Original Article

Chebulagic acid from Terminalia chebula Gangadharan Leela Shyni1

Sasidharan Kavitha1
enhances insulin mediated glucose uptake in Sasidharan Indu1
Anil Das Arya2
3T3-L1 adipocytes via PPARc signaling pathway Sasidharan Suseela Anusree1
Vadavanath
Prabhakaran Vineetha1
Sankar Vandana1
Andikannu Sundaresan1
Kozhiparambil
Gopalan Raghu1*

1
Biochemistry and Cell Culture Laboratory, Agroprocessing and Natural
Products Division, Council of Scientific and Industrial Research-National Institute
for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram,
Kerala 695019, India
2
Computational Modelling and Simulation Division, Council of Scientific and
Industrial Research-National Institute for Interdisciplinary Science and
Technology (CSIR-NIIST), Thiruvananthapuram, Kerala 695019, India

Summary
The thiazolidinedione (TZDs) class of drugs are very effective for
the treatment of type 2 diabetes mellitus (T2DM). But due to the
adverse effects of synthetic TZDs, their use is strictly regu- lated.
The therapeutic actions of TZDs are mediated via modu- lation
of peroxisome proliferator-activated receptor gamma (PPARc).
Naturally occurring PPARc modulators are more desir- able as
they lack the serious adverse effects caused by TZDs. This has
prompted the exploitation of medicinal plants used in traditional
medicine, for their potential PPARc activity. In the present work,
we studied chebulagic acid (CHA) isolated from fruits of
Terminalia chebula with respect to its effect on adipo- genesis,
glucose transport, and endocrine function of adipo- cyte. The
mRNA expression profile of PPARc target gene CCAAT/enhancer-
binding protein alpha (C/EBP-a) was analyzed
by qRT-PCR. The putative binding mode and the potential ligand-
target interactions of CH A, with PPARc was analyzed using
docking software (Autodock and iGEMDOCKv2). The results
showed that CH A enhances PPARc signaling and adipo- genesis
dose dependently but in a moderate way, less than rosiglitazone.
GLUT4 expression and adiponectin secretion was increased by
CHA treatment. The mRNA expression of PPARc target gene
C/EBP-a was increased in CH A-treated adipocytes. The
comparison of results of various parameters of adipogene- sis,
insulin sensitivity, endocrine function and molecular dock- ing
experiments of roziglitazone and chebulagic acid indicate that
the latter behaves like partial PPARc agonist which could be
exploited for phytoceutical development against T2DM.
V
C 2014 BioFactors, 00(00):000–000, 2014.

Keywords: type 2 diabetes; PPARc; GLUT4; chebulagic acid; C/EBP-a;

adiponectin; adipogenesis

Abbreviations: TZDs, thiazolidenediones; PPARg, peroxisome proliferator-activated receptor gamma; C/EBP-a, CCAAT/enhancer-binding protein alpha;
T2DM, type 2 diabetes mellitus
V
C 2014 International Union of Biochemistry and Molecular Biology

Volume 00, Number 00, Month/Month 2014, Pages 00–00

*Address for correspondence: K. G. Raghu, Biochemistry and Cell Culture Laboratory, Agroprocessing and Natural Product Division, Council of Scientific
and Industrial Research-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, Kerala 695019, India.
E-mail: raghukgopal2009@gmail.com
Received 4 September 2014; accepted 8 November 2014
DOI 10.1002/biof.1193
Published online 00 Month 2014 in Wiley Online Library
(wileyonlinelibrary.com)

BioFactors 1

1. Introduction
Type 2 diabetes mellitus (T2DM) results from the combination of
resistance to insulin action, inadequate insulin secretion, and
excessive or inappropriate glucagon secretion. The world- wide
prevalence of T2DM is rapidly increasing and the number of
patients is expected to become ~550 million by 2030 [1]. Obesity
is also another major factor which leads to release of free fatty
acids (FFA) from visceral adipose tissue that leads to insulin
resistance, and have been implicated in b-cell dysfunc- tion.
Chronic hyperglycemia of diabetes is responsible for long term
organ damage especially eyes, kidneys, nerves, and other
cardiovascular disorders [2]. Considering these reasons, the
market for the oral T2DM agents is growing faster.
Peroxisome proliferator activated receptors (PPAR) have
drawn increased attention as a drug discovery target by regu-
lating glucose and lipid metabolism [3]. PPAR and its subtypes
belong to the superfamily of nuclear receptors that function as
transcription factors activated by several ligands. PPARs serve
as vital targets and play an important role in treating obesity,
atherogenic dyslipidemia, hypertension, and insulin resistance
[4]. There are three PPAR subtypes, commonly designated as
PPARa, PPARc, and PPARd(b). PPARa is highly expressed in
tissues with high rates of mitochondrial fatty acid oxidation, such
as liver, heart, muscle, kidney, and cells of the arterial wall, and
it is activated by fibrates, fatty acids and eicosa- noids.
Thiazolidinediones (TZDs) are a group of PPARc ago- nists, a
class of anti-diabetic drugs, which include rosiglita- zone and
pioglitazone. The activation of PPARc by TZDs leads to the
redistribution of fat from visceral to subcutaneous adi- pose
tissue, increased trapping of fatty acids in adipose tissue, and a
modified secretion of hormones from adipose tissue, all factors
known to improve insulin sensitivity [5]. However,
administration of TZDs has been associated with unexpected side
effects such as weight gain, edema, and hepatotoxicity [6,7].
Recently, identification of new PPARc partial agonists that
do not present the adverse side effects caused by PPARc full
agonists have received considerable attention for developing
powerful drugs against diabetes. The ligands acting as partial
agonists induce submaximal receptor activation and have been
demonstrated to retain beneficial anti-diabetic properties with
reduced side effects. Despite the lower activation of the recep- tor,
they are still able to induce PPARc target genes responsi- ble for
anti-hyperglycemia and insulin sensitivity, but not for the
unwanted PPARc actions [8,9]. The reason for this is not entirely
understood. However, it is conceivable that partial agonists may
elicit a different conformation of the receptor– ligand complex
that leads—by altered recruitment of tran- scriptional co-
activators and repressors—to a more restricted set of expressed
target genes compared to full agonists [10]. Natural products
encompass a broad structural diversity of secondary metabolites,
which often represent privileged struc- tures serving a variety of
biological functions [11]. Chen et al. (2013) reported that 6-
hydroxydaidzein (6-HD), an isoflavone
2 Terminalia chebula Enhances Insulin-mediated Glucose Uptake
BioFactors
from fermented soybeans accelerated adipocyte differentiation
and improved insulin sensitivity by increasing the expression of
C/EBPa and facilitating the translocation of GLUT4 via the
activation of PPARc [12]. Studies conducted by Shin et al. (2010)
showed that esculetin from Fraxinus rhynchophylla sig- nificantly
blocked the induction of PPARc protein expression and inhibited
adipocyte differentiation induced by troglitazone, a PPARc
agonist [13]. Anusree et al. (2013) have conducted studies on
punicic acid (PA), a polyunsaturated fatty acid mainly found in
pomegranate seed oil and found moderate adipogenic potential
with enhanced glucose uptake and adipo- nectin secretion [14]. In
addition, there are reports of nutra- ceutical properties of some
plant extracts relevant to diabetes via their adipogenic potential
[15,16]. All these reports reveal the potential of natural resources
to provide partial PPARc agonist for pharmaceutical purpose.
Terminalia chebula Retz. (Combretaceae) is a major con-
stituent of triphala, a popular ayurvedic formulation commonly
used to treat metabolic disorders in ayurvedic medicine [17]. The
major chemical constituents of this plant are chebulagic acid,
ellagic acid, gallic acid, ellagitannins, and allotannins [18]. Lee
et al. (2006) has reported that chebulagic acid, iso- lated from the
ripe fruits of Terminalia chebula possess strong antioxidant
properties in rat hepatocytes [19]. Also Yang et al. (2013)
reported that, the chemical constituents of the fruits of
Terminalia chebula showed the enhancement of PPARa and/or
PPARc signaling in HepG2 cells [20]. However, the molecular
mechanism behind the PPARc agonistic effect of chebulagic acid
remained unexplored. This study was carried out to eluci- date
cellular mechanism of action of chebulagic acid (CHA) as a
partial PPARc agonist in 3T3-L1 preadipocytes.
2. Materials and methods
2.1. Reagents and Antibodies
Dexamethasone, insulin, protease inhibitor cocktail, rosiglita-
zone and 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]- D-
glucose (2-NBDG) were from Sigma–Aldrich (USA). Adiponec- tin
ELISA kit was from Cayman chemical (U.S.A). Dulbecco’s
Modified Eagle’s Medium (DMEM), bovine calf serum (BCS),
fetal bovine serum (FBS), phosphate-buffered saline (PBS), Pen-
icillin/streptomycin and trypsin were from Himedia (Mumbai,
India). Mouse monoclonal antibodies for PPARc (sc7273), GLUT4
(sc53566), b-actin (sc8432), GAPDH (sc365062), and bovine anti-
mouse IgG-HRP secondary antibody (sc2371) were purchased
from Santa Cruz Biotechnology (USA).
2.2. Isolation of Chebulagic Acid
The dried fruit of Terminalia chebula (100 g) was crushed into
powder with a mixer, followed by extraction (two times) with
methanol (1 L) in total for 24 H, with continuous stirring at
room temperature, and filtration. Evaporation of the solvent
under reduced pressure yielded the methanol extract (30 g).
This extract was suspended in water (250 mL) and re-
extracted with hexane (100 mL 3 3) and then with chloroform

(100 mL 3 3). Then the water fraction was lyophilized. The
lyophilized water fraction (5 g) was applied on a Sephadex LH-
20 column (3.53 45 cm2), with a water–methanol gradient, to
give four fractions. The eluted solvent volume and yield of each
fraction were as follows: fr. 1: water (200 mL) (0.8 g), fr. 2: 40%
methanol in water (200 mL) (0.4 g), fr. 3: 60% metha-
nol in water (300 mL) (2.0 g), fr. 4: 50% acetone in water (300
mL) (1.64 g). Fraction 3 (2 g) was subjected to MCI gel CH20P
column [1.5 3 15 cm2, water–methanol] four fractions were
collected: fraction 3(1) (0% methanol), fraction 3(2)- 10%
methanol- water, fraction 3(3)- 50% methanol-water and frac-
tion 3(4)- 100% methanol-water. Fraction 3(3) was further
purified by reverse phase HPLC using water (A) and acetoni-
trile (B) as solvents (Conditions: solvent program 0-30 min
100% A - 40% A; column- Gemini column 250 3 4.6 mM; oven
temperature 30○C; detector-Photo Diode Array Detector; detec-
tion wavelength 217 nM). The sample was identified as chebu-
lagic acid by comparison with standard spectral data. Purity
was determined to be ≤95%. Stock solution was prepared (10
mM) in DMSO for biological testing and the highest solvent
concentration was 0.1%.
2.3. Cell Cultures
For standard adipocyte differentiation 3T3-L1 cells were grown
to confluence in DMEM with 10% calf serum and 2 days post-
confluence differentiation was induced by DMEM with 10% fetal
bovine serum (FBS), 1 lM dexamethasone (DEX), 0.5 mM meth-
ylisobutylxanthine (MIX) and 10 lg mL21 insulin with either
vehicle (0.1% DMSO), positive control [10 lM rosiglitazone, or
CHA in DMSO (10, 50, and 100 lM)]. From day 4 to the end of the
experiment at day 8, the cells were re-fed every second day with
DMEM with 10% fetal bovine serum (FBS), 10 lg mL21 insulin
and with either vehicle (0.1% DMSO), positive control (10 lM
rosiglitazone), or CHA in DMSO (10, 50, 100 lM) [21].
2.4. In Vitro Cytotoxicity Assay (MTT Assay)
MTT assay was carried out as described by Quan et al. (2012)
[22] with slight modifications. Cells in exponential growth phase
were seeded at a density of 1 3 104 cells per well in 96- well plates
and incubated in culture medium. The cells were then treated
with various doses (1–500 lM) of CHA. After 48 H, the cells were
incubated in the dark with 20 lL of MTT solu- tion (5 mg
MTT/mL in PBS) for 4 H at 37○C. The formazan crystals thus
formed were dissolved in DMSO and the plates were read after
45 min in a microplate reader (Biotek Synergy 4, USA) at 570
nm.
2.5. Test for Adipogenic Potential, Oil-Red-O Staining,
and Quantification of Lipid Accumulation
The 3T3-L1 cells were seeded at a density of 1 3 106 cells/well
in six-well plates and grown and differentiated for 8 days as
per the protocol described earlier. On the 8th day of differen-
tiation, the lipid droplets were stained and quantified. Differ-
entiated adipocytes were stained with oil-red-O using the
method of Kasturi and Joshi (1982) with some modification
[23]. Cells were washed twice with PBS and fixed with 70%
ethanol for 30 min. Fixed cells were stained with oil-red-O
solution (0.5% oil-red-O in isopropanol diluted with water (3:2)
followed by filtration) for 1 H at room temperature and washed
twice with distilled water. The images of stained cells were
captured with a camera attached to a phase contrast microscope
(Nikon Eclipse TS 100, Japan) at 403 magnifica- tion. For the
quantification of triglyceride content in the cells, 100 lL of 100%
isopropanol was added to each well. It was gently mixed for
10–15 min and the absorbance was read at 490 nm with a plate
reader (Biotek, USA).
2.6. Western Blot Analysis
The 3T3-L1 cells at a density of 1 3 106 cells/well in six-well
plates were grown and differentiated for 8 days as per the pro-
tocol described earlier. At the end of treatment, cells were washed
twice with ice cold PBS and lysed in ice-cold lysis buffer (50 mM
Tris, 150 mM sodium chloride, 1% Triton X-100 and protease
inhibitor cocktail, pH 8.0). The protein content of the lysate was
measured using BSA protein assay kit (Thermo scientific, USA).
The lysate containing 30 lg of protein was subjected to SDS-
PAGE on 8% gel and transferred on to a poly- vinylidene
difluoride (PVDF) membrane by using Trans-Blot TurboTM (Bio-
Rad). The membrane was incubated for 1hr at room temperature
in blocking buffer (5% skim milk), washed three times with tris
buffered saline with tween 20 (TBST) (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.05% tween-20) and probed over night at 4oC with
primary antibody against GLUT4, PPARc or GAPDH (dilution;
1:500, (Santa Cruz Biotech- nology, CA,USA)). After washing
three times in TBST for 5 min each, the membrane was incubated
with horseradish peroxi- dase (HRP) conjugated secondary
antibody (dilution 1:1000, Santa Cruz Biotechnology, CA, USA)
for 1 H. After washing three times in TBST, the bands were
detected by staining with 3,3-diaminobenzidine, and the relative
intensity of bands were quantified using Bio-Rad Quantity One
version 4.5 software in a Bio-Rad gel doc. The quantity of PPARc
and GLUT4 in cell lysate was normalized with the content of
GAPDH.
2.7. Enzyme-linked Immunosorbent Assay (ELISA)
Indirect ELISA was carried out using specific primary antibody
(1:500 dilution) and HRP-conjugated secondary antibody
(1:1000 dilution) [24]. The amounts of PPARc and GLUT4
expressed in the cells were quantitated by ELISA. Cell lysate
precoated on to ELISA plates (96 well, NUNC) served as anti-
gen. It was incubated with antibodies against PPAR-c and
GLUT4 for 2 H. It was then washed with PBS-tween 20,
treated with HRP-conjugated secondary antibody and the color
was developed using o-dianisidine as the substrate. The con-
centrations of these antigens were estimated by measuring the
absorbance of the coloured HRP product spectrophotometri-
cally at 490 nm in a plate reader (Biotek, USA).
2.8. Quantification of Adiponectin by ELISA
The 3T3-L1 preadipocytes were differentiated in the presence of
CHA (10, 50, 100 lM), and rosiglitazone (10 lM) in six-well
plates for 8 days. Supernatants were taken from these plates on
3
 BioFactors

(A) Structure of Chebulagic acid (B) Effect of CHA on 3T3-L1 cell viability. 3T3-L1 cells were treated with various doses (1–500 lM)
FIG 1 of CHA. Cell viability was measured by the MTT assay after 48 H. Values are expressed as mean 6 SEM of three independ- ent
experiments with duplicates. * Indicates significant difference compared to vehicle control P 0.05; ≤ MTT assay: methyl thiazol
tetrazolium assay.

the 8th day of differentiation to check for the enzyme secretion.
Adiponectin levels were determined using a solid-phase sand-
wich ELISA kit (Cayman Chemical, USA) and the concentrations
were calculated following the manufacturer’s instructions.
2.9. Determination of mRNA Level Expression of
CCAAT/Enhancer-binding Protein Alpha (C/EBPa) by
Real-time Quantitative PCR
Total RNA was isolated from 3T3-L1 cells on the 7th day of
differentiation, using TRIzol reagent (Sigma–Aldrich). The first
strand cDNA was synthesized using cDNA synthesis kit (Life
Technologies, USA). The gene expression levels were analyzed by
quantitative real-time RT-PCR, conducted using the CFX96 Real
Time PCR system (Bio-rad, USA). The primers used in the
experiments are C/EBPa: Sense: 50 -AGACATCAGCGCCTACA
TCG-30 Antisense:30 TGTAGGTGCATGGTGGTCTG-50 . b-actin:
Sense:50 -AGTACCCCATTGAACGC-30 Antisense: 30 -TGTCAGCAA
TGCCTGGGTAC-50 . Briefly, the reaction solution (20-lL final
volume) contained 3 mM MgCl2, 2 lL of SYBR Green qPCR
kit IQ Supermix (Bio-Rad, USA) and 100 pM of each primer.
Amplification of each gene was performed on a C1000 Ther- mal
cycler (Bio-Rad, USA) and expression was normalized to
housekeeping gene (b-actin). The standard amplification program
included 30 cycles of three steps each, which involved heating
the product to 95○C with a 20 s hold, annealing to 60○C with a 20
s hold, and extending to 72○C with a 10 s hold. The mRNA levels
of the gene were normal- ized using b-actin as the internal
control. The fold change in the target gene relative to the b-actin
endogenous control gene is determined by:
Fold Change ¼ 2-DðDCTÞ
where DCT 5 CT, target 2 CT, b-actin and D(DCT) 5 DCT, stimulated
2 DCT, control
Ct (threshold cycle) is the intersection between an amplifi-
cation curve and a threshold line.
2.10. In Vitro Competitive Binding Assay
The binding of CHA on PPARc ligand binding domain was stud-
ied using a Lanthascreen TR-FRET PPARc competitive binding
assay kit from Invitrogen according to the kit protocol. A mix-
ture of 0.5 nM of GST (glutathione S-transferase)-tagged PPARc
ligand binding domain (GST–PPARc–LBD), 5 nM Tb ant- GST-
antibody, 5 nM Fluormone Pan-PPAR Green, and serial dilutions
of CHA, GW9662 or rosiglitazone were mixed and incubated in
the dark for 2 H. The TR-FRET signals were measured by
excitation at 340 nm and emission at 520 nm for fluorescein and
excitation at 340 nm and emission at 490 nm for terbium in a
Tecan multiplate reader (Tecan Infnite 200PRO, Mannedorf,
Switzerland). The ratiometric emission 520/490 was plotted
against the concentrations of the com- pound to determine the
IC50 value of the compounds. DMSO was used as the no ligand
control.
2.11. Glucose Uptake Assay
a
The glucose uptake assay was performed as previously
described with slight modification [25]. Briefly, 3T3-L1 cells
were seeded in six-well plates at a density of 1 3 106 cells/well
and differentiated using differentiation media for 7 days. On
the 8th day the medium was replaced with glucose and
serum-free DMEM containing insulin (10 lg mL21), indicated
concentrations of CHA (10, 50, 100 lM) and rosiglitazone (10
lM) and incubated for 24 H. After 24 H the medium was
removed and cells were then incubated in low glucose medium
again in the presence of different concentrations of CHA and
rosiglitazone for 3 H. Cells were stimulated with insulin (10
lg mL21) for 10 min and then treated with 100 lM 2-NBDG
for 1 H. The amount of 2-NBDG taken up by the cells was

4 Terminalia chebula Enhances Insulin-mediated Glucose Uptake

 CHA promotes preadipocyte differentiation by activating PPARc: Two-day post confluent 3T3-L1 preadipocytes (day 0) were
FIG 2 induced to differentiate with 10, 50, 100 lM CHA or 10 lM rosiglitazone and was replaced every 2 days along with the relevant
media cocktail up to day 8. The assays were performed on day 8. (A) Representative microscopic images of differentiated 3T3-
L1 adipocytes treated with CHA or rosiglitazone (10 lM). (a) Vehicle control (b) 10 lM rosiglitazone (c) 10 lM CHA (d) 50 lM CHA
(e) 100 lM CHA. (B) Cells stained with oil-red-O. (a) Vehicle control (b) 10 lM rosiglitazone (c) 10 lM CHA (d) 50 lM CHA
(e) 100 lM CHA treated. (C) Absorbance was spectrophotometrically determined at 490 nm after oil-red-O staining. Results are
mean 6 SEM, (n 5 6). (D) The cell lysates were subjected to SDS-PAGE. Immunoblot of PPAR c1, PPAR c2 was performed. The
amount of PPAR c1, PPAR c2, or GAPDH was detected with antibodies against PPARc, or GAPDH, respectively. (E)The relative
intensity of each band was quantified with GAPDH. Data are presented as mean 6 SEM; n 5 6 (F) Expression of PPARc protein
assayed through indirect ELISA method. The values given are the average of three experiments with duplicates 6 SEM. VC— vehicle
control; RG—rosiglitazone;*—Indicates significant difference compared to vehicle control, **—indicates significant dif- ference
compared to rosiglitazone treated group-P ≤ 0.05.

measured in the blue filter using a BD FACS AriaTM II flow

cytometer (BD Bioscience, USA). Data from 10,000 single cell
events were collected.
2.12. Molecular Docking
Docking experiment of CHA into the PPARc ligand binding
domain was done using the softwares Autodock 4.2 and iGEM-
DOCK [26,27]. These docking softwares were used to find the
appropriate binding and conformations of the ligand to the
receptor. The 3D model of PPARc was retrieved from the Broo-
khaven Protein Data Bank (http://www.rcsb.org/pdb; PDB ID
2Q5S). CHA (ID: 219296) structure was downloaded from
Chemspider (http://www.chemspider.com) and converted to
5
 BioFactors

ware iGEMDOCK. The binding energy of the ligand-PPARc was

analyzed using the software Autodock 4.2.

2.13. Statistical Analysis

All data are expressed as mean 6 SEM from three independent
experiments done in duplicates. The differences between treat-
ments in comparison with control were assessed using one way
ANOVA following assessment of normality (SPSS v9.0; SPSS,
Chicago, IL). Statistical significance was defined as P ≤ 0.05.

CHA up regulates adiponectin secretion in 3T3-L1
FIG 3 cells.3T3-L1 cells were differentiated and treated as
described earlier up to day 8. Adiponectin levels in the
conditioned medium on the 8th day were meas- ured
by ELISA. Data are presented as means 6 SEM (*P 0.05,
≤
**P 0.01) ≤ independent experiments.
from three
.pdb file using Chem3D-Pro 10. The docking fitness of the ligand
molecules to PPARc and the amino acids of receptor (PPARc)
involved in interaction was predicted using the soft-
3. Results
3.1. Effect of CHA on 3T3-L1 Preadipocytes Cell
Viability
The effect of CHA on adipocytes viability was confirmed by MTT
assay. 91% cell viability was observed when cells were pretreated
with 500 lM CHA for 48 H (Fig. 1B).
3.2. CHA is a Partial PPARc Agonist with Modest
Adipogenic Activity
To examine the effect of CHA on differentiation, 3T3-L1 preadi-
pocytes were induced to differentiate in the presence of CHA.
Rosiglitazone was used as a positive control in all experiments.
Microscopic examination revealed that CHA promoted the
differentiation of 3T3-L1 preadipocytes (Fig. 2A). The number

CHA enhances GLUT4 expression in 3T3-L1 cells. GLUT4 expression is determined by western blot and indirect ELISA with anti- body
FIG 4 against GLUT4 using cell lysates of differentiated 3T3-L1 adipocytes. (A) The cell lysates were subjected to SDS-PAGE. Immunoblot
of GLUT4 was performed. The amount of GLUT4 or GAPDH was detected with antibodies against GLUT4 or GAPDH, respectively
(B) The relative intensity of each band of GLUT4 protein to its own GAPDH was quantified. Data are pre- sented as mean 6 SEM; n
5 3 (C) Expression of GLUT4 protein was assayed through indirect ELISA method. The values given are the average of three
experiments with duplicates 6 SEM. VC-vehicle control; RG-rosiglitazone;*—indicates significant differ- ence compared to VC, **—
Indicates significant difference compared to rosiglitazone treated group—P ≤ 0.05.
6 Terminalia chebula Enhances Insulin-mediated Glucose Uptake

CHA upregulates mRNA levels of transcription regu-
FIG 5 lator C/EBPa in differentiated 3T3-L1 adipocytes. The
3T3-L1 adipocytes were differentiated along with
indicated concentrations of CHA (10, 50, and 100 lM) or
10 lM rosiglitazone up to day 8. After treatment, RT-
PCR was performed on isolated total RNA. The mRNA
results are expressed as difference in fold change in
treated cells compared to vehicle control. Data are
mean 6 SEM values for three independent experiments
performed in duplicates. *P ≤ 0 .05,
**P ≤ .01 versus control.
of lipid droplets, evaluated by oil-red-O staining, increased in CHA
treated cells (Fig. 2B). However, 100 lM of CHA induced
adipogenesis was less than that of 10 lM rosiglitazone (Figs.
2Ae and 2Be), indicating that CHA is less potent than rosiglita-
zone in promoting adipogenesis.
To further investigate the effect of CHA on differentiation, we
examined the protein expression of PPARc using ELISA (Fig.
2E). The results revealed upregulation in the protein level of
PPARc in rosiglitazone (P ≤ 0.05) and CHA treated groups; albeit
the latter showed a less significant expression. Further the cell
lysates were subjected to western blotting with anti- PPARc or
GAPDH antibody. Both isoforms of PPARc, PPARc1, and PPARc2
were expressed in the samples. The protein expression of PPARc2
which is more related to insulin sensitiv- ity and adipogensity
[28] was upregulated in both rosiglitazone and CHA treated
groups (Figs. 2C and 2D). The activity profile of CHA on PPARc2
expression was dose dependent but less than rosiglitazone.
PPARc1 expression in rosiglitazone and CHA treated groups
were comparable.
3.3. CHA Increases Adiponectin Secretion in 3T3-L1
Adipocytes
To examine the effect of CHA on adiponectin secretion in mature
adipocytes, we treated 3T3-L1 adipocytes with CHA or
rosiglitazone from day 4 to day 8 on alternate days. The cellu- lar
level of adiponectin was found to increase with CHA, in a dose
dependent manner. The level was increased significantly (P ≤
0.05) similar to rosiglitazone (Fig. 3).
3.4. CHA Elevated GLUT4 Protein Expression
Because PPARc agonist also regulates glucose metabolism, we
investigated the effect of CHA on glucose transport in 3T3-L1 adi-
pocytes. Expression of GLUT4 was analyzed using western blot
(Figs. 4A and 4B) and ELISA (Fig. 4C). Both western blot and
ELISA showed a same trend in increase in GLUT4 protein when
treated with 10, 50, and 100 lM concentrations of CHA compared
to vehicle control. But compared to rosiglitazone it was less.
3.5. CHA Increased mRNA Expression of C/EBP-a, a
PPARc Target, in Differentiated Adipocytes
We examined mRNA expression profile of PPARc target gene
C/EBP-a after CHA (10, 50, and 100 lM concentrations) and
rosiglitazone (10 lM) treatment in differentiated 3T3-L1 adipo-
cytes by qRT-PCR. As shown in Fig. 5, the expression level of
C/EBP-a was upregulated in the CH A treated groups and the
expression levels were comparable to rosiglitazone group.
3.6. Competitive Binding Affinity of CHA to PPAR c
The ligand/receptor interaction was studied using in itro Lan-
thascreen TR-FRET competitive binding assay. GST-purified
PPAR c LBD (0.5 nM) was incubated with Tb anti-GST- antibody,
Fluormone Pan-PPAR Green (5 nM) and CHA, and then terbium
emission and FRET signals were measured at
CHA binds to PPARc LBD as evident from PPARc
FIG 6 competitive binding assay. To verify direct binding of
CHA to PPARc, a Lanthascreen TR-FRET PPARc com-
petitive binding assay was performed. PPARc ago- nists
rosiglitazone and PPARc antagonist GW9662 (0–
10 lM), were used as comparative controls. The
reaction mixture contained 0.5 nM PPARc-LBD (GST), 5
nM Terbium-tagged anti-GST antibody, 5 nM Fluo-
rmone Pan-PPAR Green, 5 mM dithiothreitol, and
varying concentrations of CHA (2 nM–10 lM) and
rosiglitazone (2 nM–10 lM). After 3-h incubation in the
dark, TR-FRET measurements were made in a Tecan
multiplate reader. Data are mean 6 SEM val- ues for
three independent experiments performed in
duplicates. Statistical significance is indicated by *, P
≤ 0.05 compared with the TR-FRET ratio of vehicle.
7
 BioFactors

Measurement of glucose uptake using 2- NBDG in 3T3-L1 cells by flow cytometer. The representative histogram shows mean
FIG 7 fluorescence level (530 nm emission), which indicates the glucose uptake activity. FITC histograms in (A) control, (B) rosiglita-
zone, (C) CHA 10 lM, (D) CHA 50 lM and (E) CHA 100lM.

2nd H after the reaction. The IC50 value of CHA was 7.5lM, 50, and 100 lM of CHA was 32.2 6 0.5, 35.8 6 1.1, 38.6 6 0.2
whereas for rosiglitazone and GW 9662 it was 53.09 and indicating an increased glucose uptake (10.2-, 13.8-, and 16.6-
12.88 nM, respectively (Fig. 6). fold increase, respectively).

3.7. CHA Increases Insulin-stimulated Glucose Uptake
in 3T3-L1 Cells
As shown in Fig. 7, the insulin-stimulated glucose uptake in 3T3-
L1 cells tend to increase with CHA (10, 50, 100 lM) and
rosiglitazone (10 lM) when compared to vehicle control. Rosi-
glitazone caused an increase of 27.4 fold in glucose uptake (Table
1). The mean fluorescence level of cells treated with 10,
3.8. Docking of CHA to PPARc
To determine the putative binding mode and the potential ligand-
target interactions of CHA, it was docked to the PPARc of PDB
entry 2Q5S using Autodock and iGEMDOCKv2. Auto- dock 4.2
has estimated the free binding energy of CHA to be
26.85 kcal mol21 showing the moderate binding affinity into
the binding site (Figs. 8A and 8B). The docking energy
8 Terminalia chebula Enhances Insulin-mediated Glucose Uptake
 effects caused by PPARc full agonists. The problematic side
effects of PPARc full agonists mainly include weight gain or fluid
Effect of CHA on insulin-stimulated glucose uptake
TABLE 1 in 3T3-L1 cells retention. A substantial portion of the therapeutic benefits of full
and partial PPARc agonists occurs through the inhibition of the
PPARc phosphorylation at Ser273. Thus, an effective partial
Mean fluorescence Fold agonist of PPARc would have weak or low transactiva- tion
Compound level (%)a Increaseb activity while maintaining the stimulation of glucose uptake [30].
Yang et al. (2013) reported PPARc and PPARa agonistic
Control 22.0 6 0.65
potential of the compounds isolated from Terminalia species
Rosiglitazone (10 lM) 49.4 6 2.1* 27.4 [20]. It was found that CH A, corilagin, and three gallotannins
namely 2,3,6-tri-O-galloyl-b-D-glucose, 1,2,3,6-tetra-O-galloyl-
Chebulagic acid (10 lM) 32.2 6 0.5** 10.2
b-D-glucose, and 1,2,3,4,6-penta-O-galloyl-b-D-glucose from
Chebulagic acid (50 lM) 35.8 6 1.1** 13.8 Terminalia chebula enhances PPARc mediated glucose uptake
in HepG2 cell lines . However to confirm the use of CH A as a
Chebulagic acid (100 lM) 38.6 6 0.2** 16.6
therapeutic agent for diabetes its efficiency to enhance glucose
transport in adipocytes has to be studied and this has not been
a
Values are mean 6 SEM of three replicates; *—indicates significant reported yet. So in the present study we tried to elucidate the
difference compared to control, ** indicates significant difference molecular mechanism behind the PPARc agonistic effect of
≤
compared to rosiglitazone treated group—P 0.05.
b
Fold increase was calculated in comparison to control.
chebulagic acid in 3T3 L1 preadipocytes. In adipose tissue
c
Positive control. PPARc is expressed as two isoforms. One of the isoforms,
PPARc1, is expressed in many tissues and cell types, including
obtained using iGEMDOCKv2 is 278.3345 kcal mol21. The best white and brown adipose tissue, skeletal muscle, liver, pancre-
docking pose of CHA as obtained from Autodock 4.2 is shown in atic beta cells, macrophages, colon, bone and placenta. The
Fig. 8A. The Docking modes of CHA with PPAR c as obtained from expression of the other splice variant, PPARc2, is restricted to
iGEMDOCKv2 is shown in Fig. 8B. The results of Auto- dock 4.2 white and brown adipose tissue under physiological conditions
showed hydrogen bonding between PPARc and CHA at residues [31,32]. PPARc2 is not only the more adipogenic isoform in itro
GLU369, ASP362, LYS358, and GLU448 (Fig. 8C). The but is also the only PPARc isoform regulated at the tran-
interactions between PPAR c and CHA are shown in the scriptional level by nutrition [32–35]. Furthermore, expression
interaction table (Table 2) as obtained from iGEMDOCKv2. In of PPARc2 in the obese state suggests that PPARc2 may have a
addition, no hydrogen bond interaction between compound and role in insulin resistance. Zhang et al. [36] showed that
residues HIS323, TYR323, and HIS449 in the LBD of PPARc PPARc2-null mice were insulin resistant. Recently, it has been
(typical of PPARc full agonists) was predicted [29]. shown that over expression of PPARc2 is accompanied by
increasing energy expenditure and improved insulin sensitivity
[37]. In correlation with these findings our results showed that
CHA at a dose of 100 lM concentration upregulated PPARc2
4. DISCUSSION indicating its potential to improve insulin sensitivity. However
Although there are successful examples of the discovery of new since PPARc2 is the more adipogenic isoform among the two
PPARc agonists as drug candidates for diabetic complica- tions, isoforms of PPARc, its lesser expression in CH A treated group
it has recently been of great interest to identify new PPARc
partial agonists that do not present the adverse side

Docking configurations of CHA in to PPARc. Docking simulation was performed to identify interaction between PPARc and CHA
FIG 8 using the Software: iGEMDOCKv2.1 and Autodock 4.2. (A) and (B) Docking modes of CH A on human PPARc using Autodock
4.2. and iGEMDOCKv2, respectively (C) H-bonding between residues of PPARc and CHA as obtained from Autodock 4.2.
9
 BioFactors

V-S- V-M- V-M- LEU- than rosiglitazone treated group suggests that it increases the
PHE- PHE- 353 368 energy expenditure and thus improves insulin sensitivity [37].

Table depicts the pharmacological interaction of CH A and PPARc in the post-screening analysis. In the table, energy represents the binding energy; V-M—Vander vaals inter-
21.94 26.56 215.3
Insulin causes the rapid translocation of GLUT4 from
intracellular sites to the plasma membrane of fat and muscle
cells [38,39]. Various studies reveal that the increase in
expression of GLUT4 in adipocytes, as assessed by immuno-
blotting shows that GLUT4 in control and insulin-treated cells
368

accounts for a significant portion of increase in the rate of glu-

cose transport in response to insulin [38,40,41]. Our results
indicate that CHA increases the GLUT4 expression in 3T3-L1
23.05
V-M-
LEU-
353

adipocytes which account for its increased rate of glucose

transport in response to insulin. However, elucidations of action
of CHA are not in agreement between research groups. A
21.53
PHE-
V-S-

352

particular study by Yang et al., suggested an insulin depend- ent

effect whereas others showed stimulation of insulin secre- tion or
insulin sensitizing effect [20,42]. The exact molecular mechanism
25.83
V-M-
PHE-
352

underlying the effect of CHA with respect to its anti-diabetic

property is not fully known. To explore a yet unidentified
mechanism, we analyzed related signal transduc- tion pathways
20.20
MET-
V-S-

348

that are activated by this bioactive compound.

Initial cytotoxicity evaluation of CHA proved the safety of this
bioactive compound for biological applications. The adipo- genesis
V-M-
MET-

27.2

induced by rosiglitazone was however much greater when

348

compared to adipogenic potential of CHA treated cells. Yang et al.

(2013) had also reported similar results to claim partial PPARc
23.41
PHE-
V-S-

347

agonistic property of CHA [20]. In our present study we conducted

series of investigations such as protein level expression analysis,
GLUT4 receptor expression analysis and adiponectin secretion to
20.52
PHE-
V-M-

347

reveal the molecular basis of its anti-diabetic property.

Interaction table and fitness scores of CHA from iGEMDOCKv2.1

Adipose tissue is emerging as a very robust platform for

pharmaceutical research due to its pleiotropic nature in meta-
20.03
V-M-
VAL- LEU-
340

bolic syndromes [43]. Their endocrine function is very relevant in

this area. These adipocytes take part very effectively in car-
bohydrate and fatty acid metabolism through its endocrine
action with main chain; V-S—Vander vaals interaction with side chain.
24.3720.68
339 339
V-M- V-S-

function. It secretes around 75 adipokines including adiponec- tin

[44]. Adiponectin is very important in the biology of adipo- cytes
VAL-

and very essential for keeping the cell insulin sensitive [45]. We
found CHA enhances adiponectin secretion showing a novel
pathway of its involvement in metabolic syndromes. Up
21.04
MET-
V-S-

regulation of glucose transport in adipocytes by CHA clearly

334

reveals that this bioactive compound acts through GLUT4 for

insulin mediated glucose uptake. Our results show that CHA acts
24.71
V-M-
MET-

as a partial PPARc agonist and confers anti-diabetic activ- ity

334

without bringing undesirable side effects like weight gain in i o

as opposed to full agonists like TZDs.
V-M-
LEU-

20.6
333

Muscles and adipose tissues are insulin responsive and

express the insulin sensitive glucose transporter, GLUT4.
GLUT4, translocated from intracellular vesicles to the plasma
21.20
V-M-
ILE-
249

membrane in response to insulin, causes increased glucose

transport in to muscle and fat cells [46]. Insulin resistance is one
TABLE 2

of the most important factors causing insulin dependent diabetes

2Q5S-CHA

mellitus. Therefore the need for modulators to acti- vate insulin

COMD

mediated glucose uptake in such insulin respon- sive tissues is

high. 3T3-L1 adipocytes are the most preferred

10 Terminalia chebula Enhances Insulin-mediated Glucose Uptake


cell lines to study insulin stimulated glucose uptake as most of
the available muscle cell lines are not insulin sensitive in terms
of glucose transport. These cells behave like primary adipocytes
in many aspects and provide an excellent model system to study
insulin action and signaling [47]. The 3T3-L1 adipocytes when
given differentiation medium containing insu- lin, IBMX and
DEX in the presence of serum, become termi- nally differentiated
adipocytes. They progressively acquire morphological and
biochemical characteristics of mature white adipocytes [48].
The present study also showed that CHA up regulated the
mRNA expression of C/EBPa, a PPARc target in adipocytes, but
to a lesser extent compared to rosiglitazone. In addition com-
petitive ligand binding analysis also revealed that CHA have high
affinity to PPARc. It enhances glucose transport and increased
adiponectin levels. Serum levels of adiponectin are inversely
proportional to obesity, diabetes, and other insulin- resistant
states [49]. So the increase in adiponectin secretion in CHA
treated adipocytes, as revealed in our present study, is beneficial
for the control and management of metabolic syn- dromes. In this
regard, this is the first report made on CHA. In addition it also
enhanced GLUT4 translocation showing the mechanism behind
its glucose uptake enhancing property.
Overall results conclude the beneficial properties of CHA on
insulin stimulated glucose uptake based on various molecu- lar,
biochemical and virtual molecular docking studies on insu- lin
sensitivity in in itro system. This could further be eval- uated
with an aim to develop very effective, low cost and safe
nutraceuticals in future for the control and management of
diabetes.
5. Acknowledgement
The authors are thankful to the CSIR 12th 5-year plan project
“NaPAHA” (CSC 0130) for partial financial assistance. They
thank the Director, CSIR-NIIST and Head, Agroprocessing and
Natural Products Division, CSIR-NIIST for providing necessary
facilities. Dr. Shyni G.L. is grateful to Department of Biotech-
nology (DBT, New Delhi) for the Postdoctoral fellowship.
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