|Year : 2019 | Volume
| Issue : 3 | Page : 304-309
Study of antidiabetic properties of Uvaria narum leaf extract through glucose uptake and glucose transporter 4 expression studies in 3T3L1 cell line model
Murad Alsawalha1, PB Janardhana2, KR Padma3, Shiva Shankar Reddy2, Abeer M Al-Subaie4, Srinivasa Rao Bolla5, Vishnu Priya Veeraraghavan6, Joel P Joseph2, Surapaneni Krishna Mohan7
1 Department of Chemical and Process Engineering Technology, Jubail Industrial College (JIC), P. O. Box 10099, Jubail Industrial City 31961, Kingdom of Saudi Arabia
2 Department of Biotechnology, Stellixir Biotech Private Limited, Bengaluru, Karnataka, India
3 Sri Padmavati Mahila Visvavidyalayam, Padmavathi Nagar, Near West Railway Station, Tirupati, Chittoor (D.t), Andhra Pradesh, India
4 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, P. O. Box 2435, Dammam 31441, Kingdom of Saudi Arabia
5 Department of Anatomy, College of Medicine, Imam Abdulrahman Bin Faisal University, P. O. Box 2114, Dammam 31451, Kingdom of Saudi Arabia
6 Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India
7 Department of Medical Biochemistry, College of Applied Medical Sciences - Jubail (CAMSJ), Imam Abdulrahman Bin Faisal University, P. O. Box 4030, Jubail Industrial City, Al Jubail 35816, Kingdom of Saudi Arabia
|Date of Web Publication||22-Aug-2019|
Dr. Surapaneni Krishna Mohan
Department of Medical Biochemistry, College of Applied Medical Sciences - Jubail, Imam Abdulrahman Bin Faisal University, P.O.Box 4030, Jubail Industrial City, Al Jubail 35816
Kingdom of Saudi Arabia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Uvaria narum (UN) is known to have antipyretic, antimicrobial, anti-inflammatory and antimalarial properties. The antidiabetic properties of UN remains unexplored. The current study has been aimed at understanding the antidiabetic property of UN extract on an in vitro model using 3T3-L1 cell line. Methods: Methanolic extract of UN was prepared, and its cytotoxic effect on 3T3-L1 cells was assessed. Glucose uptake and glucose transporter 4 (GLUT4) translocation in 3T3-L1 cell line on treatment with the extract was evaluated against a standard drug, metformin. α-glucosidase and α-amylase inhibition activities of the extract were also assayed with acarbose as the standard drug. Results: Treatment with UN extract had no cytotoxic effect on the cells. UN extract showed a good percentage inhibition of α-amylase and α-glucosidase activities. UN extract showed 71.31% inhibition and the control drug Acarbose exhibited 88.54% inhibition in α-amylase activity. Furthermore, the extract showed 79.11% inhibition when Acarbose exhibited 87.35% inhibition in α-glucosidase activity. IC50values were also determined. Further, on treatment with the extract, 75.49% of 3T3-L1 cells took up glucose and 70.67% had GLUT4 expression. Conclusion: UN extract enhances glucose uptake and GLUT4 expression, inhibits α-amylase and α-glucosidase activities, thereby demonstrating the antidiabetic properties in vitro.
Keywords: 3T3-L1, antidiabetic activity, glucose transporter 4, Uvaria narum, α-amylase, α-glucosidase
|How to cite this article:|
Alsawalha M, Janardhana P B, Padma K R, Reddy SS, Al-Subaie AM, Bolla SR, Veeraraghavan VP, Joseph JP, Mohan SK. Study of antidiabetic properties of Uvaria narum leaf extract through glucose uptake and glucose transporter 4 expression studies in 3T3L1 cell line model. Phcog Res 2019;11:304-9
|How to cite this URL:|
Alsawalha M, Janardhana P B, Padma K R, Reddy SS, Al-Subaie AM, Bolla SR, Veeraraghavan VP, Joseph JP, Mohan SK. Study of antidiabetic properties of Uvaria narum leaf extract through glucose uptake and glucose transporter 4 expression studies in 3T3L1 cell line model. Phcog Res [serial online] 2019 [cited 2019 Sep 21];11:304-9. Available from: http://www.phcogres.com/text.asp?2019/11/3/304/265060
- Uvaria narum (UN) is a medicinal plant belongs to Annonaceae (custard apple) family and broadly disseminated in the foothills of Western Ghats
- The in vitro antidiabetic activities of UN leaf extract in 3T3-L1 cell line model have been studied
- We have performed biochemical studies such as α-amylase and α-glucosidase inhibition assays, glucose uptake, and glucose transporter 4 (GLUT4) expression studies in 3T3 L1 cell line, as part of in vitro antidiabetic activity
- Exposure to different concentrations of UN leaf extract for 24 h has not revealed any toxicity on 3T3 L1 cells
- methanolic leaf extract of UN has a major reduction effect on the activity of α-amylase enzyme and α-glucosidase enzyme
- Further on stimulation with plant extract, 75.49% of cells and 70.67% of cells displayed glucose uptake and GLUT4 expression in 3T3 L1 cells, respectively
- The UN leaves extract has shown promising antidiabetic activity and could be used as a potential source of antidiabetic agents.
Abbreviations Used: UN: Uvaria narum , DMSO: Dimethyl sulfoxide, DMEM: Dulbecco's Modified Eagle's medium, FBS: Fetal bovine serum, D-PBS: Dulbecco's phosphate-buffered saline, GLUT4: Glucose transporter 4, FITC: Fluorescein isothiocyanate, PNPG-p-nitrophenyl-α-dglucopyranoside, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 2-NBDG: 2-(N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose, SD: Standard deviation.
| Introduction|| |
Diabetes mellitus (DM) is an endocrine metabolic disorder that manifests hyperglycemia (enhanced blood glucose level), hyperlipidemia (augmented lipid level), hyperaminoacidemia, and hypoinsulinemia (reduced insulin level) conditions. Recurring hyperglycemia can lead to damage, dysfunction, and failure of different organs – the eyes, nerves, kidneys, and heart., The number of diabetes cases have been spiraling with projections reaching >300 million cases by the end of 2025.,,
The onset of type 2 diabetes, the prevalent form of DM, is characterized by insulin resistance and could lead to other complications, including cardiovascular risk factors such as hypertension, dyslipidemia, and prothrombotic factors.,, Existent therapeutic regimens known to have adverse effects on prolonged use.,, Conventionally, ethnomedicinal plants have been known to possess phytochemicals that have therapeutic effects., In the recent times, medicinal and nutraceutical herbs have been recognized for their holistic effects on the health of an individual.,
In diabetic patients, morbidity and mortality are due to the inability of target cells such as skeletal muscle cells, adipocytes, and liver cells to effectively use glucose from the blood.,, In a healthy individual, insulin stimulation increases glucose transport in muscle cells by 3 or 4 folds. A major part of this outcome is related to the net translocation of the glucose transporter 4 (GLUT4) from the intracellular compartment to the cell surface, where it facilitates the uptake of glucose and its reduction.,,
Uvaria narum (UN) is an ethnomedicinal plant that belongs to Annonaceae (custard apple) family and is found in the foothills of the Western Ghats and the Central Provinces of India. The leaves of the plant have been traditionally used to treat several diseases including diabetes.
In this study, we investigated the in vitro antidiabetic activity of the leaf extract of UN, using biochemical assays and flow cytometric analyses, on 3T3-L1 cells.
| Materials and Methods|| |
Chemicals and reagents
Dulbecco's Modified Eagle's medium (DMEM) without glucose (#AL186, Himedia), fetal bovine serum (#RM10432, Himedia), DMEM high glucose (#AL219A, Himedia), Dulbecco's phosphate-buffered saline (D-PBS) (#TL1006, Himedia), Acarbose (#A8980, Sigma), metformin (#PHR 1084, Sigma), 2-(N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) (Invitrogen: Cat no. 13195), mouse anti-anti GLUT4–fluorescein isothiocyanate (FITC) antibody (#NBP1-49533F, Novus Biologicals), dimethyl sulfoxide (DMSO) (#PHR1309, Sigma), 3-(4,5-dimethythiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) Reagent (# 4060 Himedia), FACS Calibur (BD Biosciences, USA), Microplate reader (#EC800, Biotek).
Collection of plant material
Fresh leaves of UN were collected from local areas of Mangalore, Karnataka, India [Figure 1].
Preparation of extract
The shade-dried leaves were cut, pulverized, and stored in airtight containers. The leaves were extracted with methanol by the soxhlation process. The extract was concentrated using Rotary Evaporator (Hahn vapor, hs-2005 v) under reduced pressure at a temperature not exceeding 50°C. The filtrate was freeze-dried, and the extract was dissolved in DMSO for the assays
3T3-L1 cell line was procured from NCCS, Pune, and cultured in DMEM (high glucose) supplemented with 10% FBS, 10,000 units of Penicillin G, and 10,000 μg/mL streptomycin sulfate and 10 mM HEPES. The cultures were maintained at 37°C with 5% CO2 in a humidified incubator.
MTT assay was performed to test for the cytotoxic effect of the extract. MTT assay is a colorimetric assay that measures the reduction of yellow-colored MTT by mitochondrial succinate dehydrogenase to an insoluble, dark purple formazan product., Reduction of MTT thus becomes a readout of the number of viable cells in the culture. 96 well plates were seeded with 3T3-L1 cells at an initial density of 20 × 104 cells per well. The cells were allowed to adhere overnight. Cells were then treated with varying concentrations of plant extract (25–400 μg/mL) for 24 h. Posttreatment, 0.5 mg/mL of MTT reagent was added to the cells and the cells were incubated at 37°C temperature for 2 h. Later, MTT reagent was removed and the formazan crystals were dissolved by adding 20 μL of DMSO. Absorbance at 570 nm was measured using microplate reader and percentage viability of the cells was calculated using the formula:
Glucose uptake assay
3T3-L1 cells were seeded into a six-well plate at an initial seeding density of 2 × 105 cells/well. Overnight culture of cells was washed with D-PBS and then treated for 2 h with 100 μg/mL of the extract or 100 μM the standard drug, metformin, as the control. The cells were then further incubated with 2-NBDG for 2 h. Cells were then trypsinized, washed with PBS, and resuspended in 0.5 ml of D-PBS. Cellular uptake of 2-NBDG was measured by flow cytometry (FACS Calibur, BD Biosciences) and the data were analyzed using Cell Quest Pro software.
Glucose uptake and glucose transporter 4 translocation studies
GLUT4 expression levels were analyzed using flow cytometry. 3T3-L1 cells were seeded in a six-well plate at an initial density of 2 × 105 cells/2 ml and were cultured overnight. The spent medium was aspirated, and the cells were treated with 100 μg/mL of UN extract or 100 μM of positive control, metformin, for 24 h. Posttreatment, the cells were washed with D-PBS, trypsinized, and resuspended in 0.5 ml of D-PBS. The cells were then incubated with Mouse Anti Glut4–FITC antibody (#NBP1-49533F, Novus Biologicals) for 30 min in the dark. Unbounded antibody was washed with D-PBS, and the cells were assessed for GLUT4 expression in a BD FACSCalibur Flow Cytometer in FL1 channel. The data were analyzed with Cell Quest Pro software.
α-amylase inhibition assay
The α-amylase inhibitory activity was estimated using a previously described method. UN extracts of different concentrations ranging from 31.25 to 500 μg/mL in DMSO were prepared. About 100 μL of α-amylase and UN extract were mixed and incubated in microtubes for 10 min at 37°C. 100 μL of 1% soluble starch dissolved in buffer A was added to each microtube, and the mixture was incubated for 30 min at 37°C. 200 μL of dinitrosalicylic acid color reagent was added to arrest the reaction, and the microtubes were then incubated at 100°C for 5 min. The samples were cooled to room temperature and 50 μL of the reaction mixture was transferred to the wells of 96-well microplate. This was further diluted with 150 μL of distilled water and the absorbance at 540 nm was measured in a microplate reader (EC800, Biotek). Percentage inhibition was calculated using the formula stated below, and the results were compared to the inhibitory action of the control drug, acarbose.
α-glucosidase inhibition assay
The α-glucosidase enzyme inhibitory activity of UN extract was carried out based on the procedure described by Shibano et al. with slight modifications. UN extract of concentrations ranging from 31.25 to 500 μg/mL was prepared. These were mixed with 50 μL of 0.1M phosphate buffer (pH 7.0), 25 μL of 0.5 mM 4-nitrophenyl-α-D-glucopyranoside (dissolved in 0.1M phosphate buffer, with pH of 7.0), and 25 μL of α-Glucosidase (0.1 U/mL). The reaction mixture was incubated for 30 min at 37°C. The reactions were terminated with 100 μL of 0.2M sodium carbonate. Absorbance at 410 nm was read in the microplate reader (EC800, Biotek). The results were compared with the positive control, Acarbose. Percent inhibition of α-glucosidase was calculated using the below-stated formula. IC50 values were also determined.
IC50 values in enzyme inhibition assays were determined using linear regression graph (concentration versus percentage enzyme inhibition). All the experiments were conducted in triplicates, and the results are expressed as mean percentage inhibition ± standard deviation (n =3). Statistical significance was determined by one-way analysis of variance, followed by Bonferroni post hoc test for multiple comparisons, and P < 0.05 was considered statistically significant. All statistical analyses and IC50 values determination were carried out in GraphPad Prism (version 3.1) software (San Diego, CA).
| Results|| |
α-Amylase activity inhibition
Inhibition of α-amylase activity by UN extract was tested against the control drug acarbose. UN leaf extract inhibited α-amylase activity by 19.60% at 31.25 μg/mL and 71.31% at 500 μg/mL concentration. The IC50 value of the extract was found to be 253.61 μg/mL. The control drug acarbose showed 37.15% inhibition of α-amylase activity at 31.25 μg/mL and 88.54% at 500 μg/mL concentration. The IC50 value of acarbose was found to be 40.66 μg/mL [Table 1] and [Figure 2].
|Figure 2: Inhibition of α-amylase against different concentrations of Uvaria narum ; the IC50value of the extract was 253.61 ± 3.54 μg/mL. Data were presented as mean ± standard deviation|
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Inhibition of α-glucosidase activity
UN extract exhibited 19.74% inhibition of α-glucosidase activity at 31.25 μg/mL and 79.11% at 500 μg/mL, respectively. Its IC50 was found to be 208.60 μg/mL. The control drug acarbose showed 34.96% inhibition in α-glucosidase activity at 31.25 μg/mL and 87.35% at 500 μg/mL concentrations, respectively. Its IC50 was found to be 43.53 μg/mL [Table 2] and [Figure 3].
|Figure 3: Inhibition of α-glucosidase against logarithm of different concentrations of Uvaria narum ; the IC50value of the extract was 208.60 ± 7.25 μg/mL. Data were presented as mean ± standard deviation|
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3T3-L1 cells were treated with various concentrations (25 μg–400 μg/mL) of UN extract and were assayed for their cytotoxic effect. The extract did not show any cytotoxic effect. The concentrations of the extract used and the respective percent cell viability were tabulated and plotted [Table 3] and [Figure 4], [Figure 5].
|Table 3: Percent viability of the cells treated with various concentrations of Uvaria narum extract|
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|Figure 4: The effect of Uvaria narum on 3T3L1 cell line viability as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method. Each bar graph represents percentage viability of 3T3L1 cell line against 25, 50, 100, 200, and 400 μg/mL concentrations of Uvaria narum extract after 24h exposure. The data were shown as mean ± standard deviation of triplicate experiments|
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|Figure 5: Images of 3T3 L1 cell line in inverted light microscopy after the exposure to Uvaria narum extract. From “A” to “C” where (a) control (untreated cells), (b) Standard metformin drug, and (c) 400μg concentration of Uvaria narum extract. After incubation of 24 h Uvaria narum extract displayed no toxicity|
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Glucose uptake and glucose transporter 4 expression analysis
GLUT4 expression on UN extract treatment was analyzed using flow cytometry. UN extract (100 μg/mL) treatment increased GLUT4 expression. Metformin (100 μM) was used as the control. Flow cytometric analysis revealed that 70.67% of 3T3-L1 cells treated with UN extract expressed GLUT4 and 99.97% of metformin-treated cells expressed GLUT4 [Figure 6] and [Figure 7].
|Figure 6: Glucose transporter 4 expression upon exposure of 3T3L1 cells to 100 μg/mL of plant extract and 100 μM of metformin. Each bar graph represents the % of cells expressing glucose transporter 4. Data were represented as mean ± standard deviation of triplicate experiments|
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|Figure 7: Overlaid expression of glucose transporter 4 in untreated 3T3L1 cells (black color line) and standard drug-treated cells (metformin 100 μM) (red color line) and test compound-treated cells (orange color line)|
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Glucose uptake studies
2-NBDG, a fluorescent deoxyglucose analog, was used to detect the glucose uptake in 3T3-L1 cells cultured in the presence of antidiabetic drugs. Flow cytometric analysis of the UN extract treated 3T3-L1 cells showed that 75.49% had taken up 2-NBDG. Furthermore, 99.98% of the cells treated with the control drug, metformin, had taken up 2-NBDG [Figure 8] and [Figure 9].
|Figure 8:Overlaid expression of given untreated 3T3L1 cells (black color line) and standard drug-treated cells (metformin 100 μM) (red color line) and test compound-treated cells (orange color line) against the 2-(N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose stain|
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|Figure 9: Each bar graph represents the % of cells taken up the 2-(N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose in the presence of 100 μg/ml of Uvaria narum and 100 μM of metformin. The data were represented as mean ± standard deviation of triplicate experiments|
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| Discussion|| |
The major enzymes that metabolize carbohydrates in the small intestine are pancreatic α-amylase and α-glucosidase enzymes, which convert polysaccharides to monosaccharides. Antidiabetic drugs reduce the action of these enzymes and regulate the postprandial blood glucose level in type 2 diabetic patients. The mechanisms of control of glucose levels vary with the drug. Some drugs stimulate insulin secretion or synthesis by the pancreatic beta-cells, others help in regenerating damaged pancreatic beta-cells, still others increase insulin sensitivity triggering glucose uptake by fat and muscle cells, and certain others modify the activity of enzymes pertinent to glucose metabolism to reduce the absorption of sugars from the gut., Contemporary antidiabetic drugs that specifically inhibit the activity of enzymes include acarbose, voglibose, and miglitol. However, usage of these drugs can have adverse effects such as flatulence and abdominal bloating.
Natural compounds from ethnomedicinal plants that do not manifest such effects could be used to treat type 2 diabetes. Such medicinal plants play a vital role in herbal medicine, particularly in treating ailments such as diabetes.,,, UN, an ethnomedicinal plant that belongs Annonaceae (Custard apple) family is a primary plant with several varieties, many of which have been traditionally used for medicinal and ethnobotanical purposes.,, In this study, we investigated the antidiabetic properties of UN.
Our results showed that methanolic extract of UN effectively inhibited α-amylase and α-glucosidase activities. These inhibitory effects were estimated with acarbose as the standard drug. Furthermore, UN had no cytotoxic effects on the cells. The implications of this study corroborate the previous studies on inhibitory effect of other natural compounds on α-amylase and α-glucosidase activities.,,
Cellular uptake of glucose from blood plays a crucial role in the reduction of DM. This is generally mediated by GLUT4 in the cell. On stimulation with antidiabetic drugs, GLUT4 is translocated from their intracellular sites to the cell surface. Insulin induces the translocation of GLUT4 through phosphatidylinositol-3-kinase pathway. PKB/Akt-mediated stimulation of glucose transport by insulin has also been reported in rat adipocytes and L6 muscle cells. Metformin is one of the standard antidiabetic drugs that enhance glucose uptake by inducing the translocation of GLUT4. This drug is known to act through AMP-activated protein kinase pathway. In the current study, metformin was used as the positive control for glucose uptake and GLUT4 expression studies. Of all the 3T3-L1 cells treated with UN extract, 75.49% took up 2-NBDG (a glucose analog) and 70.67% expressed GLUT4. These findings suggest that the leaf extract of UN could enhance cellular glucose uptake by inducing GLUT4 translocation. However, further studies must be performed to understand the exact mechanism of action.
| Conclusion|| |
The methanolic extract of UN inhibited the activity of the enzymes α-amylase and α-glucosidase, enhanced glucose uptake, and GLUT4 translocation in 3T3-L1 cells. These results were comparable to the action of acarbose and metformin. Further, the extract had no cytotoxic effect on the cells. In conclusion, UN extract has potential antidiabetic applications. Further work must be done to identify lead molecules from this plant.
All the authors acknowledge the services rendered by Stellixir Biotech Pvt. Ltd, Bengaluru, India, in carrying out the in vitro cell line work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3]