Biological evaluation of (3β)-STIGMAST-5-EN-3-OL as potent anti-diabetic agent in regulating glucose transport using in vitro model

2.1. Chemicals and reagents 

Unless otherwise stated, materials were obtained from Sigma–Aldrich, St. Louis. All cell culture solutions and supplements were purchased from Life Technologies Inc. (Gaithersburg, MD, USA). 2-Deoxy d-[1-3H] glucose and hybond C membrane were obtained from Amersham Pharmacia Biotech, (Buckinghamshire, UK). DMEM, Trizol reagent and MMLV reverse transcriptase, dNTP, Taq polymerase and synthesized primers were obtained from GIBCO BRL, (Carlsbad, CA, USA). Wortmannin, Mowiol, anti-GLUT4 and anti-actin antibodies were obtained from Calbiochem (Darmstadt, Germany). Rosiglitazone was a kind gift from Dr. Reddy’s Laboratories, Hyderabad. Primers were synthesized from GIBCO BRL (Carlsbad, CA, USA). IR, Phospho-IR, IRS-1, phospho-IRS-1 antibodies were procured from Santa cruz. Anti-phospho AKT and p85 PI3K were obtained from BD Pharmingen (San Diego, CA, USA) Alkaline phosphatase-conjugated secondary antibodies, Horse radish peroxidise-conjugated secondary antibodies and anti-goat IgG-FITC were obtained from Santa Cruz Biotechnology, USA. All other HPLC and analytical grade solvents and silica gel required for column purification were obtained from SISCO Research Laboratories, India.

2.2. Cell culture of L6 myotubes 

L6, a differentiating monolayer myoblast culture (obtained from ATCC-CRL-1458) was maintained in DMEM (Dulbecco’s modified Eagle’s medium) with 10% FBS and supplemented with penicillin (120units/mL), streptomycin (75μg/mL), gentamycin (160μg/mL) and amphotericin B (3μg/mL) in a 5% CO2 environment. For differentiation, the L6 cells were transferred to DMEM with 2% FBS for 4days, post-confluence. The extent of differentiation was established by observing the multinucleation of cells. Once differentiated, the cells were incubated 24h with high glucose medium (the above mentioned DMEM medium with a supplement of 25mM/L glucose) for the cells to acquire an insulin resistant state [13], [14].

2.3. Measurement of 2-deoxy-d-[1-3H] glucose uptake 

L6 myoblasts grown in 24-well plates (BD Falcon) were serum starved for 5h and were incubated with the plant extracts and compound for 24h and subjected to glucose uptake assay as reported [15]. Glucose uptake values were corrected for non-specific uptake in the presence of 10μM cytochalasin B, (5–8% of total uptake). All the assays were performed in duplicates and repeated three times, for concordance. Results have been expressed as a percentage of glucose uptake with respect to the solvent control. Rosiglitazone (50μM) was used as the positive control.

For inhibitor studies, L6 myotubes were treated with Genistein −50μM [16] and Wortmannin −100nM [17], 30min prior to the incubation with the plant extracts and compound followed by the glucose uptake assay.

2.4. Extraction of A. vasica ethyl acetate leaf extract and isolation of (3β)-stigmast-5-en-3-ol 

The leaves of A. vasica after authentication were dried, powdered and sequentially extracted using organic solvents including hexane, dichloromethane, ethyl acetate and methanol by cold maceration as previously reported [18]. All the extracts were checked for glucose uptake efficacy. The ethyl acetate extract showed maximum glucose uptake activity compared to the other extracts, and was selected for purification. A Column of silica gel 100–200 mesh size was packed with hexane. The sample (active ethyl acetate extract) was loaded as dried slurry of silica gel (1:20 by ratio). The column was eluted with a gradual increase in polarity, using ethyl acetate. The eluted fractions were tested for glucose uptake activity. The active fraction was purified by additional column chromatography and its purity established by HPLC. The structure of the active compound was determined by 1H, 13C, DEPT 90, DEPT 135, COSY and ESI-MS. The compound was structurally characterized as (3β)-stigmast-5-en-3-ol with elemental composition and mass of C29H50O and 414 respectively.

2.5. Reverse transcriptase-polymerase chain reaction 

RT-PCR was carried out as described previously [19]. The isolated mRNA were converted to cDNA by reverse transcription and subjected to PCR using specific primers as follows: IRTK, F: 5′ ATC TGG ATC CCC CTG ATA ACT GTC 3′; R: 5′ ATG TGG GTG TAG GGG ATG TGT TCA 3′ (Genbank accession number NM_017071.1), GLUT 4, F: 5′ CGG GAC GTG GAG CTG GCC GAG GAG 3′; R: 5′ CCC CCT CAG CAG CGA GTG A 3′ [20], PI3K p85α regulatory subunit, F: 5’ TGA CGC TTT CAA ACG CTA TC 3′; R : 5′ CAG AGA GTA CTC TTG CAT TC 3′ [21], PKC α , F: 5′ ACC TGA AGC TGG ACA ACG 3′; R: 5′ GCT ACT CCT ACG GTA TTT 3′(Genbank accession number NM 001105713.1) and GAPDH, F: 5′ CCA CCC ATG GCA AAT TCC ATG GCA 3′; R: 5′ TCT AGA CGG CAG GTC AGG TCC ACC 3′ [22]. PCR products were resolved in agarose gels, stained with ethidium bromide and photographed. Rosiglitazone and Insulin were used at 50μM and 100nM respectively to determine the efficacy. The expression levels were quantitated by scanning on a gel documentation and analysis system (Chemi Imager 4400, Alpha Innotech Corporation).

2.6. Immunoprecipitation 

L6. myotubes were treated with optimum concentrations of A. vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol for indicated time periods after serum starvation for 8h. The cell lysates were prepared as reported [23]. The lysates were immunoprecipitated using Protein A Sepharose beads with specific phosphorylated forms of anti-IRβ and anti-IRS-1 [24]. The immunoprecipitated lysates were then transferred onto nitrocellulose membrane.

2.7. Western blot 

The immunoblots were probed with the respective primary antibody (IR-β, IRS-1, p85PI3K, pAKT and AKT), followed by specific secondary antibody and visualized using a chromogenic substrate. The density of the protein bands were quantitated by scanning the blots on a gel documentation and analysis system (Chemi Imager 4400, Alpha Innotech Corporation).

2.8. Subcellular membrane fractionation 

Subcellular membrane fractions were obtained using the differential ultra-centrifugation method, as described previously [15], with minor modifications. Briefly, L6 myotubes after treatment with plant extract and compound were washed and resuspended in buffer I (250mM/L Sucrose, 5mM NaN3, 20mM HEPES, 200μM/L PMSF, 1μM/L Pepstatin, 1μM/L Aprotinin and 2mM/L EGTA, pH 7.4). Cell lysate was homogenized using 20 strokes of a Dounce homogenizer (0.5 cycles, 10 pulses; 2min each and lag time of 1min for each pulse). The cell homogenates were centrifuged at 750g for 5min at 4°C to remove cell debris. The Plasma membrane (PM) fraction was obtained by centrifugation of the resulting supernatant at 30,000g for 40min at 4°C. The resultant pellet was resuspended in Buffer I to constitute the PM fraction. Supernatant was removed and centrifuged at 100,000g for 75min at 4°C to generate the cytosol fraction from the obtained pellet. The light microsome (LM) pellet resuspended in buffer I and PM fraction were assayed for soluble protein content by Bradford’s assay. 5′ nucleosidase and NADPH cyt-c reductase were used as the marker enzymes for PM and LM respectively. The membrane fractions were subjected to electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-GLUT4 antibody.

2.9. Immunofluorescence analysis and confocal microscopy 

L6 myotubes (60% of differentiated cells) grown on coverslips, after treatment with the crude and pure at indicated time points were washed with 1X PBS. The cells were fixed with 3.7% formaldehyde in PBS for 10min at room temperature, and permeabilized in PBS containing 0.01% Triton-X for 10min. The cells were then blocked with 3% FBS in PBS for 10min and incubated with anti-goat GLUT 4 antibody (1:250 dilution, sc-1607) in 3% FBS in PBS for 1h at 4°C. This was followed by incubation with 1:100 dilution of donkey anti-goat IgG-FITC (sc-2024) for 1h at 4°C. All coverslips were washed with PBS thrice during each incubation step and finally mounted on slide with Mowiol for confocal microscopy. Fluorescence was measured with Leica TCS SP2 inverted spectral confocal microscope (Germany) equipped with 60× objective with excitation and emission of 468nm and 543nm (for FITC) respectively.

2.10. Statistical analysis 

Mean±SEM were represented. Statistically significant differences between groups were analysed using Student’s t-test. The statistical significance was evaluated using one way ANOVA to determine differences among groups and Tukey’s multiple comparison tests to determine significant differences between groups. These were applied using SPSS version 11.0 (SPSS, Cary, NC, USA) and the criterion for statistical significance was P<0.05.

3.1. (3β)-stigmast-5-en-3-ol stimulates glucose uptake in L6 myotubes 

To examine the effect of ethyl acetate extract and its bioactive compound, (3β)-stigmast-5-en-3-ol on glucose transport , L6 myotubes were treated with varying concentrations of the extract and the compound at three different time points (12, 24 and 36h). A dose- and time-dependent increase in glucose uptake activity was observed on treatment with the crude extract as well as the purified compound. At 24h, maximal effect was achieved which sustained even at 36h (data not shown for 12 and 36h). The ethyl acetate extract and (3β)-stigmast-5-en-3-ol significantly stimulated basal glucose uptake and the optimal dose with maximum activity was found to be 1μg/mL (102.9% of vehicle control, p<0.05) and 100ng/ml (113.6% of vehicle control, p<0.05) respectively (Fig. 1), and these concentrations were used for further studies.

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Fig. 1. Comparative analysis of Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol on 2-deoxy-d-[1-3H] glucose uptake. Dose response analysis at 24h and the structure of (3β)-stigmast-5-en-3-ol were displayed. The results were expressed as percentage glucose uptake with respect to the vehicle control. The positive control, Rosiglitazone (50μM) showed 132.2% uptake. The values are mean of ±S.E., n=3 in duplicates. (∗), P<0.05 as compared with vehicle control group.

3.2. Activation of IR-β, IRS-1, PI3K, AKT/PKB and PKC by (3β)-stigmast-5-en-3-ol in L6 myotubes 

To determine whether ethyl acetate extract and (3β)-stigmast-5-en-3-ol are capable of modulating activation of insulin receptor, gene and protein expression studies were performed. The ethyl acetate extract and (3β)-stigmast-5-en-3-ol increased the mRNA levels of Insulin receptor from 6h onwards, and a significant increase in expression was observed at 18h (p<0.05) as shown in Fig. 2a and b. In addition, the expression of phospho-specific IR-β and IRS-1 were also checked. Our results revealed the increase of IR-β and IRS-1 tyrosine phosphorylation upon treatment with ethyl acetate extract and (3β)-stigmast-5-en-3-ol, as shown in Fig. 3a. A representative densitometric scanning for semi-quantitative analysis is shown in Fig. 3b. Taken together, these data suggested that the ethyl acetate extract and (3β)-stigmast-5-en-3-ol activated IR-β and IRS-1 similar to the positive control, insulin.

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Fig. 2. (a) Effect of Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol on IRTK, PI3K, PKC and GLUT4 mRNA expression. L6 myotubes were incubated with Insulin (100nM), Rosiglitazone (50μM), crude (1μg/mL) or pure (100ng/mL) at indicated time points for each marker. Lane 1 denotes the 100bp marker, Lanes 2–4 indicates untreated cells (control), Rosiglitazone and Insulin respectively. Lanes 5–7 and 8–10 indicates 6h, 18h and 24h of Adathoda vasica crude treated cells and (3β)-stigmast-5-en-3-ol treated cells respectively. Lane 11 shows the PCR negative control. GAPDH transcripts were used as the internal control. Insulin serves as a positive control for IRTK, PI3K, PKC and GLUT4 expressions. Rosiglitazone serves as a positive control for GLUT4 and negative control for IRTK, PI3K and PKC expressions. All the samples were run on the same gel and the dividing lines are for better understanding of the data. (b) Semi-quantitative analysis of IRTK, PI3K and GLUT4 mRNA transcripts upon treatment with Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol. The signaling intensities of IRTK, PI3K, PKC and GLUT4 transcripts were quantified using densitometric scanning. The signaling intensities were quantified arbitrarily. Bars represent mean of ±S.E., n=3 and a representative agarose gel is shown here. (∗), P<0.05 as compared with untreated control group.

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Fig. 3. (a) Effect of Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol on protein expression of pIR-β and pIRS-1. L6 myotubes were incubated with Insulin (100nM), Rosiglitazone (50μM), crude (1μg/mL) or pure (100ng/mL) at indicated time points for each marker. Whole cell lysates were immunoprecipitated using Protein A sepharose beads with phospho-specific antibodies followed by western blot as mentioned in the methods section. Lanes 1–3 indicates untreated cells (control), Rosiglitazone and Insulin treated cells. Lanes 4 & 5 and 6 & 7 indicate 18h and 24h of Adathoda vasica ethyl acetate extract treated cells and (3β)-stigmast-5-en-3-ol treated cells respectively. IR-β and IRS-1 protein expression were shown as immunoblot. (b) Semi-quantitative analysis of pIR-β, pIRS-1 protein expression upon treatment with Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol. The signaling intensities were quantified arbitrarily. Bars represent mean of ±S.E., n=3 and a representative blot is depicted here. (∗), P<0.05 as compared with untreated control group.

To examine whether (3β)-stigmast-5-en-3-ol stimulates glucose transport in a PI3K dependent manner, RT-PCR analysis and phospho-detect p85 PI3K were employed. As shown in Fig. 2a, treatment with crude and (3β)-stigmast-5-en-3-ol resulted in a time dependent increase in mRNA levels of PI3K. This data was substantiated by enhancement in the levels of p85 PI3K on treatment with the same (Fig. 4a).

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Fig. 4. (a) Effect of Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol on protein expression of p85 PI3K and pAKT. L6 myotubes were incubated with Insulin (100nM), Rosiglitazone (50μM), crude (1μg/mL) or pure (100ng/mL) at indicated time points for each marker. Whole cell lysates were immunoprecipitated using Protein A sepharose beads with phospho-specific antibodies followed by western blot for pAKT protein expression whereas for p85 PI3K whole cell lysates were used for western blot. Lane 1 indicates untreated cells (control) and Lane 2 indicates Insulin treated cells. Lanes 3–8 indicate 12h,18h and 24h of Adathoda vasica ethyl acetate extract treated cells and (3β)-stigmast-5-en-3-ol treated cells respectively. β-actin and AKT served as loading control for protein expression of p85 PI3K and pAKT respectively. (b) The signaling intensities of p85 PI3K, pAKT protein expression upon treatment with Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol were quantified arbitrarily. Bars represent mean of ±S.E., n=3 and a representative blot is depicted here. (∗), P<0.05 as compared with untreated control group.

To assess the effect of (3β)-stigmast-5-en-3-ol on PKB/AKT activation, the phosphorylation pattern was also studied. Both the crude and (3β)-stigmast-5-en-3-ol augmented the levels of phospho-AKT as shown in Fig. 4a. The next proximal kinase, PKCα was also analysed using RT-PCR. The crude as well as (3β)-stigmast-5-en-3-ol were able to significantly increase the mRNA levels at 24h (p<0.05) as indicated in Fig. 2. Collectively, our data demonstrates that the crude extract and (3β)-stigmast-5-en-3-ol activates the insulin receptor, insulin receptor substrate-1, followed by the activation of the downstream kinases like PI3K, AKT and PKC α, which accounts for their anti-diabetic potential.

3.3. Translocation of GLUT4 from light microsomes to plasma membrane by (3β)-stigmast-5-en-3-ol 

To check whether the increase in glucose uptake by (3β)-stigmast-5-en-3-ol is attributable to the translocation of GLUT4 from light microsomes (LM) to plasma membrane (PM), the translocation pattern was studied qualitatively, using confocal microscopy and semi-quantitatively determined by measuring the amount of GLUT4 in LM and PM using immunoblot. At earlier time points (Insulin-5min, 6h treatments with Rosiglitazone, A. vasica crude extract and (3β)-stigmast-5-en-3-ol), lack of detectable fluorescence was noticed suggesting low levels of GLUT4. At 12h, the presence of GLUT4 is localized near the nucleus, which exhibits enhanced fluorescence upon treatment with crude extract and (3β)-stigmast-5-en-3-ol. At 24h, ethyl acetate extract, (3β)-stigmast-5-en-3-ol, Rosiglitazone and insulin (15min) showed increased fluorescence through out the cell (Fig. 5).

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Fig. 5. Confocal immunofluorescence microscopy of FITC tagged GLUT4 in skeletal muscle cells. L6 cells (treated) were fixed onto coverslips using 3.7% formaldehyde and permeabilized with 0.01% Triton X-100. The cells were then labeled with anti-goat GLUT 4 primary antibody and after subsequent washing with 1X PBS, fluorescence stained with donkey anti-goat IgG-FITC secondary antibody. The coverslips after thorough washing were mounted on the slides using Mowiol for confocal microscopy. Panel (a) refers to untreated cells and panel (b and c) indicates the fluorescence pattern of the cells treated with Insulin for 5min and 15min respectively. (d–f), (g–i) and (j–l) represents the cells treated with 6h, 12h and 24h of Rosiglitazone, Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol respectively. 600× magnification.

Fig. 6a explains the translocation pattern of GLUT4 protein in response to crude and (3β)-stigmast-5-en-3-ol from LM to PM. Our results revealed that (3β)-stigmast-5-en-3-ol significantly augmented GLUT4 protein in PM content (p<0.01) like the ethyl acetate crude extract. The translocation pattern was comparable with that of the positive control, insulin. Semi-quantitative analysis is illustrated in Fig. 6b.

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Fig. 6. (a) GLUT4 translocation pattern of the cells treated with Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol using subcellular membrane fractions. Serum-starved L6 myotubes were treated with Insulin for 15min, crude and pure for 24h, followed by sub-cellular membrane fractionation as mentioned in the methods section. The resultant LM and PM fractions were immunoblotted with anti-GLUT4 antibody and the translocation pattern analyzed. Total GLUT4 content were also shown. (b) Representative densitometry analysis of GLUT4 translocation. The signaling intensities of GLUT4 protein expression in LM & PM were detected and quantitated using densitometric scanning. The signaling intensities were quantified arbitrarily and the data was expressed as percentage over control. Bars represent mean of ±SE., n=3 and a representative blot is depicted here. (∗∗), P<0.01 as compared with untreated control group.

3.4. Inhibition of Insulin receptor and PI3K blocks the glucose uptake mechanism 

To confirm whether (3β)-stigmast-5-en-3-ol enhances glucose transport the same way as insulin, the involvement of insulin receptor and PI3K was further checked using inhibitor studies. As shown in Fig. 7a, pretreatment with genistein (specific inhibitor for Insulin receptor tyrosine kinase [16]) followed by incubation with ethyl acetate extract or (3β)-stigmast-5-en-3-ol resulted in decrease in glucose uptake up to 67.20±7% and 49.68±3% respectively. Likewise, pretreatment with wortmannin (a specific PI3K inhibitor [17]) and subsequent incubation with ethyl acetate extract or (3β)-stigmast-5-en-3-ol resulted in a marked decrease in glucose uptake of up to 50.67±3% and 41.35±6% respectively, as shown in Fig. 7b.

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Fig. 7. Glucose uptake patterns of Adathoda vasica ethyl acetate extract and (3β)-stigmast-5-en-3-ol in presence of Genistein-IRTK inhibitor (a) and Wortmannin- PI3K inhibitor (b). L6 myotubes were treated with Genistein – 50μM (a) and Wortmannin – 100nM (b), 30min prior to the incubation with the crude (1μg/mL), pure (100ng/mL) or insulin (100 nM), followed by the 2-deoxy-d-[1-3H] glucose uptake assay. The results were expressed as percentage of glucose uptake with respect to the vehicle control. The values are mean of ±S.E., n=3 in duplicates. (∗), P<0.05 as compared with vehicle control group.