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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 52  |  Page : 834-839  

Triphala, regulates adipogenesis through modulation of expression of adipogenic genes in 3T3-L1 Cell line


1 Obesity Research Laboratory, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India
2 Cancer Lab, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra, India

Date of Submission20-Apr-2017
Date of Acceptance27-May-2017
Date of Web Publication31-Jan-2018

Correspondence Address:
Ruchika Kaul Ghanekar
Cancer Lab, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra
India
Supriya Bhalerao
Obesity Research Laboratory, Interactive Research School for Health Affairs, Bharati Vidyapeeth University, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.224329

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   Abstract 


Background: Triphala, an Ayurvedic polyherbal formulation, is used for the treatment of various diseases including obesity. Objective: The present study was planned to evaluate the anti-adipogenic potential of aqueous extract of Triphala (TPaq) using 3T3-L1 adipocyte cell line model. Methods: The effect of aqueous extract of Triphala (TPaq) was tested on the viability of 3T3- L1 cells by MTT assay. The cells were treated with a cocktail of dexamethasone (DEX), isobutylmethylxanthine (IBMX) and insulin to induce adipogenesis. The cells were treated either with the induction cocktail or with the cocktail containing different concentrations (1, 10 and100 μg/ml) of TPaq. Intracellular lipid content was analyzed using Oil O Red stain and was quantified after extracting with isopropanol at 500 nm wavelength. The expression of early (PPAR-γ and C/EBP-α) and late (GLUT4 and FAS) phase adipogenic genes was studied by real time PCR. Results: TPaqdid not affect the viability of 3T3-L1 cell line. Interestingly, TPaqinduced a concentration dependant decrease in the intracellular lipid content and expression of both early and late phase adipogenic genes. This decrease was statistically significant compared to cells treated with only induction cocktail. Conclusion: These results suggested that Triphala regulated lipid accumulation by down regulating expression of adipogenic genes, resulting into prevention of adipogenesis.
Abbreviations used: TPaq: Aqueous extract of Triphala; DMEM: Dulbecco's Modified Eagle's medium; FBS: Fetal Bovine Serum; IBMX: Isobutyl methylxanthine; DMX: Dexamethasone; MTT: [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay; PPARγ: Peroxisome proliferator-activated receptor; C/EBP:Enhancer binding protein α, FAS:Fatty acid synthase; Glut-4: Glucose phosphate transporter 4.

Keywords: Adipocyte, Obesity, Lipid accumulation, 3T3-L1


How to cite this article:
Banjare J, Raina P, Mansara P, Ghanekar RK, Bhalerao S. Triphala, regulates adipogenesis through modulation of expression of adipogenic genes in 3T3-L1 Cell line. Phcog Mag 2017;13, Suppl S4:834-9

How to cite this URL:
Banjare J, Raina P, Mansara P, Ghanekar RK, Bhalerao S. Triphala, regulates adipogenesis through modulation of expression of adipogenic genes in 3T3-L1 Cell line. Phcog Mag [serial online] 2017 [cited 2019 Nov 22];13, Suppl S4:834-9. Available from: http://www.phcog.com/text.asp?2017/13/52/834/224329





Summary

  • The purpose of this study was to evaluate the effect of an ayurvedic polyherbal drug Triphala on adipogenesis using 3T3-L1 cell line. The results suggested that Triphala regulated lipid accumulation by downregulating expression of adipogenic genes, resulting into the prevention of adipogenesis.



   Introduction Top


The cause of obesity is related to formation of adipocytes (fat cell) through adipogenesis which leads to enhanced lipid accumulation. Adipogenesis involves differentiation of preadipocytes into mature adipocytes by clonal cell expansion.[1] The process is highly regulated by various transcriptional factors from early to terminal phase of adipogenesis.[2] 3T3-L1 cell line is a well-defined in vitro model for studying adipogenesis and glucose uptake mechanism. The differentiation of 3T3-L1 cell requires synergistic activity of multiple transcription factors and adipogenic modulatory factors including peroxisome proliferator-activated receptor-gamma, CCAAT/enhancer-binding protein (C/EBP), fatty acid synthase, and so on.[3]

Various medicinal plants have been studied for their inhibitory effect on adipogenesis. In the present study, we selected Triphala, an ayurvedic polyherbal formulation comprising of equiproportional fruit parts of Terminalia chebula, Terminalia bellerica, and Phyllanthus emblica. Our previous report on drug usage of Triphala by ayurvedic practitioners and the survey of marketed antiobesity herbal formulations have demonstrated wide use of Triphala in the management of obesity.[4] The ingredients of Triphala have been studied individually or in combination with other medicinal plants for antioxidant, antidiabetic, antihypercholesteremic, and antiobesity activities.[5]Phyllanthus emblica has been reported to induce minimal adipocyte differentiation and stimulate glucose uptake.[6]T. bellerica and T. chebula have been found to be effective in reducing lipid accumulation in adipocytes.[7] However, the whole formulation has not yet been studied for its effect on adipogenesis. With this background, we have evaluated the antiadipogenic activity of aqueous extract of Triphala (TPaq) by studying its effect on lipid accumulation and expression of early- and late-phase adipogenic genes in 3T3-L1 fibroblast cell line.


   Materials and Methods Top


Materials

3T3-L1 cells were obtained from National Centre for Cellular Science (NCCS, Pune, India). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other chemicals used in study such as trypsin, insulin, isobutylmethylxanthine (IBMX), dexamethasone, isopropanol, and oil O red stain were purchased from Sigma-Aldrich, US. The primers and reagent used for gene expression study were purchased from Invitrogen, Waltham, Massachusetts, USA.

Cell culture

3T3-L1 cell line was maintained in DMEM containing 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. Once the cells were 80% confluent, differentiation to adipocytes was induced using a cocktail of 0.57 μg/ml of insulin, 0.5 mM of IBMX, and 0.25 μM of dexamethasone for 48 h. This was followed by culturing of cells in DMEM supplemented with 10% FBS with or without aqueous extract of Triphala (TPaq) and the media was changed after every 2 days up to 8 days.

Drug

Standardized TPaq was procured from Pharmanza Herbal Pvt. Ltd. Petlad, Gujarat, India [Appendix 1]. Different concentrations of TPaq, namely, 1, 10, and 100 μg/ml were prepared by dissolving the powdered extract (5 mg/μg) in sterile distilled water. The doses were prepared in DMEM according to the concentration of the stock solution.



Cell viability

The effect of TP on viability of 3T3-L1 cells was studied using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. 3T3-L1 preadipocytes were grown in a 96-well plate with 1, 10, and 100 μg/mL concentrations of TPaq for 24 h. Cell viability was also determined on day 4 and on day 8 of the experiments in 3T3 cells treated with induction cocktail and 1, 10, and 100 μg/mL concentrations of TPaq. Cells were incubated with MTT for 24 h [8] to develop crystals. Insoluble formazan crystals were then dissolved in isopropanol, and absorbance was measured between 570 and 630 nm using microplate reader (FLUOstar Omega, BMG LABTECH GMBH Ortenberg, German).

Cell morphology

Cell differentiation and formation of lipid droplets was seen under inverted microscope from day one onward. On the last day of experiment, cells were washed gently with Phosphate Buffered Saline (PBS) twice, fixed with 10% fresh formaldehyde in PBS for 30 min at room temperature, and stained by oil O red solution with 70% isopropanol and water (1:1) for 1 h. After staining of lipid droplets with water, the oil O red solution was removed and the plates were rinsed with water and dried. Images were taken on fluorescent microscope (FLoid cell imaging station, life technologies, India).

Quantification of lipid accumulation

The intracellular lipid accumulation of the control and treated cells was analyzed using ImageJ software, and the density histograms of images were plotted. Further, the dye retained in the cells was eluted with isopropanol and quantified by measuring optical absorbance at 500 nm using microplate reader (FLUOstar Omega, BMG LABTECH GMBH Ortenberg, German).

Quantitative real-time polymerase chain reaction

For studying the effect of TPaq on expression of adipogenic genes, 2 early-phase genes, namely, peroxisome proliferator-activated receptor-gamma (PPARγ) and C/EBP-α and 2 late-phase genes, namely, glucose transporter receptor 4 (GLUT4) and fatty acid synthase (FAS) of adipogenesis were selected. Total RNA was isolated on the 8th day of the experiment using standard laboratory protocol. Two μg of total RNA were used to synthesize cDNA using reverse transcriptase. After reverse transcription, samples were examined by SYBR premix Ex Taq using Takara Thermal Cycler Dice Real Time System (Otsu, Shiga, Japan). Each cDNA was amplified (95°C for 5 s, 58°C–64°C for 10 s, and 72°C for 20 s for 40 cycles) using gene-specific primers designed from sequences acquired from NCBI nucleotide sequence database [Table 1]. All reactions were done in triplicates and the gene expressions were normalized using glyceraldehyde-3-phosphate dehydrogenase as reference gene.
Table 1: Primers sequence used for reverse transcription-polymerase chain reaction

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Statistics

The data were shown as mean ± standard deviation and analyzed using one-way ANOVA test with Tukey's multiple comparison test. P < 0.05 was considered as level of significance. The data were analyzed using GraphPad Prism 6 Software, San Diego, California, USA.


   Results Top


Cell viability studies with aqueous extract of Triphala

Initially, 3T3-L1 cells were treated with different concentrations (1–100 μg/mL) of TPaq for a period of 24 h to test its effect of on cell viability. TPaqper se was found to be nontoxic to the cells at the tested concentrations [Figure 1]a. Further, the effect TPaq on the cell viability of 3T3-L1 was observed after the 4th and 8th days of treatment with the induction cocktail (containing insulin, IBMX, and dexamethasone) alone [Figure 1]b and [Figure 1]c. The viability of the cells was not affected after treatment with induction cocktail containing TPaq. However, at 100 μg/ml, there was a significant (P< 0.001) reduction in the percentage of viable cells (74.71 ± 3.86%) after 8 days.
Figure 1: Effect of true positive on viability of 3T3-L1 preadipocytes cells (a) Only true-positive treatment (b) on day 4 when incubated with induction cocktail (c) on day 8 when incubated with induction cocktail *P < 0.01, **P < 0.01, and ***P < 0.001 as compared to control using one-way ANOVA followed by Tukey's posttest

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Changes in cell morphology with aqueous extract of Triphala

The control cells revealed spindle-like appearance of fibroblasts under inverted microscope. Contrarily, the cells treated with induction cocktail alone and with different concentrations of TPaq showed the presence of mature spherical-shaped adipocytes with lipid droplets. The extent of lipid droplets was less in TPaq-treated cells compared to induction cocktail-treated cells [Figure 2].
Figure 2: Cell morphology of control (c), induction (i), concentration of Triphala 1, 10, and 100 μg/ml

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Aqueous extract of Triphala reduced lipid accumulation

On day 8 of experiment, cells treated with induction cocktail showed ~2.2-fold increase in lipid content as compared to control cells (P< 0.001) [Figure 3]a. The cells treated with TPaq showed ~1.43-, 1.67-, and 2.5-fold decreases in lipid content at 1, 10, and 100 μg/ml concentrations, respectively, compared to the cells treated with induction cocktail alone. The decrease was statistically significant (P< 0.001).
Figure 3: Intracellular lipid accumulation using (a) ImageJ software density histogram (b) isopropanol extraction method. ***P < 0.001 as compared to control, ##P < 0.01, ###P < 0.001 as compared to induction using one-way ANOVA followed by Tukey's posttest

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The intracellular lipid accumulation was quantified further. The cells treated with induction cocktail resulted in ~2-fold increase in lipid content compared to untreated control cells (P< 0.001). On the other hand, the cells treated with TPaq showed ~1.33-, 1.45-, and 1.78-fold decrease in lipid content at 1, 10, and 100 μg/ml concentrations, respectively, which was statistically significant (P< 0.001) compared to the cells treated with induction cocktail alone [Figure 3]b.

Aqueous extract of Triphala downregulated adipogenic gene expression

The mechanism of TPaq was studied using relative mRNA expression of genes involved in adipogenic process. All the four studied genes, namely, PPAR-γ, C/EBP-α, GLUT4, and FAS showed ~20-fold increase in mRNA expression after 8 days of treatment with induction cocktail compared to the untreated control cells (P< 0.001). The treatment with TPaq decreased the mRNA expression of all the genes in a concentration-dependent manner.

The expression of PPAR-γ was decreased by ~5, 11, and 30 folds at 1, 10, and 100 μg/ml concentrations, respectively, compared to cells treated with induction cocktail alone [Figure 4]a. Interestingly, expression of C/EBPα was decreased by ~8, 12, and 60 folds at 1, 10, and 100 μg/ml concentrations, respectively. This decrease was significant statistically (P< 0.001) at all studied concentrations [Figure 4]b.
Figure 4: Relative mRNA expression of (a) peroxisome proliferator-activated receptor-gamma, (b) C/enhancer-binding protein-alpha, (c) glucose transporter receptor 4 and (d) fatty acid synthase. ***P < 0.001 as compared to induction using one-way ANOVA followed by Tukey's posttest

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TPaq treatment reduced mRNA expression of GLUT4 by ~3, 16, and 45 folds [Figure 4]c whereas expression of FAS was found to be decreased by ~5, 10, and 30 folds at 1, 10, and 100 μg/ml concentrations, respectively [Figure 4]d. This reduction was statistically significant (P< 0.001).


   Discussion Top


The present study reports the antiadipogenic potential of TPaq in 3T3-L1 cell line model. The cells treated with TPaq reduced lipid accumulation and downregulated expression of adipogenic genes. Various plants such as Curcuma longa, Moringa oleifera, Murraya koenigii,[9] D-seco limonoids of Swietenia m ahogani,[10] Platyphylloside isolated from Betula platyphylla,[11] and yanggyuksanhwa-tang [12] have been reported to exhibit antiadipogenic activity in 3T3 cell line model. It has been proposed that different phytoconstituents that target different stages of the adipocyte life cycle might prove beneficial for decreasing lipid accumulation, inducing apoptosis, or by inhibiting adipogenesis or both.[13]

Triphala is the most common formulation used by ayurvedic physicians and is marketed as one of the formulations against obesity. A previous study using high-fat diet-induced obesity in animal model has reported lipid-lowering activity of Triphala.[14] Even though the individual ingredients of Triphala have been studied for their antiadipogenic potential in vitro, the whole formulation has not been tested for its effect on adipogenesis.

The programmed differentiation of preadipocytes to fully differentiated adipocytes with increased lipid accumulation throughout the adipogenic process is accompanied by an increase in the expression of various transcription factors and adipocyte-specific genes.[15]

During differentiation, the action of adipogenic genes, which include members of the C/EBP family (C/EBP-α, -β, and -δ) and PPARγ, induces adipogenesis.[16] C/EBP-β is expressed early in the adipocyte differentiation program, and it initiates mitotic clonal expansion. In response to an adipogenic induction, C/EBP-β and-δ are first activated to promote PPARγ and C/EBPα expression.[17] The transcription factor PPARγ is a master regulator of adipocyte differentiation, and its activation is both necessary and sufficient for adipocyte differentiation.[18] The activation of C/EBPα and PPARγ leads to terminal differentiation by inducing the transactivation of many adipocyte genes encoding proteins and enzymes responsible for maintaining the adipocyte phenotype such as FAS.[19] In the present study, TPaq downregulated mRNA expression of both PPARγ and C/EBPα. Phyllanthus emblica, one of the ingredients of Triphala, has been shown to inhibit PPARγ.[20] Interestingly, the whole formulation modulates these two important genes involved in regulation of adipogenesis.

A number of genes are involved in adipocyte lipid accumulation. FAS regulates de novo lipogenesis from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate and is expressed at high levels in adipose tissue, liver, and lung.[21] GLUT4 is the insulin-regulated glucose transporter found primarily in adipose tissues and striated muscle (skeletal and cardiac). Its expression is increased during adipocyte differentiation, and it maintains glucose homeostasis in differentiated and insulin-responsive cells.[22] We demonstrated that TPaq downregulated mRNA expression of GLUT4 and FAS which are responsible for lipid accumulation in adipocytes.

All these data suggest that Triphala could be explored as potential drug candidate in regulating obesity.


   Conclusion Top


Triphala significantly decreased the adipogenesis in 3T3-L1 cells by reducing lipid accumulation and inhibiting the expression of adipogenic genes. These results confirm the antiobesity potential of Triphala.

Acknowledgment

We would like to acknowledge Director, IRSHA and ministry of AYUSH for providing financial support for the study.

Financial support and sponsorship

Ministry of AYUSH.

Conflicts of interest

There are no conflicts of interest.



 
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