v/v) and filtered through a 0.22 ␮m Millipore membrane filter. A 20 ␮l sample volume was injected into a MS C-18 XTerra column (5 ␮m, 250 mm × 4.6 mm) and eluted by a mobile phase consisting of 0.1% (v/v) formic acid in H2 O (A) and ACN (B). The gradient was: 0 min, 10% B; 30 min, 40% B; 35 min, 50% B; 37 min, 80% B. The flow-rate was 1 ml/min. A LC–MS system consisting of Waters 2767 Sample Manager, 2525 binary gradient pump, CFO and coupled to a single quadrupole ZQ mass spectrometer (Micromass 4000) operating in ESI positive and negative mode of ionization was used. Data acquisition was achieved using the MassLynx software ver 4.0. The optimum values of MS analyses were as follows: ESI negative mode of ionization; capillary voltage, 3.87 kV; cone voltage, 40 V; dissolvation temperature, 350 ◦ C; source temperature, 100 ◦ C; extractor, 2 V; RF lens, 0.2 V; nebulizing gas, 500 L/h; cone gas 70 L/h. High-purity nitrogen was used as nebulizer and cone gas. MS analyses were performed in mass range of 100–1500 m/z. Photodiode array detector (2996) was used to scan the sample in the range of 200–400 nm. 2.4. Myogenic cell cultures

The fresh roots of Helicteres isora Linn. (family: Sterculiaceae) were collected from the Jamnagar district, Gujarat, India and authenticated by the taxonomist of the Gujarat Ayurved University, Jamnagar. The sample with voucher no. 5049 was kept in the Department of Pharmacognosy, L.M. College of Pharmacy. 2.2. Isolation method of saponins and sapogenin The saponins were isolated from the n-butanol fraction. To this end, root powder (100 g) was sequentially extracted with petroleum ether, ethyl acetate and n-butanol (4× 200 ml). The pooled n-butanol fractions were dried under reduced pressure (BF, yield 2.15%). For the separation of saponins 2 g BF was dissolved in methanol (100 ml), kept refrigerated and treated with diethyl ether (200 ml) to obtain precipitates of glycosides, which were separated by centrifugation. The precipitates of glycosides (saponins) were purified by reprecipitation and recrystallization to yield creamish brown colored powder (yield 216 mg), which gave positive chemical tests for saponins and negative tests for other classes of glycosides. Acid hydrolysis of saponins: for the isolation of sapogenins the saponins (100 mg) were dissolved in methanol:water (80:20) and hydrolysed with 1N H2 SO4 for 4 h, followed by neutralization with sodium carbonate. The sapogenins were extracted using chloroform and dried (yield: 45 mg). 100 mg sapogenins were loaded in silica (70 g, grade 8; 200–400#) glass columns (49 cm × 3 cm), and column chromatography was performed using gradient elution with toluene and acetone to yield fractions (8 ml each) as follows: toluene → fractions 1–15, toluene:acetone (97.5:2.5) → fractions 15–45, toluene:acetone (96:4) → fractions 46–143, toluene:acetone (95:5) → fractions 144–154 and toluene:acetone (94:6) → fractions 154–210. Different fractions were monitored by thin layer chromatography (TLC) using the solvent system toluene:acetone (3:1). Fractions 46–140 giving a single spot on TLC (Rf 0.43), containing the single sapogenin were pooled and concentrated to dryness followed by purification of fraction by recrystallization (yield 19 mg). 2.3. High performance liquid chromatography–mass spectrometry (HPLC–MS fingerprint) analysis HPLC grade acetonitrile (ACN) and methanol (J.T. Baker, Philipsburg, PA, USA), analytical grade formic acid (Ranchem, Delhi, India), purified water from Millipore Milli-Q system, and MSC-18 XTerra column (Waters, Milford, MA, USA) were used for the experiment.

Myoblasts from the murine muscle-derived C2C12 cell line were obtained from the American Type Culture Collection (Manassas, VA). Undifferentiated cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, 4.5 mg/ml glucose) supplemented with 10% heat-inactivated fetal calf serum, 1% l-glutamine, 1% penicillin/streptomycin and 25 mM Hepes at 37 ◦ C in the presence of 5% CO2 . For biochemical analysis, the cells were grown on 6well plates (BD Biosciences) at a density of 5 × 105 cells/well in 4 ml of growth medium. Two days after plating, the cells reached 80–90% confluency (day 0). Differentiation was then induced by shifting the growth medium to differentiation medium, consisting of DMEM supplemented with 0.5% heat-inactivated fetal calf serum, 1% l-glutamine, 1% penicillin/streptomycin and 25 mM Hepes. The differentiation medium was changed after every 24 h and the differentiation was allowed to continue for 6 days. All experiments were performed 6 days after the beginning of differentiation. The differentiated cells were incubated with 100 ␮g/ml saponins or sapogenin for the required time periods (RNA isolation: 24 h and 48 h; Western blot analysis: 1 h, 3 h, 6 h, 12 h and 24 h). 2.5. Western blot analysis The expression levels of each protein were analyzed by Western blotting. In brief, cells were washed twice with ice cold phosphate-buffered saline (PBS) and cells were lysed with cell lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3 VO4 , 1 ␮g/ml leupeptin and 1 mM PMSF, added immediately prior to use). The extracts were centrifuged at 14,000 × g for 10 min at 4 ◦ C to remove insoluble material. The protein concentration of the supernatant was determined and 1:5 Laemmli sample buffer added. Total protein (50–100 ␮g) was subjected to 7.5–10% SDS-PAGE (1:30, bis:acrylamide). Proteins were transferred to a nitrocellulose or polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp.), and the membranes were then blocked for 2 h at room temperature with 5% non- fat dried milk in tris-buffered saline (NFDM/TBS) containing 0.1% Tween 20. Immunoblotting was carried out with an overnight incubation at 4 ◦ C with either 5% BSA/TBS- or 3% NFDM/TBS-containing Glut4 antibody (1:1000) (Abcam), PI3Kinase p85 antibody (1:1000) (Upstate), phosphoAkt (Ser473) antibody (1:1000), Akt antibody (1:1000), AMPK ␣

388

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

antibody (1:1000), phospho-AMPK ␣ (Thr172) antibody (1:1000), phospho-GSK-3␣/␤ (Ser21/9) antibody (1:1000), or GSK-3␤ antibody (1:1000). A GAPDH antibody (1:1000) (Cell Signaling Technology, Inc., New England Biolabs) was used as a loading control. For detection of phospho-PI3Kinase, the total cell protein was immunoprecipitated using a p-Tyr (PY20) antibody (Santa Cruz, Biotech) (5 ␮g/500 ␮g total protein) and the immunoprecipitation kit (protein A) from Roche (Mannheim, Germany) according to the manufacturer’s protocol. The precipitate was then subjected to Western blot analysis using PI3Kinase p85 antibody (1:1000) (Upstate). Specific protein bands were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase and a Super Signal Chemiluminescence detection procedure (GE Healthcare, UK). Specific Bands were quantitated by Quantity one software (Bio rad gel doc system, Chemidoc XRS). Levels of each protein were expressed as the ratio of signal intensity for the target protein relative to that of GAPDH or target phospho-protein relative to the total protein. Three independent experiments were performed for each condition. 2.6. Effect on insulin sensitivity C2C12 cells were incubated with insulin (100 nM) in the presence or absence of saponins (100 ␮g/ml). Total cell lysates were prepared for each condition after different incubation times. Western blot analysis was performed for phospho-Akt (Ser473) as described above. Sapogenin was not used for these experiments due to the paucity of this compound. In a different set of experiments C2C12 cells were exposed to saponins (100 ␮g/ml) with or without pretreatment with 100 nM of the PI3 kinase inhibitor Wortmannin for 30 min. To evaluate the effect of Wortmannin on the effect of saponins total cell lysates were prepared for each condition, and Western blot analysis was performed for phospho-Akt (Ser473) as described above. 2.7. Immunofluorescence and image analysis For immunofluorescent staining, cells were grown on 30-mm glass coverslips (neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany) in 6-well plates (5 × 105 cells/well/coverslip) or 15-mm glass coverslips (neoLab Migge Laborbedarf-Vertriebs GmbH) in 12-well plates (2 × 105 cells/well/coverslip). Two days after plating, the cells reached 80–90% confluency (day 0). Differentiation was then induced by shifting the growth medium to differentiation medium, consisting of DMEM supplemented with 0.5% heat-inactivated fetal calf serum, 1% l-glutamine, 1% penicillin/streptomycin and 25 mM Hepes. The differentiation medium was changed after every 24 h, and the differentiation was allowed to continue for 6 days. All experiments were performed 6 days after the beginning of differentiation. For determination of phospho-Akt (Ser473) and to study the Glut4 translocation, the cells were serumstarved for 4 h and then treated with test compounds for 30 min, 1 h, 2 h, 4 h or 6 h. After experimental treatments, the cells were washed with PBS and fixed for 20 min in 4% paraformaldehyde diluted (1:1) with PBS/0.3% Triton. The cells were washed, and then blocked in 5% normal goat serum/1× PBS/0.3% Triton for 1 h at room temperature. The cells were incubated overnight at 4 ◦ C with rabbit antiphospho-Akt (Ser473) (1:200, Cell Signaling Technology) or rabbit anti-Glut4 (1:300, Abcam). After incubation, the cells were rinsed three times with PBS for 10 min each. Then, they were incubated with the secondary antibody FITC goat anti-rabbit (1:500, Invitrogen) for 1.5 h at room temperature. Thereafter, they were rinsed three times with PBS for 5 min each and incubated with rhodamine phalloidin (1:40, Invitrogen) for 20 min. The nuclei were stained

with DRAQ-5 dye (1:500, Biostatus, Leicestershire, UK) for 5 min at 37 ◦ C. The slides were mounted with Prolong antifade reagent (Invitrogen). The images were taken on a Zeiss LSM 5 EXCITER Confocal Laser Scanning Microscope, equipped with a 405–633 nm laser (Carl Zeiss MicroImaging GmbH, Germany) using a water immersion Plan-Neofluar 63/1.3 NA DIC. For microscopic analysis, three independent experiments were performed for each condition. 2.8. RT-PCR and semiquantitation of the expression of Glut4 and PPAR gamma The C2C12 cells were treated with saponins or sapogenin (100 ␮g/ml) for 24 h or 48 h. The cells were washed twice with ice cold PBS and total RNA was isolated using peqGOLD Total RNA Kit from PEQLAB Biotechnologies GmbH, following the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically. First, strand cDNA was synthesized using 2 ␮g total RNA and the Transcriptor High Fidelity cDNA synthesis kit from Roche. The cDNA concentration was determined spectrophotometrically and equal amounts of cDNA from each sample were further coamplified with gene specific primers (Invitrogen) as follows: Glut4 (F-ACTCTTGCCACACAGGCTCT, R-AATGGAGACTGATGCGCTCT, product size: 174 bp); PPAR gamma (F-GGATTCATGACCAGGGAGTTCCTC, R-GCGGTCTCCACTGAGAATAATGAC, product size: 156 bp); and GAPDH (F-AAGGTCATCCCAGAGCTGAA, R-TGTGAGGGAGATGCTCAGTG, product size: 445 bp) used as control. The Faststart Taq DNA Polymerase Kit from Roche was used according to the manufacturer’s protocol. The linearity of the polymerase chain reaction (PCR) was tested by amplification of 500 ng of cDNA per reaction from 15–40 cycles. The linearity range was found to be between cycles 15 and 35. In no case, the amount of cDNA used for PCR exceeded 500 ng per reaction. The samples were amplified for 30 cycles by using the following parameters: 95 ◦ C/4 min/1 cycle, followed by 95 ◦ C/1 min, 56 ◦ C/30 s and 72 ◦ C/1 min—30 cycles, and final extension at 72 ◦ C/7 min. PCR products (2 ␮l) were electrophoresed on 1.5% agarose gel with TAE buffer and photographed. Bands were quantitated by Quantity one software (Chemidoc XRS). Levels of mRNA were expressed as the ratio of the signal intensity for the target gene relative to that of GAPDH. 2.9. Cell viability assay (WST-1 assay) Cell viability was measured after 24 h and 48 h incubation with the saponins and sapogenin, utilizing the cell proliferation reagent WST-1 from Roche according to the manufacturer’s protocol. In brief, the C2C12 cells were cultured in 96-well plates, in normal cell culture medium (2 × 104 cells in 100 ␮l culture medium per well). The cells were incubated with saponins (100 ␮g/ml) or sapogenin (100 ␮g/ml) for 24 h and 48 h. At the end of the incubation period 10 ␮l/well WST-1 solution was added to each well, and the cells were incubated at 37 ◦ C in 5% CO2 atmosphere for 2 h. The plates were shaken thoroughly on a shaker for 1 min and the absorbance was measured at 440 nm and 690 nm as reference in an ELISA plate reader against a reagent (100 ␮l cell culture medium and 10 ␮l WST-1 solution). 2.10. Data analysis and statistics Results are presented as arithmetic means ± SEM. Statistical difference between the means of the various conditions were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple test. Data were considered statistically significant if p < 0.05.

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

389

Fig. 1. High performance liquid chromatography–mass spectrometry (HPLC–MS fingerprint) analysis: (A) chromatogram, (B) mass chromatogram and (C) mass spectra of saponins.

3. Theory/calculation In our previous studies we identified saponins as active constituents exerting insulin-sensitizing and antihyperlipidemic effects. The insulin resistance in diabetes is associated with impaired signaling through PI3K-AKT pathway and decrease in the Glut4 transporter in the skeletal muscle. We evaluated whether saponins sensitize C2C12 cells by activating the PI3K-AKT pathway. 4. Results 4.1. High performance liquid chromatography–mass spectrometry (HPLC–MS fingerprint) analysis An efficient LC–MS method has been developed for the saponins of Helicteres isora. The optimum conditions for mass detection were selected for good ionization of each compound in the extract. The chromatogram showed four peaks of triterpenoid glycosides. Mass spectra of the peaks of the glycosides (saponins) matched with the reported spectra of cucurbitacin B and its isoforms. Mass spectra of the aglycone matched with that of cucurbitacin B (data not shown). The qualitative estimation of all four peaks was done on the basis of percentage area of each peak. All the constituents of

the saponins well detected and exhibited quasi-molecular ionization in ESI negative mode of ionization. Peak 1 (tR 14.52 min, area 10.05%, cucurbitacin B, m/z 541 with loss of water); peak 2 (tR 18.57 min, area 74.58%, cucurbitacin B glucoside, m/z 721), peak 3 (tR 21.23 min, area 13.97%, isocucurbitacin m/z 705) and peak 4 (tR 23.18 min, area 1.17%, isocucurbitacin glycoside, m/z 703) (Fig. 1). Cucurbitacin B glycoside is the major compound (approx. 2/3rd) in the saponins of Helicteres isora and it is assumed to be the main compound responsible for the biological activity. The effects of saponins and sapogenin were studied in C2C12 myotubes. Prior to the experiments, the cells had to undergo differentiation. As shown in Fig. 2A, undifferentiated C2C12cells – undifferentiated myoblasts – appeared as elongated or star-shaped, flat, mononucleated cells, isolated or occasionally clustered. C2C12 cells cultured for 3–4 days in differentiation medium, progressively became thick, long and multinucleated myotubes covered with microvilli. They lost stress fibers and adhesion to the underlying substrate. They underwent actin redistribution followed by the spatial organization of thick and thin myofilaments. The differentiated C2C12 cells showed characteristic features of myotubes: a progressive cell elongation was evident, with thick and thin myofilament organization, followed by the appearance of myotubes with characteristic nuclear indentations and clefts (Fig. 2B).

390

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

Fig. 2. Immunoflourescence analysis pictures of undifferentiated and differentiated C2C12 cells fixed and subjected to immunofluorescent staining for actin with rhodamine phalloidin (red) and nucleus with DRAQ-5 dye (blue): (A) undifferentiated C2C12 cells and (B) differentiatiated C2C12 cells. (For interpretation of the references to color in the citation of this figure, the reader is referred to the web version of the article.)

4.2. Western blot analysis According to Western blotting, exposure of differentiated C2C12 skeletal muscle cells to saponins (100 ␮g/ml) or sapogenin from Helicteres isora was followed by an increase in the phosphorylation of the phosphatidylinositol 3 kinase (PI3K). The phospho-PI3K protein levels relative to the total PI3K protein levels increased in a time-dependent manner following treatment with either, saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) (Fig. 3). A phospho-AMPK (Thr172) band could not be detected upon treatment with either saponins or sapogenin, and the total AMPK protein levels were sim-

Fig. 3. Effect of saponins and sapogenin incubation on phosho-PI3K protein levels expressed as relative phospho-PI3K density to total PI3K. These are representative immunoblots obtained from three independent experiments. The ratio of phosphoPI3K protein to total PI3K protein was expressed as the mean ± SEM of three independent experiments.

ilar after all incubation periods measured, indicating that neither saponins nor sapogenin had an appreciable effect on the AMPK pathway. Activation of PI3K is expected to result in phosphorylation of Akt. A time-dependent increase in the levels of phosphoAkt (Ser473) relative to the Akt total protein levels was indeed observed following treatment with either, saponins (100 ␮g/ml) or sapogenin (100 ␮g/ml). The relative increase in the ratio phosphoAkt (Ser473)/total Akt reached statistical significance (p < 0.05) 12 h following exposure to saponins and 6 h following exposure to sapogenin (Fig. 4). Activation of Akt is expected to stimulate phosphorylation of glycogensynthase kinase GSK-3␤. As illustrated in Fig. 5, treatment with either, saponins (100 ␮g/ml) or sapogenin (100 ␮g/ml) indeed

Fig. 4. Effect of saponins and sapogenin incubation on phosho-AKT (Ser473) protein levels expressed as relative phospho-AKT density to total AKT. These are representative immunoblots obtained from three independent experiments. The ratio of phospho-AKT (Ser473) protein to total AKT protein was expressed as the mean ± SEM of three independent experiments. **Significantly different from 0 h (p < 0.05).

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

Fig. 5. Effect of saponins and sapogenin incubation on phospho-GSK-3␣/␤ (Ser21/9) protein levels expressed as relative phospho-GSK-3␣/␤ (Ser21/9) density to total GSK-3␤. These are representative immunoblots obtained from three independent experiments. The ratio of phospho-GSK-3␣ (Ser21) protein to total GSK-3␤ protein was expressed as the mean ± SEM of three independent experiments. *Significantly different from 0 h (p < 0.05) and **significantly different from 0 h (p < 0.01).

increased the phospho-GSK-3␣/␤ (Ser21/9) protein levels relative to the GSK-3␤ total protein levels. The relative increase in phosphoGSK-3␣/␤ reached statistical significance (p < 0.05) 6 h following incubation with saponins and 6 h (p < 0.01) and 12 h (p < 0.05) following incubation with sapogenin.

391

Fig. 7. Effect of wortmannin on AKT activation by saponins. Representative immunoblots obtained from three independent experiments. The ratio of phosphoAKT (Ser473) protein to GAPDH protein was expressed as the mean ± SEM of three independent experiments. *Significantly different from 0 h (p < 0.05).

to insulin. Morover, as shown in Fig. 7, the stimulating effect of saponins on phosphorylation of Akt (Ser473) was inhibited by pretreatment with the PI3K inhibitor Wortmannin (100 nM), indicating that saponins increase insulin sensitivity by activating the PI3K/Akt signaling pathway. 4.4. Effect on Glut4

As a next step, we studied whether or not saponins exert an insulin-sensitizing effect. To this end, the C2C12 cells were incubated with insulin (100 nM) in the presence or absence of saponins (100 ␮g/ml). As shown in Fig. 6, the presence of saponins augmented the stimulation of Akt phosphorylation (Ser473) by insulin, indicating that saponins increased the sensitivity of C2C12 cells

Akt increases skeletal muscle glucose uptake by enhancing the activity of the glucose carrier Glut4. Western blotting was thus performed to elucidate an effect of saponins and sapogenin on the Glut4 protein abundance in C2C12 skeletal muscle cells. As shown in Fig. 8, a 48 h incubation with saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) indeed increased the Glut4 protein levels relative to the GAPDH protein levels. The relative increase in the Glut4 abundance reached statistical significance (p < 0.05) 48 h after incubation with saponins (Fig. 8).

Fig. 6. Effect of saponins on insulin sensitivity. Representative immunoblots obtained from three independent experiments. The ratio of phospho-AKT (Ser473) protein to GAPDH protein was expressed as the mean ± SEM of three independent experiments. In: insulin and SA: saponins. *Significantly different from 0 h (p < 0.05); **significantly different from 0 h (p < 0.01) and # significantly different from 0 h (p < 0.001).

Fig. 8. Effect of saponins and sapogenin incubation on Glut4 protein levels expressed as relative Glut4 density to GAPDH. These are representative immunoblots obtained from three independent experiments. The ratio of Glut4 protein to GAPDH protein was expressed as the mean ± SEM of three independent experiments. CON: control; SA: saponins and SG: sapogenin. *Significantly different from CON (p < 0.05).

4.3. Effect on insulin sensitivity

392

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

viability 24 h or 48 h after treatment with saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml).

5. Discussion

Fig. 9. Effect of saponins and sapogenin incubation on the expression levels of (A) and (B) Glut4, (C) and (D) PPAR gamma. The expression levels are expressed as relative signal intensity of target gene to GAPDH. Representative pictures obtained from three independent experiments. The relative signal intensity was expressed as the mean ± SEM of three independent experiments. CON: control; SA: saponins and SG: sapogenin. *Significantly different from CON (p < 0.05) and **significantly different from CON (p < 0.01).

The increase in Glut4 protein abundance could have at least in part been secondary to stimulation of transcription. Thus, RT-PCR was utilized to determine the Glut4 transcript levels. As illustrated in Fig. 9, the incubation with saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) indeed increased the transcript levels of Glut4 relative to that of the house keeping gene GAPDH. The increase reached statistical significance 48 h after incubation with saponins (p < 0.01) and sapogenin (p < 0.05). Moreover, incubation with saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) increased the transcript levels of PPAR gamma, an effect reaching statistical significance (p < 0.05) 48 h following incubation with sapogenin (Fig. 9). 4.5. Immunofluorescence and image analysis The influence of saponins (p < 0.01) and sapogenin on Akt and Glut4 was further analyzed by immunofluorescence. As confirmed in Fig. 10 saponins (100 ␮g/ml) and sapogenin (100 ␮g/ml) showed activation of phospho-Akt (Ser473) in a time-dependent manner. An increase in the fluorescence of phospho-Akt (Ser473) was detected after 1 h, 2 h, 4 h and 6 h incubation with saponins and sapogenin (Fig. 10). Translocation of the glucose transporter Glut4 to the myotube cell surface from the intracellular compartment was observed after a 2 h, 4 h and 6 h incubation with saponins and sapogenin (Fig. 11). To explore, whether the observed effects may have reflected alterations in cell viability, the cell proliferation reagent WST-1 was employed. As shown in Fig. 12, there was no difference in the absorbance of the control cells and cells treated with either saponins or sapogenin for 24 h or 48 h. These results indicate that under the chosen experimental conditions, there is no change in cell

The LC–MS spectra reveal the presence of triterpenoid glycosides in the saponin-rich sample. From the evaluation of the mass spectra and comparison with the reported spectra, the glycoside appears to be related to the cucurbitacin B glucoside and its isoforms in structure. The peaks were identified as cucurbitacin B, m/z 541 with loss of water; cucurbitacin B glucoside; isocucurbitacin and traces of the isoform of isocucurbitacin glycoside. Cucurbitacin isoforms and related glycosides have been reported in previous studies to possess significant antidiabetic activity (Guerrero-Analco et al., 2005, 2007). Interestingly, CuB is used as an antidiabetic drug in China (Wakimoto et al., 2008). Further analytical studies are required to confirm more structural details of the saponins. The results of the present study indicate that saponins and sapogenin activate the PI3K/AKT pathway in C2C12 skeletal muscle cells. The phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway is a crucial signaling cascade triggered by insulin and growth factors. The PI3K pathway serves to protect cells against apoptosis, an effect important for the survival of tumor cells. Moreover, the pathway plays a pivotal role for the metabolic effects of insulin, and impaired signaling through PI3K may predispose to the development of diabetes. The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central node in cell signaling downstream of growth factors, cytokines, and other cellular stimuli and is one of the most important and versatile protein kinases. Aberrant loss or gain of Akt activation underlies the pathophysiological properties of a variety of complex diseases, including type-2 diabetes and cancer (Manning and Cantley, 2007). In response to growth factors, Akt signaling regulates nutrient uptake and metabolism in a cell-intrinsic and cell-type-specific manner through a variety of downstream targets (Manning and Cantley, 2007). One of the most important physiological functions of Akt is to acutely stimulate glucose uptake in response to insulin (Calera et al., 1998). Akt2, the primary isoform in insulin-responsive tissues, has been found to associate with glucose transporter 4 (Glut4)-containing vesicles upon insulin stimulation of adipocytes (Calera et al., 1998), and Akt activation leads to Glut4 translocation to the plasma membrane (Kohn et al., 1996). The Rab-GAP AS160 (also known as TBC1 domain family member 4; TBC1D4) is an important direct target of Akt involved in this process (Eguez et al., 2005; Sano et al., 2007). Five putative Akt sites are phosphorylated on AS160 in response to insulin, of which S588 and T642 score the highest using the Scansite program (Sano et al., 2003). Mutation of these two sites to alanine significantly block insulin-stimulated Glut4 translocation (Sano et al., 2003). The other candidate Akt substrates involved in various steps of Glut4 translocation have been identified, including PIKfyve (Berwick et al., 2004). Glut1 is the main glucose transporter in most cell types, and unlike Glut4, it appears to be regulated primarily through alterations in expression levels. Activation of mTORC1, through Akt-mediated phosphorylation of TSC2 and PRAS40, can contribute to both HIF␣-dependent transcription of the Glut1 gene and cap-dependent translation of Glut1 mRNA (Taha et al., 1999; Zelzer et al., 1998). Akt activation can also alter glucose and lipid metabolism within cells. Upon entry into the cell, glucose is converted to glucose 6-phosphate through the action of hexokinases. Akt has been demonstrated to stimulate the association of hexokinase isoforms with the mitochondria, where they more readily phosphorylate glucose, but the direct target of Akt is currently unknown (Robey and Hay, 2006). Glucose 6-phosphate can be stored by conversion to glycogen or catabolized to produce cellular energy through

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

393

Fig. 10. Immunofluorescence pictures of C2C12 cells subjected to immunofluorescent staining for actin with rhodamine phalloidin (red), nucleus with DRAQ-5 dye (blue) and anti-phaphospho-Akt (Ser 473) which was visualized with FITC-conjugated secondary antibody (green): (A) control cells 0 min, (B) C2C12 cells treated with saponins (100 ␮g/ml) for different time points and (C) C2C12 cells treated with sapogenin (100 ␮g/ml) for different time points. Fluorescence signals were detected under confocal microscopy. Images are representative fields of view from three independent experiments. (For interpretation of the references to color in the citation of this figure, the reader is referred to the web version of the article.)

glycolysis. The Akt signaling can regulate both of these processes. Particularly important in muscle and liver, Akt-mediated phosphorylation and inhibition of GSK3 prevents it from phosphorylating and inhibiting its namesake substrate glycogen synthase, thereby stimulating glycogen synthesis (Whiteman et al., 2002). Akt activation also increases the rate of glycolysis (Elstrom et al., 2004), and this probably contributes to the excessive flux through glycolysis in tumor cells. The ability of Akt to enhance the rate of glycoly-

sis is due, at least in part, to its ability to promote the expression of glycolytic enzymes through HIF␣ (Lum et al., 2007; Majumder et al., 2004; Semenza et al., 1994). As described above, phosphorylation of substrates by GSK3 often targets them for proteasomal degradation, and GSK3 has been shown to promote degradation of the sterol regulatory element-binding proteins (SREBPs), which are transcription factors that turn on the expression of genes involved in cholesterol and fatty acid biosynthesis (Sundqvist et al., 2005).

394

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

Fig. 11. Immunofluorescence pictures of C2C12 cells subjected to immunofluorescent staining for actin with rhodamine phalloidin (red), nucleus with DRAQ-5 dye (blue) and anti-Glut4 which was visualized with FITC-conjugated secondary antibody (green): (A) control cells 0 min, (B) C2C12 cells treated with saponins (100 ␮g/ml) for different time points and (C) C2C12 cells treated with sapogenin (100 ␮g/ml) for different time points. Fluorescence signals were detected under confocal microscopy. Images are representative fields of view from three independent experiments. (For interpretation of the references to color in the citation of this figure, the reader is referred to the web version of the article.)

Therefore, Akt-mediated inhibition of GSK3 promotes SREBP stability and enhances lipid production. As shown here, incubation with saponins increase the phosphoAkt (Ser473) protein levels relative to the Akt total protein in a time-dependent manner. Similarly, increase in the phospho-

Fig. 12. Cell viability assay (WST-1) (A) C2C12 cells. Data are presented as means ± SEM, n = 8. CON: control cells; SA: saponins and SG: sapogenin.

GSK-3␣/␤ (Ser21/9) protein relative to GSK-3␤ total protein was observed, which is the substrate of Akt. Activation of Akt was also observed in immunofluorescence. There was an increase in the fluorescence of the phospho-Akt (Ser473) following incubation with saponins and the sapogenin. These results suggested that the saponin fraction showed comparatively significant effect as the sapogenin fraction on the activation of PI3K/AKT pathway. Hence, the activation of Akt and the subsequent phosphorylation and inactivation of GSK-3␣/␤ is responsible for the observed antihyperglycemic and antihyperlipidemic effects of the saponins in HFD-STZ diabetic rats and C57BL/KsJ-db/db mice (Bhavsar et al., 2009). According to immunofluorescence, saponins and sapogenin fostered the translocation of the glucose transporter Glut4 from the intracellular compartment to the myotube cell surface. The ratelimiting step in the uptake and metabolism of glucose by insulin in target cells is glucose transport, which is mediated by specific glucose transporters of the plasma membrane. In normal muscle cells and adipocytes, the glucose transporter isoform is Glut4, a 12-

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

transmembrane domain protein that facilitates transport of glucose in the direction of the glucose gradient (Barrett et al., 1999; Douen et al., 1990). Insulin promotes Glut4 incorporation into the plasma membrane, and this translocation from intracellular compartments appears to fail due to insulin resistance of type II diabetes (Garvey et al., 1998; King et al., 1992; Klip et al., 1990; Zierath et al., 1996). Mice with an adipose-specific knockout of the GLUT4 glucose transporter have impaired insulin sensitivity in muscle and liver (Abel et al., 2001). Interestingly, food deprivation also causes a form of insulin resistance and is associated with a decrease in adipose Glut4 expression (Sivitz et al., 1989). Insulin resistance in type II diabetes is manifested by decreased insulin-stimulated glucose transport and metabolism in adipocytes and skeletal muscle resulting in down-regulation of the major insulin-responsive glucose transporter, Glut4 (Kellerer et al., 1999). The molecular basis of insulin resistance depends on impaired insulin signal transduction with key defects in the glucose transport. The skeletal muscle plays a pivotal role in energy balance and is the primary tissue for insulin-stimulated glucose uptake and disposal (Smith and Muscat, 2005). In our Western blot study on C2C12 cells saponins and sapogenin increased the Glut4 protein as well as mRNA expression levels. According to the present observations, saponins exerted their antihyperglycemic effect in HFD-STZ diabetic rats and obese C57BL/KsJ-db/db mice (Bhavsar et al., 2009) at least in part due to Akt-dependent stimulation of glucose uptake into skeletal muscle. Our results provide the molecular basis for understanding the antidiabetic and antihyperlipidemic effects of the active constituents saponins and the isolated sapogenin. Saponins and sapogenin may be considered as potential lead molecules for antidiabetic and antihyperlipidemic effects and should be studied further for the strucural details by means of other analytical methods. 6. Conclusions Saponins and sapogenin stimulate glucose transport at least in part by PI3K/Akt-mediated translocation of glucose transporter Glut4 to the myotube cell membrane and enhance glycogen synthesis through phosphorylation and inactivation of GSK-3␣ and ␤. The observed effects of saponins and sapogenin on PI3K/Akt and GSK3␣ and ␤ provide a molecular explanation of the antihyperglycemic effect of those valuable nutrients. Conflict of interests The authors disclose that there are no conflicts of interests. Acknowledgements They acknowledge the technical assistance of E. Faber and the meticulous preparation of the manuscript by T. Loch and L. Subasic. This study was supported by the Deutsche Forschungsgemeinschaft (GK 1302) and by a Deutscher Akademischer Austauschdienst DAAD fellowship to S.K. Bhavsar. References Abel, E.D., Peroni, O., Kim, J.K., Kim, Y.B., Boss, O., Hadro, E., Minnemann, T., Shulman, G.I., Kahn, B.B., 2001. Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature 409, 729–733. American Diabetes Association, 2008. Diagnosis and Classification of Diabetes Mellitus, vol. 31. Diabetes Care, pp. S55–S60. Aquino, R., De Simone, F., De Tommasi, N., Piacente, S., Pizza, C., 1995. Structure and Biological Activity of Sesquiterpene and Diterpene Derivatives from Medicinal Plants. In: Phytochemistry of Plants Used in Traditional Medicine. Oxford University Press, New York, pp. 249–278. Barrett, M.P., Walmsley, A.R., Gould, G.W., 1999. Structure and function of facilitative sugar transporters. Current Opinion in Cell Biology 11, 496–502.

395

Berwick, D.C., Dell, G.C., Welsh, G.I., Heesom, K.J., Hers, I., Fletcher, L.M., Cooke, F.T., Tavare, J.M., 2004. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles. Journal of Cell Science 117, 5985–5993. Bhavsar, S.K., Singh, S., Giri, S., Jain, M.R., Santani, D.D., 2009. Effect of saponins from Helicteres isora on lipid and glucose metabolism regulating genes expression. Journal of Ethnopharmacology 124, 426–433. Calera, M.R., Martinez, C., Liu, H., Jack, A.K., Birnbaum, M.J., Pilch, P.F., 1998. Insulin increases the association of Akt-2 with Glut4-containing vesicles. The Journal of Biological Chemistry 273, 7201–7204. Chakrabarti, R., Vikramadithyan, R.K., Mullangi, R., Sharma, V.M., Jagadheshan, H., Rao, Y.N., Sairam, P., Rajagopalan, R., 2002. Antidiabetic and hypolipidemic activity of Helicteres isora in animal models. Journal of Ethnopharmacology 81, 343–349. Douen, A.G., Ramlal, T., Rastogi, S., Bilan, P.J., Cartee, G.D., Vranic, M., Holloszy, J.O., Klip, A., 1990. Exercise induces recruitment of the “insulin-responsive glucose transporter”. Evidence for distinct intracellular insulin- and exercise-recruitable transporter pools in skeletal muscle. The Journal of Biological Chemistry 265, 13427–13430. Eguez, L., Lee, A., Chavez, J.A., Miinea, C.P., Kane, S., Lienhard, G.E., McGraw, T.E., 2005. Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein. Cell Metabolism 2, 263–272. Elstrom, R.L., Bauer, D.E., Buzzai, M., Karnauskas, R., Harris, M.H., Plas, D.R., Zhuang, H., Cinalli, R.M., Alavi, A., Rudin, C.M., Thompson, C.B., 2004. Akt stimulates aerobic glycolysis in cancer cells. Cancer Research 64, 3892–3899. Garvey, W.T., Maianu, L., Zhu, J.H., Brechtel-Hook, G., Wallace, P., Baron, A.D., 1998. Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance. Journal of Clinical Investigation 101, 2377–2386. Guerrero-Analco, J., Medina-Campos, O., Brindis, F., Bye, R., Pedraza-Chaverri, J., Navarrete, A., Mata, R., 2007. Antidiabetic properties of selected Mexican copalchis of the Rubiaceae family. Phytochemistry 68, 2087–2095. Guerrero-Analco, J.A., Hersch-Martinez, P., Pedraza-Chaverri, J., Navarrete, A., Mata, R., 2005. Antihyperglycemic effect of constituents from Hintonia standleyana in streptozotocin-induced diabetic rats. Planta Medica 71, 1099–1105. Kellerer, M., Lammers, R., Haring, H.U., 1999. Insulin signal transduction: possible mechanisms for insulin resistance. Experimental and Clinical Endocrinology & Diabetes 107, 97–106. King, P.A., Horton, E.D., Hirshman, M.F., Horton, E.S., 1992. Insulin resistance in obese Zucker rat (fa/fa) skeletal muscle is associated with a failure of glucose transporter translocation. Journal of Clinical Investigation 90, 1568–1575. Kirtikar, K.R., Basu, B.D., 1995. An ICS: Indian Medicinal Plants. International 611 Book Distributors, Dehradun, India, pp. 371–372. Klip, A., Ramlal, T., Bilan, P.J., Cartee, G.D., Gulve, E.A., Holloszy, J.O., 1990. Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle. Biochemical and Biophysical Research Communication 172, 728–736. Kohn, A.D., Summers, S.A., Birnbaum, M.J., Roth, R.A., 1996. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. The Journal of Biological Chemistry 271, 31372–31378. Kumar, G., Banu, G.S., Murugesan, A.G., Pandian, M.R., 2006a. Hypoglycaemic effect of Helicteres isora bark extract in rats. Journal of Ethnopharmacology 107, 304–307. Kumar, G., Banu, S., Murugesan, A.G., 2009a. Influence of Helicteres isora administration for diabetes mellitus: its effect on erythrocyte membrane and antioxidant status. Food and Chemical Toxicology 47, 1803–1809. Kumar, G., Banu, S., Murugesan, A.G., 2009b. Influence of Helicteres isora administration for diabetes mellitus: the effect on changes in tissue fatty acid composition. Food and Chemical Toxicology 47, 1826–1830. Kumar, G., Murugesan, A.G., 2008. Hypolipidaemic activity of Helicteres isora L. bark extracts in streptozotocin induced diabetic rats. Journal of Ethnopharmacology 116, 161–166. Kumar, G., Murugesan, A.G., Rajasekara, P.M., 2006b. Effect of Helicteres isora bark extract on blood glucose and hepatic enzymes in experimental diabetes. Pharmazie 61, 353–355. Lum, J.J., Bui, T., Gruber, M., Gordan, J.D., DeBerardinis, R.J., Covello, K.L., Simon, M.C., Thompson, C.B., 2007. The transcription factor HIF-1alpha plays a critical role in the growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes and Development 21, 1037–1049. Majumder, P.K., Febbo, P.G., Bikoff, R., Berger, R., Xue, Q., McMahon, L.M., Manola, J., Brugarolas, J., McDonnell, T.J., Golub, T.R., Loda, M., Lane, H.A., Sellers, W.R., 2004. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nature Medicine 10, 594–601. Manning, B.D., Cantley, L.C., 2007. AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274. Muoio, D.M., Newgard, C.B., 2008. Mechanisms of disease: molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nature Reviews: Molecular Cell Biology 9, 193–205. Robey, R.B., Hay, N., 2006. Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt. Oncogene 25, 4683–4696. Sano, H., Eguez, L., Teruel, M.N., Fukuda, M., Chuang, T.D., Chavez, J.A., Lienhard, G.E., McGraw, T.E., 2007. Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane. Cell Metabolism 5, 293–303. Sano, H., Kane, S., Sano, E., Miinea, C.P., Asara, J.M., Lane, W.S., Garner, C.W., Lienhard, G.E., 2003. Insulin-stimulated phosphorylation of a Rab GTPase-activating

396

S.K. Bhavsar et al. / Journal of Ethnopharmacology 126 (2009) 386–396

protein regulates GLUT4 translocation. The Journal of Biological Chemistry 278, 14599–14602. Semenza, G.L., Roth, P.H., Fang, H.M., Wang, G.L., 1994. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. The Journal of Biological Chemistry 269, 23757–23763. Sivitz, W.I., DeSautel, S.L., Kayano, T., Bell, G.I., Pessin, J.E., 1989. Regulation of glucose transporter messenger RNA in insulin-deficient states. Nature 340, 72–74. Smith, A.G., Muscat, G.E., 2005. Skeletal muscle and nuclear hormone receptors: implications for cardiovascular and metabolic disease. The International Journal of Biochemistry and Cell Biology 37, 2047–2063. Sundqvist, A., Bengoechea-Alonso, M.T., Ye, X., Lukiyanchuk, V., Jin, J., Harper, J.W., Ericsson, J., 2005. Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF (Fbw7). Cell Metabolism 1, 379–391. Taha, C., Liu, Z., Jin, J., Al Hasani, H., Sonenberg, N., Klip, A., 1999. Opposite translational control of GLUT1 and GLUT4 glucose transporter mRNAs in response to insulin. Role of mammalian target of rapamycin, protein kinase b, and phosphatidylinositol 3-kinase in GLUT1 mRNA translation. The Journal of Biological Chemistry 274, 33085–33091. Venkatesh, S., Dayanand, R.G., Reddy, Y.S., Sathyavathy, D., Madhava, R.B., 2004. Effect of Helicteres isora root extracts on glucose tolerance in glucose-induced hyperglycemic rats. Fitoterapia 75, 364–367. Wakimoto, N., Yin, D., O’Kelly, J., Haritunians, T., Karlan, B., Said, J., Xing, H., Koeffler, H.P., 2008. Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo. Cancer Science 99, 1793–1797.

Whiteman, E.L., Cho, H., Birnbaum, M.J., 2002. Role of Akt/protein kinase B in metabolism. Trends in Endocrinology and Metabolism 13, 444–451. Zelzer, E., Levy, Y., Kahana, C., Shilo, B.Z., Rubinstein, M., Cohen, B., 1998. Insulin induces transcription of target genes through the hypoxia-inducible factor HIF1alpha/ARNT. EMBO Journal 17, 5085–5094. Zierath, J.R., He, L., Guma, A., Odegoard, W.E., Klip, A., Wallberg-Henriksson, H., 1996. Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM. Diabetologia 39, 1180–1189.

Glossary High performance liquid chromatography–mass spectrometry (HPLC–MS fingerprint): An analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC–MS is a powerful technique used for many applications which has very high sensitivity and specificity. Reverse transcription polymerase chain reaction (RT-PCR): A variant of polymerase chain reaction (PCR), a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed “amplification”. In RT-PCR, however, the RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional or real-time PCR.