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Chemico-Biological Interactions 174 (2008) 6978

Contents lists available atScienceDirect

Chemico-Biological Interactions

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h e m b i o i n t

Tannins present inCichorium intybusenhance glucose

uptake and inhibit adipogenesis in 3T3-L1 adipocytes

through PTP1B inhibition

V.S. Muthusamy a, S. Anand a, K.N. Sangeetha a, S. Sujatha a,Balakrishnan Arun b, B.S. Lakshmi a,

a Centre for Biotechnology, Tissue Culture and Drug Discovery Lab, Anna University, Chennai 600025, Tamilnadu, Indiab Department of Pharmacology, Nicholas Piramal Research Centre, Mumbai 400063, India

a r t i c l e i n f o

Article history:

Received 21 December 2007

Received in revised form 12 April 2008

Accepted 15 April 2008

Available online 24 April 2008

Keywords:

Cichorium intybus

Tannins

Insulin resistance

AdipogenesisPTP1B

a b s t r a c t

Insulin resistance is a fundamental aspect for theetiologyof non-insulin dependentdiabetes

mellitus (NIDDM) and has links with a wide array of secondary disorders including weight

gain and obesity. The present study analyzes the effect ofCichorium intybus methanolic

(CME)extract on glucose transportand adipocyte differentiation in 3T3-L1 cells by studying

the radiolabelled glucose uptake and lipidaccumulation assays, respectively.By performing

detannification (CME/DT),the role of tannins present in CME on both theactivitieswaseval-

uated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a

dose-dependent response. Glucose uptake profile in thepresence of PI3Kand IRTKinhibitors

(Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME

inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptakein inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME.

Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression anal-

ysis revealed the unique behavior of CME and CME/DT. The duality exhibited byC. intybus

through adipogenesis inhibition and PPAR up regulation is of interest. Current observationconcludes that the activities possessed by C. intybusare highly desirable for the treatment

of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1

adipocytes.

2008 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: CME,Cichorium intybusmethanolic extract; CME/DT,Cichorium intybusdetannified methanolic extract; DMEM, Dulbeccos Modified Eagle

Medium; IBMX, 3-isobutyl-1-methyl xanthine; DEX, dexamethasone; DMSO, dimethylsulphoxide; FBS, foetal bovine serum; KRPH, krebs ringer phosphate

HEPESbuffer;HEPES, N-2-hydroxyethypiperazine- N-2-ethanesulfonic acid;SDS, sodium dodecyl sulphate;LDH, lactate dehydrogenase;Rosy, rosiglitazone;

PI3K, phosphatidylinositol 3-kinase; GLUT4, glucose transporter 4; PPAR, peroxisome proliferator activator receptor gamma; PTP1B, protein tyrosinephosphatase 1B; C/EBP, CCAAT/enhancerbindingprotein; SREBP, sterol regulatory element binding protein;IRS, insulin receptorsubstrate;WT, Wortmannin;

GS, Genistein; ALP, alkaline phosphatase; NBT, nitroblue tetrazolium; BCIP, 5-bromo-4-chloro-3-indolylphosphate; IDV, integrated density value. Corresponding author. Tel.: +91 44 22350772; fax: +91 44 22350299.

E-mailaddresses: [email protected](V.S. Muthusamy),anand [email protected](S. Anand),sangee22 [email protected]

(K.N. Sangeetha),[email protected](S. Sujatha),[email protected](B. Arun),[email protected](B.S. Lakshmi).

0009-2797/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2008.04.016
http://www.sciencedirect.com/science/journal/00092797mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_9/dx.doi.org/10.1016/j.cbi.2008.04.016http://localhost/var/www/apps/conversion/tmp/scratch_9/dx.doi.org/10.1016/j.cbi.2008.04.016mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://www.sciencedirect.com/science/journal/00092797


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70 V.S. Muthusamy et al. / Chemico-Biological Interactions 174 (2008) 6978

1. Introduction

Obesity combined with insulin resistance is a major

risk factor for the prevalence of non-insulin dependent

diabetes mellitus (NIDDM). Adipocyte is the only appar-

ent link between diabetes and obesity, which stores excess

glucose in the form of fat by changing dramatically in its

size in accordance with metabolic needs. Since the role

of adipocyte in energy homeostasis is vital, its dysfunc-

tion can negatively influence other systems [1]. The loss

of insulin action selectively in adipose tissue leads to sec-

ondary disorders of diabetes through the malfunctioning

of adipocyte function, changes in adipogenesis, alterations

in glucose and lipid metabolism, and protein expression

[2]. Now, wide varieties of pharmacological drugs are being

used for NIDDM treatment. However, currently available

anti-diabetic drugs (e.g., thiazolidinedione) promote obe-

sity and hyperandrogenemia, while reducing bloodglucose

[3].In the current scenario, the strategy to reduce hyper-

glycemia without increasing adiposity or with reduction of

bodyweight constitutes a preferred one for the drug devel-

opment of NIDDM.

Botanicals are thought of offering strong potential with

minimal side affects particularly against metabolic syn-

dromes as most of their efficacies are from a mixture of

active molecules acting at the same time [4]. The plant

selected for the present study is Cichorium intybus (Fam-

ily: Compositeae) a salad crop known as Chicory, French

endive, and succory, native of Asia and this plant is mainly

cultivated for its roots that contains a high content of

well-known fructopolymer inulin. C. intybus is known to

contain anthocyanins, phenols[5],sesquiterpenes such as

guaianolides, eudesmanolides, and germacranolides[6].It

is also cultivated for its food valuable sprouts. Earlier inves-

tigations have reported that the ethanolic extract of C.

intybus has anti-diabetic and hypolipidemic activities in

streptozotocin-induced diabetic rats [7]. It is being used by

traditional healers of South Indian villages for the treat-

ment of NIDDM (through personal communication with

Tamil Nadu Medicinal Plants Corporation Limited, Kolli

Hills, South India).

Thepresentstudy was commencedwith theaim of eval-

uating the methanolic (CME) and detannified methanolic

extract (CME/DT) ofC. intybusleaves for anti-diabetic and

adipogenesis inhibition potential since the two different

activities working together seem to be an ideal combina-

tion for the treatment of insulin resistance and obesity.

Tannins are plant polyphenols with the molecular weight

of 5003000 Da. Tannins are widely found in food plants

and arebroadly applied for various industrial needs. In bio-

logical systems, tannins are known to be a potent metal

ion chelators, protein precipitating agents and antioxidants

[8]. The importance of tannins in plant based drug dis-

covery approach is meager because of its high molecular

weight and complex nature [9]. Nevertheless, Liu et al. [10]

reported that tannins are significant molecules in banaba

extract (Lagerstroemia speciosa) which exhibit anti-diabetic

and adipogenesis inhibition activities in 3T3-L1 cells. Since

the leaves ofC. intybushave been reported for presence of

tannins, the detannification analysis was initiated to eval-uate the biological significance of tannins present in the

plant. In addition, we have examined the mechanisms by

which CME and CME/DT mediates the glucose transport

and adipogenesis inhibition activities.

2. Materials and methods

2.1. Chemicals and reagents

All cell culture solutions and supplements were pur-

chased from Life Technologies Inc. (Gaithersburg, MD,

U.S.A.). Dulbeccos Modified Eagle Medium (DMEM) was

obtained from GIBCO, BRL (Carlsbad, CA, U.S.A.). 2-Deoxy-

d-3[H] glucose was obtained from Amersham Pharmacia

Biotech (Buckinghamshire, U.K.). TRIzol reagent and MMLV

reverse transcriptase, dNTP, Taq polymerase was obtained

from GIBCO BRL (Carlsbad, CA, U.S.A.), USA and New

England Biolabs (Herts, U.K.), respectively. Insulin, 3-

isobutyl-1-methyl xanthine (IBMX), dexamethasone (DEX)

and Genistein were obtained from Sigma (Andover, U.K.).

Wortmannin was obtained from Calbiochem (Darmstadt,

Germany). Insulin and Protein A Sepharose beads were

obtained from Sigma (Andover, U.K.). IR and IRS antibodies

were procuredfrom BD Pharmingen (San Diego, CA, U.S.A.).

Rosiglitazone was a kind gift from Dr. Reddys Laboratories,

Hyderabad. Recombinant human PTP1B was obtained from

Biomol Research Laboratories, Inc. (PA, U.S.A.). Primers

were synthesized from GIBCO, BRL (Carlsbad, CA, U.S.A.).

All fine chemicals were obtained from Sigma (Andover,

U.K.). All other chemicals and organic solvents used were

of the highest analytical grade. Leaves ofC. intybus were

collectedfrom Trichy, Tamilnadu, India. Thecollected mate-

rial has been authenticated and the vouchers are stored at

the Department of Life sciences, Plant breeding and tissue

culture laboratory, Bharathidasan University, Trichy, Tamil-

nadu, India.

2.2. Cell culture of 3T3-L1 adipocytes

3T3-L1 preadipocytes (obtained from ATCC-CL-173) was

cultured in DMEM with 10% FBS and supplemented with

penicillin (120 units/ml), streptomycin (75g/ml), gen-tamycin (160g/ml) and amphotericin B (3g/ml) in 5%CO2 environment. 3T3-L1 preadipocytes grown in 24 well

plates until 2 days postconfluence and the cells were

induced by the differentiation medium (combination of

0.5mmol/l of IBMX, 0.25mol/l of DEX and 1 mg/l ofinsulin in DMEM medium with 10% FBS) to differentiate

into adipocytes. Three days after induction, the differentia-

tion medium was replaced with medium containing 1 mg/l

insulin alone. The medium was subsequently replaced

again with fresh culture medium (DMEM with 10% FBS)

after 2 days. By monitoring the formation of multinucle-

ation in cells, the extent of differentiation was measured.

2.3. Extraction and detannification

An authenticated, dried, pulverized leaves ofC. intybus

(100 g) was subjected to sequential extraction using differ-

ent solventswith increasing polarity (hexane, ethyl acetate,

and methanol) by cold maceration technique. The extractswere filtered and concentrated to drynessin vacuoat 45 C


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V.S. Muthusamy et al. / Chemico-Biological Interactions 174 (2008) 6978 71

to get a pasty mass of crude extract. The extracts were then

quantitatively reconstituted with DMSO and subjected for

2-deoxy-d-1-3[H] glucose uptake assay.

Detannified extract was prepared by previously

described gelatin procedure [11] with minor modifica-

tions. In the detannification process, 100 mg/ml extract

was prepared in methanol and centrifuged for 10 min at

4000 rpm and filtered with a 0.2

m filter; 40 mg/ml of

gelatin was added with the filtered extract in the ratio of

1:4, which was then centrifuged for 10 min at 400 0 rpm.

The supernatant was transferred to a new tube and the

pellet, which contained tannin and gelatin, was discarded.

This step was repeated twice to completely remove tannin.

The supernatant was collected in a new tube, and mixed

with same volume of 40 mg/ml gelatin, and centrifuged

at 8000 rpm for 20 min to remove residual tannin. The

resulting supernatant was transferred to a new tube and

the pellet discarded. A 5-fold volume of ethanol was added

to remove excess gelatin in the remaining extract; this

solution was then centrifuged at 8000 rpm for 10min, and

the final tannin-free supernatant was transferred to a new

tube and vacuum-dried. Methanolic (CME) and detannified

methanolic (CME/DT) extracts were subsequently tested

for glucose uptake activity.

2.4. Tannin estimation

The amount of tannins present in the extracts of

CME and CME/DT were estimated by Folin-Ciocalteu

colorimetric method [12] using Gallic acid as a stan-

dard. The quantitatively reconstituted extracts (1 mg/ml in

methanol) were incubated with Folin-phenol reagent in

the presence of alkaline medium (sodium carbonate) for

30 min at room temperature. The OD measurement was

taken at 640 nm for the estimation of tannin, and from the

calibration curve the tannin content was calculated.

2.5. Measurement of 2-deoxy-d-3[H] glucose

3T3-L1 adipocytes grown in 24-well plate (BD Falcon)

were subjected to glucose uptake as reported [13] with

minor modifications. After differentiation induction for the

stipulated period, the cellswere incubatedwithextracts for

24 h. The cells were then stimulated with Insulin (100 nM)

for 20 min followed by rinsing with Krebs Ringer phos-

phate HEPES (KRPH) solution (118 mM NaCl, 5 mM KCl,

1.3 mM CaCl2

, 1.2mM MgSO4

, 1.2 mM KH2

PO4

and 30mM

HEPESpH 7.4). The cells were subsequently pulsed for

20 min in KRPH solution containing 0.5Ci/ml 2-deoxy-d-3[H] glucose. The assay was terminated by aspirating

the medium. The cells were then washed thrice with ice-

cold KRPH solution and lysed in 0.1% SDS. The lysate was

transferred to a 96-well plate (Packard) with glass fiber

paper and air dried overnight. Theradioactivity of the sam-

ples was measured using a Top count liquid scintillation

counter (Packard, Ramsey, MN, U.S.A.). Glucose uptake was

also checked in the presence of 100 nM of Wortmannin

(WT)[14],a selective phosphatidylinositol 3-kinase (PI3K)

inhibitor and50M of Genistein (GS)[15], an IRTKinhibitor

to understand themechanism of action. All theassays wereperformed in duplicates for concordance. Results were

expressed as % glucose uptake with respect to solvent con-

trol.Rosiglitazone (50M) was used as thepositivecontrol.

2.6. Adipocyte differentiation assay

3T3-L1 preadipocytes differentiating in the presence

of CME and CME/DT were assessed for induced dif-

ferentiation over non-treated cells by measuring theaccumulation of triglycerides [16]. Triglyceride content

was measured using a commercially available kit (Adi-

poRedassay Reagent; Lonza Walkersville,Inc., Walkersville,

MD). Cells (5000 cells/well) were grown in 96-well plates.

Two days after attaining confluence, proliferation medium

was replaced with differentiation medium containing

as described above, with logarithmic doses of CME,

CME/DT ranging from 1 pg/ml to 10g/ml and Rosiglita-zone (50M). Rosiglitazone was used as a positive control.Alternatively, preadipocytes were maintained with fresh

FBS-DMEM every other day for the whole spectrum of

induction period. AdipoRed, a solution of the hydrophilic

stain Nile Red, is a reagent that enables the quantificationof intracellular lipid. Cells were washed with PBS (pH 7.4)

and 200l of PBS was added to the wells. 5l of AdipoRedreagent was added to each well. After 10 min, the plates

were placed in the fluorometer (Flouroskan Ascent, Ther-

moLab systems, Helsinki, Finland) and fluorescence was

measured with an excitation wavelength of 485 nm and

emission wavelength of 572 nm.

2.7. PTP1B Inhibition study

The phosphatase activity was assayed using 20 mM of

p-nitro phenyl phosphatase (pNPP) as substrate and car-

ried out in sodium acetate buffer, in a 96-well format[17].The reaction was initiated by the addition of recombinant

PTP1B enzyme in to CME, CME/DT and Sodium orthovana-

date (100ng, 1g and 100M, respectively) pretreatedwells and incubated for 30 min at 37 C. By measuring the

OD at 405 nm, the rate of PTP 1B-catalyzed hydrolysis of

substrate measured.The non-enzymatic hydrolysis of pNPP

was normalized by measuring theincreasein OD at 405nm

obtained in the absence of PTP1B enzyme.

2.8. Reverse transcriptase-polymerase chain reaction

RT-PCR was performed by according to the method

described previously[18]. In brief, 3T3-L1 adipocytes after

experimental incubation (15min for Insulin and 18 hr for

Rosiglitazone, CME and CME/DT) of samples were imme-

diately homogenized using total RNA isolation reagent

TRIzol reagent. The RNA obtained was then converted to

cDNA by reverse transcription and subjected to PCR with

specific primers for GLUT 4, PI3K, C/EBP, PPAR, SREBP-1c and GAPDH. PCR products were run on 1.5% Agarose

gels, stained with ethidium bromide and photographed.

PCR-products were consistent with the predicted sizes. To

determinethe efficacy, Insulin and Rosiglitazone were used

at 100nM and 50M dose, respectively, for all the markers[19,20]. The expression levels were quantitatedby scanning

on a gel documentation andanalysis system (ChemiImager4400, Alpha Innotech Corporation).


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2.9. Immunoprecipitation and Western blot analysis

Total cell lysates were prepared as reported previously

[21]with minor modifications. Anti-IRS-1 antibody (phos-

phorylated peptide of the tyrosine 891 mouse IRS) was

added to the eppendorf containing the cold lysates and

incubated at 4 C for 1h. 50l of the Protein A Sepharosebeads was activated by washing with cold lysis buffer

(500l) and centrifuged at 10,000g for 30s in twice.Finally, 50l of washed Protein A Sepharose slurry wasadded to total protein (125g) isolated form CME andCME/DT treated adipocytes and immunoprecipitated for

1h at 4 C on a rocking platform. Spin the eppendorf at

10,000gfor 1 min at 4 C for washing thrice with 500lof LysisBuffer.Afterthe last wash, 50l of 1Xlaemmli sam-ple buffer was added to bead pellet, vortexed and heated

to 90100 C for 10 min. Again, spin the sample for 5 min

at 10,000gfollowed by 10% SDS polyacrylamide gel elu-

tion. After run the gel was electrophoretically transferred

onto a nitrocellulose membrane at 120 mA for 90 min.

Whereas for IR

, total protein (100

g) isolated form CME

and CME/DT treated adipocytes were mixed with SDS sam-

ple buffer and heated at 90100 C for 10min followed by

10% SDS polyacrylamide gel elution and electrophoretical

transfer to a nitrocellulose membrane. Then, the mem-

brane was blocked by blocking agent (5% skimmed milk)

for overnight at 4 C followed by primary antibody (1:250

of anti-phospho IR) incubation for 2 h. After TBS wash-ing andsecondary antibody (ALPconjugate)incubation, the

blot was visualized using NBT/BCIP chromogenic agent and

photographed.

2.10. Assessment of cytotoxicity by lactate

dehydrogenase (LDH) release assay

Lactate dehydrogenase (LDH) release assay was per-

formed[22] using a cytotox 96 assay kit (Promega). This

assay quantitatively measures the LDH, a stable cytosolic

enzyme released during cell lysis. The assay was done with

0.2106 cells/0.2ml/well, seeded in 96-well cell culture

plates. Cells on treatment with different concentrations

(ranging from 10 ng/ml to 10g/ml) of extracts from C.intybusin 3T3-L1 adipocyte, was measured at a time point

of 24 h. 0.05% Triton X-100 was used to induce maximal

lysis. The plate was read at 492 nm in a scanning multiwell

spectrophotometer.

2.11. Statistical analysis

Data were expressed in meanS.E. Mean difference

between the groups was analyzed by one way analysis

of variance followed by Tukeys multiple comparison test

(using SPSS version 11.0 software, SPSS, Cary, NC, U.S.A.).

p < 0.05 was considered as statistically significant.

3. Results

3.1. Extraction and detannification

The shade dried leaves of C. intybus was sequen-tially extracted with hexane, ethyl acetate and methanol

Fig. 1. (a) Doseresponse glucose uptake analysisof hexane, ethyl acetate

andmethanolextracts ofC. intybus. (b) Doseresponseanalysis of CMEand

CME/DT on glucose uptake activity. Differentiated3T3-L1adipocytes,after

serum starvation were pre-incubated with described doses of extracts for

24 h. The cells were subjected to 15 min insulin stimulation (100nM) and

then 0.5Ci/ml of 2-deoxy-d-3[H] glucose was added for 20 min and theuptake was measured. The results were expressed as % change in glu-

cose uptake with respect to the solvent control (DMSO). Dose-dependent

uptake was observed with both the extracts in respect to solvent con-

trol. Data are meansS.E.,n =3. (*),p < 0.05 as compared with untreated

control group.

(with the extraction yield of 1.53, 2.21 and 10.1% w/w,

respectively). Methanolic extract ofC. intybus (CME) was

subjected to detannification and the efficiency of detan-

nification process was measured by tannin estimation.

Fig. 2. Influence of CME and CME/DT on the cytotoxicity of 3T3-L1

preadipocytes. Cytotoxic effect of CME and CME/DT measured on 3T3-

L1 cells during postconfluent mitotic clonal expansion. Postconfluent

3T3-L1 cells were treated with all indicated doses of CME and CME/DT.

After 24 h, LDH was measured in supernatants by using the formula %

LDH release = (ODSample ODcontrol)/(ODTritonX ODcontrol)100. C ytotoxi-

city was expressed as % LDH release. Data shown reflect the means S.E.of triplicates of two independent experiments.


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Fig. 3. Effect of CME and CME/DT on lipid accumulation in 3T3-L1 adipocytes after differentiation induction. (A) Two-day postconfluent 3T3-L1 cells

were differentiated according to the protocol followed by treatment with described doses of CME and CME/DT or vehicle for 2 days. Eight days after

induction of differentiation, cellswere assayed for total triglyceride content using Adipored reagent. Preadipocytes were separately maintained by protocol

as indicated. Data shown reflectthe meansS.E. of triplicates of two determinations. (*),p < 0.05 as compared with preadipocytes group. Photomicrographs

were documented to evaluate the morphological changes of adipocytes at magnification 100. (B) Control (C) differentiation-induced (D) differentiationmedium + CME (100ng/ml) (E) differentiation medium+ CME/DT (1g/ml).

The amount of tannins present in CME was found to be

2.78% w/w and after detannification (in CME/DT) the value

was come down to 0.31% w/w. The results clearly revealed

that the detannification process is more than 85% efficient.

3.2. Measurement of 2-deoxy-d-3[H] glucose

All sequential extracts of C. intybus were subjected to

radiolabelled glucose uptake assay (Fig. 1a). CME showed

maximal glucose uptake and hence it was selected forthe detannification process. A dose-dependent increase

(Fig. 1b) in glucose uptake was observed with CME and

CME/DTand concentrations of 100 ng/ml and1 g/ml wereobserved to be the optimal dose showing maximum glu-

cose uptake activity, respectively.

3.3. Assessment of cell cytotoxicity

The cell cytotoxicity of CME and CME/DT was studied at

higher doses ranging from 10 ng/ml to 10g/ml by means

of LDH release at 24 h. The result implies that the CME andCME/DT are non-toxic even at higher doses (Fig. 2).


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3.4. Adipocyte differentiation assay

Two-day postconfluence, 3T3-L1 preadipocytes were

induced for differentiation in the presence of logarith-

mic doses of CME and CME/DT ranging from 1pg/ml to

10g/ml. Intracellular lipid content was used to mea-sure the degree of adipocyte differentiation. As illustrated

in Fig. 3A, when CME is added to the preadipocytes in

the presence of differentiation medium, preadipocytes

remainedlargely undifferentiatedand thelipid contentwas

very basal. Surprisingly, in CME/DT treated preadipocytes

the lipid content was significantly increased. Cytotoxic-

ity assay by LDH release measurement revealed that the

preadipocytes treated with CME and CME/DT for the whole

spectrum of assay duration were more than 90% viable

(data not shown) and also the differences in morphology

of CME and CME/DT treated adipocytes were photomicro-

graphed at magnification 100 (Fig. 3BE).

Fig. 4. (a)Glucose transport behavior of CMEand CME/DTin thepresence

of Genistein(GS) IRTKinhibitor. (b) 3T3 adipocyteswere pretreated with

CME and CME/DT in addition of Wortmannin (WT) PI3K inhibitor at the

concentration indicated and subjected to glucose uptake assay. Inhibitors

suppressed the glucose transport of CME but not CME/DT, which showed

uptake similar to that of positive control Rosiglitazone. The results were

expressedas % change in glucose uptake withrespectto thesolventcontrol

(DMSO). Data are meansS.E. of triplicates of two independent experi-ments. (*),p < 0.05 as compared with untreated control group.

Fig. 5. Effect of CME and CME/DT on inhibition of PTP1B enzyme. The

effect of CME (100 ng/ml) and CME/DT (1g/ml) on PTP1B enzyme inhi-bition was observed. CME showed maximal inhibition of 92% similar to

positivecontrol sodium orthovanadate. CME/DT exhibited negligibleinhi-

bition of PTP1B enzyme. Data shown reflectthe meansS.E. of triplicates

of two independent experiments.

3.5. Measurement of 2-deoxy-d-3[H] glucose uptake in

the presence of PI3K and IRTK inhibitor

Our study reveals that the glucose uptake behavior of

CME, after treatment with 50M and 100nM of GS andWT, respectively, in 3T3-L1 adipocytes, arrested the insulin

mediated glucose uptake.But, the inhibitors did not control

the glucose uptake activity of CME/DT (Fig. 4aand b).

3.6. PTP1B inhibition assay

Asshownin Fig. 5, CME exhibits potent PTP1Binhibition

(92%) similar to the positive control sodium orthovanadate

(100M).But, CME/DTshowed only basal level PTP1B inhi-bition.

3.7. Effect of CME and CME/DT on insulin signaling

cascade

After demonstrating the effect of CME and CME/DT on

PTP1B inhibition, the study was further extended to deter-

mine the modulations of downstream markers involved in

glucose uptake such as IR and IRS1 at protein level bywestern blotting and GLUT4 and PI3K at transcript level by

RT-PCR. The levels of GLUT4 and PI3K were expressed after

normalization with GAPDH (Fig. 7E) expression. Relative

densitometry scanning was performed for semiquantita-

tive analysis (Figs.6C, D,7Band D).

Immunoprecipitation and western blot analysis (Fig. 6A

and B) revealed that the IR and IRS1 which are impor-tant markers involved in the early stage of insulin signaling

event, were significantly phosphorylated after cells were

treated with CME on 3T3-L1 adipocytes using phospho

specific antibodies. Whereas CME/DT treated cells showed

basal level expression of IR and IRS1. Consequently theexpression of PI3K(Fig.7A) at18 h post-treatmentof 3T3-L1

adipocytes with CME (100 ng/ml) was found to be sig-

nificant. However, the adipocytes treated with CME/DT

failed to upregulate the PI3K expression, which is similar

to that of Rosiglitazone a negative control for the PI3Kmediated glucose uptake. In GLUT4 expression our find-


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Fig. 6. Effect of CME and CME/DT on IRand IRS1 expression. (A) 3T3-L1 adipocytes pretreated with CME for 24 h showed phosphorylation of IRandIRS1 on parwith positive controlinsulin (100 nM) (B) CME/DTfailed to induce the phosphorylation. Bars represent the meansS.E. (n = 3) of densitometric

representation of IR and IRS1 expressions (C and D). (*), p < 0.05 as compared with untreated control group.

ing (Fig. 7C) shows that 3T3-L1 adipocytes treated with

CME (100 ng/ml) and CME/DT (1g/ml) is upregulated and

equivalent to positive control, Insulin (100 nM) and Rosigli-tazone (50M).

3.8. Effect of CME and CME/DT on key genes involved in

adipocytes differentiation

The effect of CME (100 ng/ml) and CME/DT (1g/ml)extracts on C/EBP, PPARand SREBP-1c expression in 3T3-L1 adipocytes is presented in Fig. 8A, C and E. CME/DT

stimulated significant expression of all the three genes

as compared to positive control Rosiglitazone (50uM)

whereas CME treated adipocytes showed basal expres-

sion.

4. Discussion

In the present study, the glucose uptake and antiadi-pogenic activities of CME and CME/DT have been observed

along with confirming the duality (PTP1B inhibitor and

PPAR agonist) of C. intybus in exhibiting anti-diabeticactivity. The crude extracts of Aegles marmelos, Syzygium

cuminiand Pterocarpus marsupium were reported for glu-

cose uptake activity via PI3K and PPAR on L6 myotubes[23,20],which supports our data for the duality observed

in glucose uptake activity of C. intybus. Pushparaj et al.

[7]described that the treatment with ethanolic extract of

C. intybus in streptozotocin-induced diabetic rats reduces

the blood glucose, triglycerides and cholesterol levels sig-

nificantly. It is a clear evidence to justify our findings on

adipogenesis inhibition.

Fig. 7. Effect of CME and CME/DT on PI3K and GLUT4 mRNA transcripts. 3T3-L1 adipocytes were incubated with Insulin (100 nM), Rosiglitazone (50M),CME(100ng/ml) andCME/DT (1g/ml) for indicatedtime.Lane 16indicates untreated control, Insulin, Rosiglitazone, CME, CME/DTtreated cells andPCRnegative control, respectively. (A) The expression levels of PI3K transcripts (248 bp), compared with control cells. Insulin and CME showed the significant

expression whereas Rosiglitazone treated cells and CME/DT treated cells showed basal expression of PI3K. (C) Illustrates the elevated levels of GLUT4

transcripts (318bp) in all the lanes (6E). The mRNA of GAPDH (588 bp) in the same sample was used as a loading reference. Bars represent the meansS.E.(n = 3) of densitometric representation of PI3K, and GLUT4 expressions (B and D). (*),p < 0.05 as compared with untreated control group.


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Fig. 8. Effect of CME and CME/DT on genes involved in adipogenesis. CME inhibited adipocyte differentiation by altering the expression of key genes

involved in adipogenesis. Maximal expression of C/EBP(224 bp), PPAR(155bp) and SREBP1c (222 bp) were observed with Rosiglitazone and CME/DTtreated cells whereas insulin and CME failed to upregulate the expression on comparison with untreated cells (A, C and E). Bars represent the meansS.E.

(n = 3) of densitometric representation of gene expressions (B, D and F). (*),p < 0.05 as compared with untreated control group.

Liu et al. [10] have reported that the tannins are the

imperative molecules in natural products especially for

treating metabolic syndromes like diabetes. In their study,

they have concluded that the tannins of banaba aqueous

extract(Lagerstroemia speciosa) are the compoundsrespon-

sible for glucose transport and adipocyte differentiation

inhibitory activities. However, in our finding, significant

glucose uptake was observed even after removing tannins

from CME. Since the observation differed from the earlier

reports, we hypothesized that the tannins are sparingly

extractable with methanol than water so that their quan-

tity and role might be insignificant in thebiological activity

of CME.

We then studied the effects of CME and CME/DT

on suppressing preadipocyte differentiation. Adipogen-

esis is often depicted by a cascade of genetic events

and are controlled by various factors. The factors which

include insulin-like growth factor I (IGF-1), macrophage

colony-stimulating factor, fatty acids, prostaglandins and

glucocorticoids promote adipogenesis by activating a

cascade of transcription factors that coordinate the dif-

ferentiation process [24]. To stimulate adipogenesis of

preadipocyte cell lines, a combination of adipogenic induc-

ers, including a glucocorticoid agonist, an agent to increase

intracellular cAMP and high concentrations of insulin to

stimulate IGF-I, is generally used[25].In adipocyte differ-

entiation assay, CME possesses inhibitory activity but not

CME/DT. This is an interesting observation, which coincides

with earlyreports on inhibitory role of tannins in adipocyte

differentiation. This led us to investigate the mechanism

used by tannins of CME in glucose uptake and adipogenesis

inhibition.

Inhibitors of insulin mediated glucose transport were

incorporated with CME and CME/DT to further substanti-

ate the signaling pathway. In normal as well as in diabetes

conditions, the protein tyrosine kinases play a crucial role

in numerous cellular signaling pathways. It is essential

to activate the tyrosine kinase by insulin, leading to the

phosphorylation of its receptors thus inducing a functional

change in the immediate signaling molecule such as PI3K

[26]. Genistein (GS) a reported insulin receptor tyro-

sine kinase (IRTK) inhibitor controls the insulin stimulated

glucose uptake[27], and Wortmannin (WT) an identi-

fied inhibitor of PI3K inhibiting the insulin-stimulated PI3K

activity and GLUT4 translocation [28] were selected and

used for the assay. The finding from the inhibitor study

suggested that the mechanism of CME is through insulin

mediatedPI3Kdependent manner whereas the mechanism

of CME/DT is through PI3K independent manner.

Current researchers have demonstrated the possible

association of PTP1B in insulin sensitivity and obesity [29].

Insulin mediated glucose uptake is negatively regulated by

protein tyrosine phosphatases (PTPs). Among several PTPs,

PTP1B has been implicated to be a key modulator of insulin

signal transduction by acting at downstream signaling

components, such as IRS1 and PI3K [30].Over expression

of PTP1B protein have been reported in insulin-resistant

conditions which are associated with obesity [31]. Since,

PTP1B plays a pivotal role in insulin resistance and adi-

pogenesis, the findings of PTP1B inhibition assay reasons

the glucose uptake and adipogenesis inhibitory activities

of tannins present in CME.

Insulin binding to its transmembrane insulin receptor

(IR) activates IRTK, which then causes phosphorylation


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of family of insulin receptor substrate (IRS) proteins on

selective tyrosine residues[32].From the insulin-signaling

cascade, it is apparent that the activation of PI3K is nec-

essary and in some cases sufficient to understand many of

insulins effects on glucose and lipid metabolism. In adi-

pose tissue, insulin exerts its normal cellular effects via

PI3K by first binding to its receptor followed by the activa-

tion of IRS[33].Finally, insulin mediated glucose uptake in

muscle and adipose tissue takes place through the translo-

cation of an integralmembrane, glucose transporter protein

GLUT4, from an intracellular compartment to thecell sur-

face[34].The IR, IRS1, PI3K and GLUT4 expression resultsconfirm that the anti-diabetic activity of CME and CME/DT

is through GLUT4 endocytosis but via bi-functional mech-

anism.

Peroxisome proliferator activator receptor (PPAR),

CCAAT/enhancer binding protein (C/EBP), and sterol reg-

ulatory element binding protein (SREBP) families are the

well-documented primary adipogenic transcription fac-

tors involved in adipocyte differentiation and among them

PPAR

is most extensively studied for its therapeutic utility

in the treatment of NIDDM[35].CME controls the expres-

sion of all the three adipogenic markers and similar results

were reported for dietary flavonoids by Chien et al. [19].

The unique expression of SREBP-1c on adipocytes treated

with insulin is reasoned by the fact that lipogenesis action

of insulin is through SREBP. Cristina et al. [36]showed a

decrease in adiposity with a down regulation of PPAR,SREBP1 and otheradipogenicgenes in animals treated with

PTP1B antisense oligonucleotide, suggesting that PTP1B

reduction might have a primary role in inhibiting adipo-

genesis and the development of obesity in these animals.

Our results on PTP1B inhibition and mRNA expression of

adipogenic genes reason the glucose uptake activity and

adipocyte differentiation activity of CME. At the same time,

the expression of adipogenic markers increased signifi-

cantly after detannification (in CME/DT). The activation of

PPAR by CME/DT explains its glucose uptake activity andGLUT4 mRNA expression and enlightens the significance of

tannins in the biological activity of CME.

The two activities, glucose uptake and adipogenesis

inhibition, were mediated by two independent factors

insulin receptor (IR) and IGF1 but controlled by the only

linkage PTP1B enzyme. Therefore, the inhibition of PTP1B

enzyme activity by the tannins present in CME rationalizes

its bi-functional activity. In theory, CME should not exhibit

adipogenesis inhibition due to the presence of PPAR

acti-

vator (from non-tannin fraction) because blends of two

independently functioning molecules will behave with the

additive mechanism of two molecules. But, our finding dif-

fersfromthehypothesis.Thisbringsustoanewproposition

that the interaction of tannins present in CME on PTP1B

inhibition may be very potent than the interaction of non-

tannins present in CME/DT with PPAR agonism.

5. Conclusion

Tannins are freely available in our daily diet and till

now the usefulness of tannins are limited to only neu-

traceutical level as a biological antioxidant. To the best ofour knowledge, it is for the very first time that we are

reporting the therapeutical significance of C. intybus and

its tannins. The ability of existing therapy to target var-

ious aspects of the insulin resistance syndrome induces

other metabolic abnormalities chiefly in lipids. Therefore,

glucose-lowering drugs deficient of adipogenic activity are

the need for the current situation. The tannins ofC. intybus

methanolic extract seem to have such a beneficial combi-

nation. IfC. intybusis identified for the presence of PTP1B

inhibitor and PPARactivator at the molecular level, thenit will also be interesting to find the possibility of par-

tial PPAR agonism/antagonism in future. The propositionwould become apparent once the CME and CME/DT are

completely evaluated. Pharmaceutical significance of the

present analysis depends on the lead molecule identifica-

tion and in vivo animal studies, which are necessary for

further exploration.

Acknowledgements

Authors would liketo thank Dr. R.B. Narayanan, Director,

Centre for Biotechnology, Anna University, Chennai, India.

The valuable suggestions of Dr. M. Ramanathan and Mr.

C. Saravanababu also acknowledged. Muthusamy.V.S is a

CSIR-Research Associate and is highly grateful to Council

of Scientific and Industrial Research, Govt. of India for the

financial assistance.

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