Roles of insulin receptor substrates in the action of green tea (-)-epigallocatechin gallate on insulin-stimulated growth of

3T3-L1 preadipocytes

國 立 中 央 大 學

生 命 科 學 研 究 所

碩 士 論 文

胰島素接受器受質在綠茶唲茶素對胰島素

刺激前脂肪細胞生長作用中扮演的角色

研 究 生：古惠珍 (Hui-Chen Ku)

指導教授：高永旭 (Yung-Hsi Kao) 博士

中 華 民 國 九 十 六 年 七 月

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研究生簽名: 古惠珍 學號：942204009

論文名稱: 胰島素接受器受質在綠茶唲茶素對胰島素刺激前脂肪細胞生長作用中扮

演的角色

指導教授姓名：高永旭

系所 ： 生命科學 所 博士班 碩士班

日期：民國 96 年 7 月 20 日

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I

Abstract Obesity is a common disease, and it was associated with risk of cancer, diabetes,

hypertention, and cardiovascular disease. Development of obesity results from increases

in the cell number and fat accumulation of adipocyts, and is regulated by endocrine and

nutritional factors. Insulin regulates mitogenesis and adipogenesis of fat cells and

stimulates lipogenesis and inhibits lipolysis. By contrast, green tea catechins (once called

vitamin P), especially (-)-epigallocatechin gallate (EGCG), inhibits proliferation and

differentiation of fat cells and induces the apoptosis of preadipocytes and adipocytes.

EGCG has been proposed as a chemopreventative for obesity and diabetes. However,

relatively little is known about the mechanism of the action of EGCG on

insulin-stimulated fat cell function. This study was designed to investigate the pathways

of EGCG's modulation of the insulin-stimulated mitogenesis of 3T3-L1 preadipocytes.

EGCG decreased insulin-induced increases in levels of phospho-insulin receptor (pIR),

phospho-insulin receptor substrates 1, 2, and 3 (pIRS1, 2, and 3), phospho-Raf1,

phospho-MEK1/2, and phospho-Erk1/2 proteins and induced further insulin-decreased

pIRS4 level. In addition, EGCG reduced the insulin-stimulated associations of IR with

IRS1 and IRS2. EGCG also reduced the association of IRS proteins with downstream

signaling proteins. Besides, pretreatment with 67-kDa laminin receptor could antagonize

the effect of EGCG on insulin-induced pMEK1/2 and pIRS2. These data suggest that

EGCG exerts its antimitogenic effects via inhibiting insulin signaling by the 67-kDa

laminin receptor-dependent pathway. Moreover, EGCG was more effective than

epicatechin, epicatechin gallate, and epigallocatechin in changing the insulin-stimulated

mitogenic signals. Results of this study may relate to the mechanism by which EGCG

modulates insulin-related preadipocytes.

II

中文摘要

肥胖是一種常見的疾病，與癌症，糖尿病，高血壓以及心血管疾病都有相關。

造成肥胖的原因是脂肪細胞數目的增加，或脂肪細胞內三酸甘油酯的堆積。根據研

究指出，內分泌與營養都會影響到肥胖的調節作用，其中胰島素在細胞分化時期會

刺激細胞進行增生和 adipogenesis，並促使細胞進行脂肪生成和抑制脂肪分解作

用。然而綠茶唲茶素(亦稱維生素 P)，尤其是綠茶表沒食子酸酯型唲茶素酸酯(英文

名稱和簡稱分別為 (-)-epigallocatechin gallate 和 EGCG)，已被證實可以抑制脂

肪細胞的增生與分化，並可促使前脂肪細胞和脂肪細胞的 apoptosis，所以可作為

預防肥胖的保健物質。因此，EGCG 是否可調節胰島素刺激前脂肪細胞的增生仍然不

清楚。在本論文中，使用 3T3-L1 前脂肪細胞株做為研究素材，我發現胰島素濃度

100 nM，胰島素可分別增加胰島素接受器、IRS1、IRS2、IRS3、Raf1、MEK 和 Erk

蛋白的磷酸化，但減少 IRS-4 蛋白的磷酸化，然而前處理 EGCG (20μM)於前脂肪細

胞可以明顯抑制胰島素所促進訊息蛋白的磷酸化及胰島素接受器與 IRS-1 和 IRS-2

兩者之間的結合作用，並且可以明顯抑制胰島素所促進 IRS 蛋白質和下游訊息蛋白

質之間的結合作用。此外，前處理層粘連蛋白接受器的抗體(67-kDa laminin

receptor；被認為是一種 EGCG 接受器)，可以中和 EGCG 抑制胰島素所促進訊息蛋

白 MEK 和 IRS2 的磷酸化。以上種種的跡象顯示，EGCG 可透過阻礙胰島素的訊息傳

導路徑而抑制前脂肪細胞的增生，並且 EGCG 可透過層粘連蛋白接受器干擾胰島素

的訊息傳導作用。此外，EGCG 比其他綠茶唲茶素干擾胰島素刺激增生更有效果。此

篇研究結果可以說明在前脂肪細胞中，EGCG 調節胰島素訊息的作用機制。

III

Acknowledgements Firstly, I thank Professor Yung-Hsi Kao for his advice in pursuing my

master degree. With his supervision and guidance, I have acquired

invaluable knowledge.

Secondly, I thank Professors Rong-Nan Huang, Shiow-Lian Catherine Jin,

Chien-Chia Wang, and Shen-Liang Chen as my committee members for my

oral examination.

I extend my grateful thanks to the lab partners in the Professor Kao’s lab

due to their technical assistance. Finally, I thank my beloved family for their

support and understanding in the course of my past and future studies.

IV

Abstract Ⅰ 中文摘要 Ⅱ Acknowledgements Ⅲ Contents Ⅳ List of figures Ⅴ Abbreviations Ⅵ Introduction 1 Materials and Methods 4 Results 8 Discussions 12 References 14 Appendix 39

V

List of figures

Fig. 1 Chemical structures of four major green tea catechins. 17 Fig. 2 Effect of EGCG on insulin-induced phosphorylation of insulin 18

receptor substrates (IRSs) and insulin receptor was dose dependent Fig. 3 Effect of EGCG on insulin-induced phosphorylation of insulin 20

receptor substrates (IRSs) and insulin receptor was time dependent

Fig. 4 Inhibitory effect of EGCG on insulin-induced phosphorylation of 22 insulin receptor substrates (IRSs) in the 3T3-L1 preadipocytes

Fig. 5 Inhibitory effect of EGCG on insulin-induced levels of 23 pRaf1, p MEK, and p ERK proteins

Fig. 6 Catechin-specific effect of green tea on insulin-stimulated signaling 25

Fig. 7 EGCG reduced insulin-stimulated interactions of insulin receptor 27 with IRS1 and IRS2 proteins

Fig. 8 Effect of EGCG on insulin-stimulated interactions of IRS1 with 28

Ras, pRaf1, and pMEK

Fig. 9 Effect of EGCG on insulin-stimulated interactions of IRS2 with 30 Ras, pRaf1, and pMEK

Fig.10 Effect of EGCG on insulin-mediated interactions of IRS3 with 32

insulin signaling proteins Fig. 11 Effect of EGCG on insulin-mediated interactions of IRS4 with 34

insulin signaling proteins

Fig. 12 Effect of EGCG on insulin-mediated interactions of IRS proteins with 36 laminin receptor

Fig. 13 Laminin receptor antibody antagonized the effects of EGCG on 38

insulin-stimulated pMEK and pIRS2 levels

VI

Abbreviations

BrdU 5-bromo-2’-deoxyuridine

BSA Bovine serum albumin

CS Calf serum

DMEM Dulbecco’s Modified Eagle Medium

DMSO Dimethyl sulfoxide

EC Epicatechin

ECG Epicatechin gallate

EGC Epigallocatechin

EGCG Epigallocatechin gallate

EDTA Ethylene Diamine Tetraacetic Acid

Erk Extracellularly regulated kinase

GTC Green tea catechin

IP Immunoprecipitation

IR Insulin receptor

IRS Insulin receptor substrate

67-kDa LR 67-kDa laminin receptor

MEK Mitogen-activated protein kinase kinase

PBS Phosphate buffered saline

PMSF Phenylmethylsulfonyl fluoride

SDS Sodium dodecyl sulfate

TEMED N,N,N,N-tetramethyl-ethylenediamine

Tween 20 Polyoxyethylenesorbitan monolaurate

1

Introduction

Insulin Insulin, a polypeptide hormone with a molecular weight of about 5800 Daltons, is

produced by β cells of pancreas. It is composed of two peptide chains referred to as the

A chain and B chain linked together by two disulfide bonds. In most species, the A chain

consists of 21 amino acids and the B chain of 30 amino acids. Insulin is a key hormone

regulating growth, development and homeostasis of glucose, fat, and protein metabolism

(1,2). Insulin resistance is a usual pathological condition in which target cells are unable

to respond to regular levels of circulating insulin. Human with insulin resistance are very

subject to developing type 2 diabetes, and insulin resistance is often associated with

several health disorders, including obesity, hypertension, chronic infection, and

cardiovascular diseases (3).

Insulin signal transduction Insulin acts by binding to its transmembrane receptor (IR), which is a tyrosine kinase

having an α2β2 configuration. Insulin binding to the α subunits induces conformational

changes and thereby resulting in stimulation of the β subunits tyrosine kinase activity

through autophosphorylation of Tyr residue in the β subunits (1,4). The activated insulin

receptor then phosphorylates substrate proteins, such as Shc, Gab-1, Cbl/CAP, and the

family of insulin receptor substrate (IRS) proteins, on selective Tyr residues that serve as

docking sites for downstream molecules. This elicits two main kinase signaling

pathways – the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase

(PI3K) pathways. These two kinase pathways mediate the metabolic and

growth-promoting functions of insulin, such as translocation of GLUT4 glucose

transporters, stimulation of glycogen and protein synthesis, and initiation of specific gene

transcription (5,6). Whether one of the pathways can explain for the the green tea

catechin regulation of insulin signaling will require further studies.

2

Insulin receptor substrate proteins The family of insulin receptor substrates contains nine members: (i) four insulin

receptor substrate (IRS) proteins (IRSI, IRS2, IRS3 and IRS4), (ii) 3 Src - Homology

-collagen (SHC) proteins, (iii) growth factor receptor bound-2 (Grb-2) associated binder-I

(Gab-I), and (iv) p62dok (4). All IRS proteins are characterized by an NH2-terminal

pleckstrin homology (PH) domain followed by a phosphotyrosine binding (PTB) domain

and a domain of variable length consisting of considerable potential tyrosine

phosphorylation sites (7-9). The molecular weights of the IRS proteins are 185 kDa for

IRSI, 190 kDa for IRS2, 60 kDa for IRS3 and 160 kDa for IRS4, respectively. To date

mice lacking IRS-1, IRS-2, IRS-3 and IRS-4 have been described (10). IRS1-deficient

mice have growth retardation and insulin resistance (11, 12). IRS2 KO mice are diabetic

(13). IRS3-null mice have no glucose metabolism or growth problem (14). Mice lacking

insulin receptor substrate 4 exhibit mild defects in growth, reproduction, and glucose

homeostasis (15). Whether any of IRS proteins is involved in the green tea catechin

regulation of insulin signaling pathway in 3T3-L1 preadipocytes was relatively little

known.

EGCG and preadipocytes Green tea, a beverage generally consumed in Asian countries, is a vital source of a

kind of flavonoids called catechins. Green tea catechins primarily include

(-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin gallate, and

(-)-epicatechin. They have different characteristic chemical structures (Fig. 1) and

biological functions (16-22). For instance, EGCG lowers the incidences of cancers (17,

18, 20), oxidative stress-induced neurodegenerative diseases (23), collagen-induced

arthritis (24), and cytokine-induced inflammation in vivo (17). In addition, EGCG can

reduce body weight and body fat (25, 26). In spite of the significance of EGCG and other

tea catechins, little is known about the mechanism of their action in regulating the

mitogenesis of preadipocytes. The fact that the EGCG receptor, the so-called 67-kDa

laminin receptor, discovered in cancer cells (27), has not been identified in fat cells and

the fact that fat cells have different isoforms of laminins (28) has also caused many

3

controversies. Thus, a complete examination of the signal element through which EGCG

performs its regulation of preadipocyte mitogenesis should help make these observations

clear.

Preliminary observations Previously we observed the followings (22): (1) Insulin-stimulated mitogenesis, as

indicated by changes in the cell number and BrdU incorporation, of 3T3-L1

preadipocytes is inhibited by EGCG in dose-dependent manner. (2) EGCG decreased

insulin-stimulated MEK1 activity in a dose-dependent manner in 3T3-L1 preadipocytes.

(3) EGCG decreased insulin-induced increases in levels of phospho-insulin receptor β

and phospho-insulin receptor substrate 1. (4) EGCG reduced the insulin-stimulated

associations of IR with IRS1. (5) EGCG was more effective than epicatechin, epicatechin

gallate, and epigallocatechin in reducing the insulin-induced increase in MEK activity.

Despite the importance of EGCG in regulating adipocyte activity, the exact mechanism

of its action in mediating insulin signaling in the mitogenesis of fat cells was still not

clear.

Objectives The present study was designed to examine the mechanism by which EGCG acts in

modulating insulin signaling in the mitogenesis of 3T3-L1 preadipocytes. Our specific

aim was to investigate whether IRS proteins other than IRS1 is involved in the EGCG

modulation of the insulin signaling pathway. Because the 67 LR was discovered as an

EGCG receptor in cancer cells, further study as to whether it mediates the EGCG

regulation of insulin signaling in preadipocytes was also addressed.

4

Materials and Methods

Chemical reagents Green tea EGCG and other catechins (>98% pure) were isolated from green tea

(Camellia sinensis) in our laboratory as described previously (28). Catechins were

dissolved in 0.1% DMSO. Other materials (i.e., penicillin G, streptomycin, etc) were

purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM),

fetal bovine serum (FBS), calf serum, penicillin-streptomycin and trypsin were purchased

from GibcoBRL of Life Technologies (New York, NY). BenchMarkTM Pre-Stained

Protein Ladder was purchased from Invitrogen Life Science Technologies (Carlsbad, CA).

TEMED, 2-mercaptoethanol, and EDTA were purchased from J. T. Baker. 1,

4-dithiothreitol, and Triton X-100 were purchased from Merck. Bio-Rad protein assay

dye reagent and Coomassie blue R-250 were purchased from Bio-Rad. Except laminin

receptor antibody (#GCA1016B95b; Genesis Biotech Inc, Taiwan), all antibodies (β-actin,

sc-1616; IRS1, sc-8038; IRS2, sc-1555; IRS3, sc-28831; IRS4, sc-28830; pIRS1,

sc-17200; pIRS1/IRS2, sc-17195-R; p-tyrosine, sc-7020; pMEK, sc-; pRaf1, sc-28005;

Ras, sc-35; laminin R, sc-20979; IGFI R, sc-462; IR,sc-09; pIR, sc-25103; donkey

anti-rabbit IgG-HRP, sc-2313; donkey anti-mouse IgG-HRP, sc-2005; donkey anti-goat

IgG-HRP, sc-2020; goat anti-rat IgG-HRP, sc-2006; normal mouse serum, sc-45051;

protein A/G plus agarose, sc-1616) were purchased from Santa Cruz Biotechnology

(Santa Cruz Biotechnology, Inc, CA). Insulin, acrylamide/ bis- acrylamide, trypan blue,

DMSO and BSA were purchased from Sigma.

Cell culture Murine 3T3-L1 cell line was purchased from American Type Culture Collection

(ATCC, Manassas, VA). The preadipocytes were grown in DMEM (pH 7.4). containing

10% heat-inactivated (56°C, 30 min) CS, 100 units/ml penicillin G, and 0.1mg/ml

streptomycin under the humidified condition of 95% air and 5% CO2 at 37°C. 3T3-L1

cells were plated at a density of 12,000-15,000 per cm2 of a 10-cm plate (29), and then

subcultured every 2-3 days before the confluent. In this study, 3T3-L1 cells at the second

5

day of culture (log phase of 3T3-L1 development) were starved for overnight in

serum-free DMEM, and then pretreated with catechins at a varying dose( 0-50 uM) and

duration( 0-24h). After the treatment, cells were stimulated with 100 nM insulin for 5

min.

Protein determination Protein content of the cell lysates was determined in duplicate by the protein-dye

binding (32) microassay method using a microtiter plate reader. The reaction mixture

contained 10 µl of lysates and 200 ul of diluted (1:4, v/v) dye solution (Bio-Rad Protein

Assay). After 10 min incubation, the absorbance of the reaction was read at 595 nm. A

linear range of BSA standard was observed from 0.25 to 5 µg per well.

Western blot analysis Western blotting analysis was performed as described by Hung et al. (30). 3T3-L1

preadipocytes were grown in 10 cm plates. The cells were washed twice with 10 mM

PBS and lysed in the lysis buffer containing 20 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1

mM Na3VO4, 0.2% Triton-X100, and 1 mM phenylmethylsulfonyl fluoride (PMSF).

Lysate was agitated for 15 min at 4°C and their supernatant were collected after the

centrifugation at 14,000 rpm and 4°C for 10 min. Protein concentration of the supernatant

was determined with Bradford reagent. Samples were then diluted by adding the same

volume of 2× sample buffer containing 100 mM Tris-HCl (pH6.8), 4% SDS, 20%

glycerol, 0.2% bromophenol blue, and 10% β-mercaptoethanol (Sigma). Equal amounts

of protein (50 µg) were electrophoresed by denaturing 12% SDS-PAGE. Proteins were

then blotted onto Immobilon-NC transfer membranes (Millipore Corporation, Bedford,

MA). The membranes were blocked with 5% nonfat dried milk in PBS-0.1% Tween 20

(polyoxyethylenesorbitan monolaurate, Sigma) for 1 h. After being washed with PBST

for 3 times, the membranes were incubated with primary antibodies (diluted in blocking

buffer) for 1 h at room temperature or overnight at 4 °C. Primary antibodies used for

Western blotting were diluted as follows: anti-pRaf1, 1:1000; anti-pMEK, 1:1000;

anti-pERK, 1:1000; anti-pIRS1, 1:500; anti-pIRS1/2, 1:1000; anti-IRS3, 1:1000;

6

anti-IRS4, 1:1000; anti-IR, 1:500; anti-pY, 1:500; anti-laminin R, 1:500; anti-Ras, 1:500;

anti-β-actin, 1:2000. Secondary antibodies (i.e. horseradish peroxidase-conjugated

donkey anti-goat IgG, donkey anti-mouse IgG, donkey anti-rabbit IgG, and goat anti-rat

IgG )were diluted in blocking buffer at a final ratio of 1:3,000. The membranes were

washed three times prior to detection using the Western Lightning chemiluminescence

Reagent Plus kit (PerkinElmer Life Science, Inc. MA). The relative amounts of these

proteins were normalized with β-actin.

Immunoprecipitation We followed the method as described by Hung et al. to immunoprecipitate insulin

receptor, IRS1, IRS2, IRS3, and IRS4 after 3T3-L1 preadipocytes were washed twice

with PBS and lysed in the lysis buffer containing 20 mM Tris-HCl (pH 7.6), 1 mM

EDTA, 1 mM Na3VO4, 0.2% Triton-X100, and 1 mM PMSF (30). Lysate was agitated

for 15 min at 4°C. After the centrifugation at a force of 14,000 rpm and 4°C for 10 min,

the supernatant was collected and determined with protein concentration by Bradford

reagent. An aliquot of the supernatant (1 mg of protein) was preincubated with 10 µl

mouse polyclonal insulin receptor antibody or preimmunized normal mouse serum (NMS;

as the control) for 1 hr at RT or overnight at 4°C. Then, they were precipitated with the

addition of 20 µl Protein A-agarose (Santa Cruz Biotech). Western blot analysis was

performed with IRS1, IRS2 , IRS3, IRS4, Ras, pRaf1, pMEK1/2, and 67-kDa laminin

receptor antibodies, respectively.

Insulin preparation Human insulin ( molecular Weight: 5807.57, molecular formula : C257H383N65O77S6,

sigma) was solubilized in 0.01N HCl solution and is sterile filtered. The final

concentration of insulin stock solution was 100 μ M. A stock solution can be stored

frozen at -20°C in a single-use aliquot. Freeze-thaw cycles should be avoided.

Statistical analysis Unpaired Student’s t test was used to examine differences between chemical-treated

7

groups and non-treated control. ANOVA and Student-Newman-Keuls multiple range test

were used to examine differences among various groups. Significance was set as P<0.05

or P<0.01. All results are presented as the mean±SE. All statistics were performed using

Sigmaplot.

8

Results

Effect of EGCG on insulin-induced phosphorylation of insulin receptor

substrates (IRSs) and insulin receptor was dose dependent in 3T3-L1

preadipocytes To test whether the inhibitory effect of EGCG on the insulin-induced phosphorylation

of IRS proteins and insulin receptor was dose dependent, we treated cells with different

concentrations of EGCG (10, 20, and 50 uM), followed by stimulation with 100 nM

insulin for 5 min. EGCG dose-dependently inhibited insulin-induced levels of

phospho-IRS1 (Fig. 2A), phospho-IRS2 (Fig. 2B), and phospho- insulin receptor (Fig.

2C).

Effect of EGCG on insulin-induced phosphorylation of insulin receptor

substrates (IRSs) and insulin receptor was time dependent in 3T3-L1

preadipocytes To know whether the inhibitory effect of EGCG on insulin-induced phosphorylation of

IRS proteins and insulin receptor was time dependent, we treated cells with EGCG for

different time periods (30 min, 60 min, 90 min, 3h, 6h, 12h and 24h), followed by

stimulation with 100 nM insulin for 5 min. EGCG time-dependently inhibited

insulin-induced levels of phospho-IRS1 (Fig. 3A), phospho-IRS2 (Fig. 3B), and phospho-

insulin receptor (Fig. 3C).

Inhibitory effect of EGCG on insulin-induced phosphorylation of insulin

receptor substrates (IRSs) in the 3T3-L1 preadipocytes We assessed whether EGCG inhibited the insulin-stimulated phosphorylation of other

IRS proteins (Fig.4). Pretreatment with 20 μM EGCG resulted in a significant decrease

in the amount of phospho-IRS-1, but not total IRS-1 protein induced by insulin. EGCG

significantly decreased insulin-induced levels of phospho-IRS2 (Fig. 4B) and

phospho-IRS3 (Fig. 4C), but further decreased insulin-inhibited phospho-IRS4 levels.

9

(Fig.4D). However, none of total IRS2 and IRS3 proteins were altered by insulin

treatment in the presence or absence of EGCG.

Inhibitory effect of EGCG on insulin-induced levels of pRaf1, pMEK, and

pERK proteins To further assess whether EGCG regulated the downstream signaling proteins of IRS,

the levels of the phosphorylated Raf1, MEK1/2, and ERK1/2 were measured (Fig.5). We

found that EGCG decreased insulin-induced levels of phospho-Raf1(Fig. 5A),

phospho-MEK1/2(Fig. 5B), and phospho-ERK1/2(Fig. 5C). However, insulin did not

alter total levels of MEK1 or ERK1/2 in the presence or absence of EGCG.

Catechin-specific effect of green tea on insulin-stimulated signaling Catechin-specific effect of green tea on insulin-stimulated signaling was assessed by

measuring the amounts of the phosphorylated forms of insulin receptor (Fig. 6A), IRS1

(Fig. 6B), IRS2 (Fig.6C), and MEK1/2 (Fig.6D). At the given concentration of 20 uM for

90 min, EGCG was generally more effective than other catechins in reducing

insulin-stimulated signaling.

EGCG reduced insulin-stimulated interactions of insulin receptor with IRS1

and IRS2 proteins For clear assessment of the effect of EGCG on insulin signaling in 3T3-L1

preadipocytes, we further determined whether EGCG regulates insulin-mediated

association of insulin receptor with IRS1 and IRS2 proteins. We found that EGCG

reduced insulin-stimulated associations of insulin receptor with IRS1 (Fig. 7A) and IRS2

(Fig. 7B).

Effect of EGCG on insulin-stimulated interactions of IRS1 with Ras, pRaf1,

and pMEK1/2 To further understand the mechanism of the molecular effects of EGCG on insulin

signaling, we assessed whether EGCG affected insulin-mediated interaction of IRS1

10

with Ras, pRaf1, and pMEK1/2. We found that EGCG could reduce insulin-stimulated

interaction of IRS-1 with either pRaf1 (Fig. 8D) or pMEK1/2 (Fig. 8E). However, EGCG

had no effect on the insulin-mediated association of IRS1 with Ras (Fig. 8C).

Effect of EGCG on insulin-stimulated interactions of IRS2 with Ras, pRaf1,

and pMEK1/2 We further examined the effect of EGCG on insulin-mediated interactions of IRS2

with Ras, pRaf1, and pMEK1/2. EGCG was observed to reduce insulin-stimulated

interactions of IRS2 with Ras (Fig. 9C) and pRaf-1 (Fig. 9D). However, EGCG had no

effect on the insulin-mediated interaction of IRS2 with pMEK (Fig. 9E).

Effect of EGCG on insulin-mediated interactions of IRS3 with insulin

signaling proteins We knew that EGCG reduced insulin-stimulated levels of phospho-insulin receptor

substrate 3 (Fig. 2C). Thus, whether EGCG also affectes the insulin-mediated association

of IRS3 with insulin signaling proteins was assessed. We found that EGCG could reduce

insulin-stimulated association of IRS3 with pRaf1 (Fig. 10C). In contrast, EGCG tended

to increase insulin-mediated interaction of IRS3 with Ras (Fig. 10B).

Effect of EGCG on insulin-mediated interactions of IRS4 with insulin

signaling proteins Insulin was found to decrease levels of phospho-IRS4 (Fig 2D). Thus, we tested

whether EGCG affected the insulin-mediated interaction of IRS4 with insulin signaling

proteins. We found that EGCG tended to increase insulin-mediated interaction of IRS4

with either Ras (Fig. 11A), pRaf1 (Fig11B), and pMEK1/2 (Fig. 11C).

Effect of EGCG on insulin-mediated interactions of IRS proteins with

67-kDa laminin receptor The 67-kDa laminin receptor (67 LR) has recently been found in cancer cells as an

EGCG receptor. To further assess whether EGCG affected the insulin-mediated

11

association of IRS proteins with 67 LR, we found EGCG could increase

insulin-stimulated interactions of IRS1 with 67 LR (Fig. 12A), IRS2 with 67 LR (Fig.

12B) and IRS4 with 67 LR (Fig. 12D), but it did not alter insulin-stimulated interactions

of IRS3 with 67 LR (Fig. 12C).

Laminin receptor antibody antagonized the effects of EGCG on

insulin-stimulated pMEK and pIRS2 levels To assesse whether EGCG affected insulin signaling through 67-kDa LR, we

pretreated preadipocytes with 67-kDa LR antiserum for 1h and then stimulated them with

EGCG for 90 min and insulin for another 5 min (Fig. 13). We found that the

EGCG-decreased insulin-increased levels of phospho-MEK1/2 (Fig. 13A) and

phospho-IRS2 (Fig. 13B) were blocked by the addition of laminin receptor antiserum.

12

Discussions

Previously we observed that insulin at 1-1000 nM could stimulate the growth of

3T3-L1 preadipocytes as indicated by increased cell number and BrdU incorporation (22).

However, the results did not demonstrate whether EGCG regulated the phosphorylation

of the different types of IRS. The present study is delineated to demonstrate that EGCG

decreases insulin-induced increases in levels of the different IRS types and insulin

receptor with dose- and time-dependent manners. EGCG decreased insulin-induced

increases in levels of pIRS1, pIRS2, and pIRS3. However, the insulin-decreased levels of

pIRS4 is further enhanced by EGCG treatment. These observations suggest that EGCG

has the selective effect on insulin signaling to a particular type of IRS. This conclusion is

also supported by the following observations.

First, EGCG reduced insulin-induced association of IRS1 with pRaf1 and pMEK1/2,

but not with Ras (Fig. 8). Second, EGCG reduced insulin-induced association of IRS-2

with Ras and pRaf-1, but not with pMEK1/2 (Fig. 9). Third, EGCG reduced

insulin-induced association of IRS3 with pRaf1, but not with Ras (Fig.10). Finally,

EGCG increasd insulin-induced association of IRS4 with Ras, pRaf1, and pMEK1/2(Fig.

11).

In this study, we found that EGCG reduced insulin-stimulated associations of insulin

receptor with IRS1 (Fig. 7A) and IRS2 (Fig. 7B). These results may be used to explain

the findings that EGCG significantly decreased insulin-induced increases in levels of

phospho-IRS1(Fig. 4B) and phospho-IRS2 (Fig. 4C). In addition, EGCG reduced

insulin-stimulated association of IRS with Ras, pRaf1, and pMEK1/2 . These results can

explain that EGCG significantly decreased insulin-induced increases in levels of

phospho-Raf1(Fig. 5A), phospho-MEK1/2(Fig. 5B), and phospho-ERK1/2(Fig. 5C).

Catechin-specific effects of green tea have been reported on the biological system and

cell functions based on the various laboratory studies (31-33). We found that, at the same

dose and duration of treatment, EGCG was more effective than other catechins to inhibit

insulin signaling in stimulating mitogenesis of 3T3-L1 preadipocytes. According to the

nature of their structures (Fig 1), the galloyl group in the C ring of EGCG seemed to be

13

responsible for its effects. Since the esterification of the galloyl group partially reflects

the hydrophobic force of EGCG (34), the hydrophobic interactions of EGCG with the

cellular components of 3T3-L1 preadipocytes, such as EGCG receptor (27), may

contribute in the inhibitory effects of EGCG on insulin-signaling. But, this will require

more thorough studies.

The 67-kDa laminin receptor has recently been found in cancer cells as an EGCG

receptor (27). It is responsible for the inhibitory effect of EGCG on growth of the

metastatic lung cancer cells more than of non-metastatic cells. Whether EGCG regulates

insulin signal pathway in 3T3-L1 preadipocytes through affecting its own receptor or

other growth factor’s receptor is still not clear. We observed herein that EGCG

stimulated the association of either IRS1, IRS2, or IRS4, but not IRS3, with the EGCG

receptor (Fig. 12). Besides, pretreatment with 67 LR antiserum can block the inhibitory

effect of EGCG on insulin-increased levels of pMEK1/2 (Fig. 13A) and pIRS2 (Fig.

13B). These results indicate the laminin receptor-dependent effect of EGCG on the

insulin signaling in preadipocytes. This study is similar to the reported findings by

Tachibana et al (27).

We can conclude that EGCG dose- and time-dependently decreased insulin-induced

increases in levels of phospho-insulin receptor (p-IR), phospho-insulin receptor substrates

1, 2, and 3 (pIRS1, pIRS2, and pIRS3), phospho-Raf1, phospho-MEK, and

phospho-Erk1/2 proteins. In addition, EGCG reduced the insulin-stimulated

associations of IR with IRS1 and IRS2. EGCG also reduced the association of these

two IRS proteins with their downstream signaling proteins. Moreover, EGCG was more

effective than epicatechin, epicatechin gallate, and epigallocatechin in changing the

insulin-stimulated mitogenic signals. Results of this study may relate to the

mechanism by which EGCG modulates insulin-related fat cell activity and body weight

as reported by Kao et al. (22) and Hung et al. (30).

14

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Fig. 1. Chemical structures of four major green tea catechins. The differences among these catechins occur in the number of hydroxyl groups, the molecular weight, and the presence of a galloyl group.

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Fig. 2. The effect of EGCG on insulin-induced phosphorylation of insulin receptor substrates (IRSs) and insulin receptor was dose dependent. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated with EGCG (10, 20, and 50 μM) for 90 min, and then treated with 100 nM insulin for 5 min. (A) pIRS1 was measured by Western blot analysis, and corrected with β-actin. (B) pIRS2 was measured by Western blot analysis. (C) pIR was measured by Western blot analysis. Data are expressed as the means ± SE of duplicates within three independent experiments (＊, P＜0.05 versus insulin)

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Fig. 3. The effect of EGCG on insulin-induced phosphorylation of insulin receptor substrates (IRSs) and insulin receptor was time dependent. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated with EGCG (20 μM) for different time periods, and then treated with 100 nM insulin for 5 min. (A) pIRS1 was measured by Western blot analysis, and corrected with β-actin. (B) pIRS2 was measured by Western blot analysis. (C) pIR was measured by Western blot analysis. Data are expressed as the means ± SE of duplicates within three independent experiments.

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Fig. 4. Inhibitory effect of EGCG on insulin-induced phosphorylation of insulin receptor substrates (IRSs) levels. 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. Cells were pretreated with EGCG (20 μM) for 90 min, and then treated with 100 nM insulin for 5 min. (A) pIRS1 was measured by Western blot analysis, and was corrected with β-actin. (B) pIRS2 was measured by Western blot analysis. and corrected with β-actin. (C) Lysates were immunoprecipitated by IRS3 and then its phosphorylation levels were measured by Western blot with phosphotyrosine (pY) antibody. (D) Lysates were immunoprecipitated by IRS4 and then its phosphorylation levels were measured by Western blot with phosphotyrosine (pY) antibody. Data are expressed as the means ± SE of duplicates within three independent experiments (＊, P＜

0.05 versus insulin)

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Fig. 5. Inhibitory effect of EGCG on insulin-induced levels of pRaf1, pMEK, and pERK proteins. Cells were pretreated with EGCG (20 uM) for 90 min, and then treated with 100 nM insulin for 5 min. (A) pRaf1 was measured by Western blot analysis, and was corrected with β-actin. (B) pMEK1/2 was measured by Western blot analysis. (C) pERK was measured by Western blot analysis. Data are expressed as the means ± SE of duplicates within three independent experiments.

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Fig. 6. Catechin-specific effect of green tea on insulin-stimulated signaling. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated with EC, ECG, EGC,or EGCG (20μM) for 90 min, and then treated with 100 nM insulin for 5 min. (A) pIR was measured by Western blot analysis, and was corrected with β-actin. (B) pIRS1 was measured by Western blot analysis. (C) pIRS2 was measured by Western blot analysis. (D) pMEK was measured by Western blot analysis.

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Fig. 7. EGCG reduced insulin-stimulated interactions of insulin receptor with IRS1 and IRS2 proteins. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated with EGCG (20 μM) for 90 min, and then treated with 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with insulin receptor, and then detected with pIRS1 and pIRS2 by Western blot analysis.

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Fig. 8. The effect of EGCG on insulin-stimulated interactions of IRS1 with Ras, pRaf1, and pMEK. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated EGCG 20 μM for 90 min, then treated 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with IRS1 then detected with IRS1 (A), pIRS1(B), Ras (C), pRaf1 (D), and pMEK1/2 (E) by Western blot analysis.

30

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Fig. 9. The effect of EGCG on insulin-stimulated interactions of IRS2 with Ras, pRaf1, pMEK. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated EGCG 20μM for 90 min, then treated 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with IRS2 then detected with IRS2 (A), pIRS2(B), Ras (C), pRaf1 (D), and pMEK1/2 (E) by Western blot analysis.

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Fig. 10. The effect of EGCG on insulin-mediated interactions of IRS3 with insulin signaling proteins. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated EGCG 20 μM for 90 min, then treated 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with IRS3 then detected with IRS3 (A), Ras(B), and pRaf1 (C) by Western blot analysis.

34

35

Fig. 11. The effect of EGCG on insulin-mediated interactions of IRS4 with insulin signaling proteins. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated EGCG 20 μM for 90 min, then treated 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with IRS4 then detected with Ras (A), pRaf1(B), and pMEK1/2 (C) by Western blot analysis.

36

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Fig. 12. The effect of EGCG on insulin-mediated interaction of IRS proteins with laminin receptor. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated EGCG 20μM for 90 min, then treated 100 nM insulin for 5 min. Cell lysates were immunoprecipitated with IRS1 (A), IRS2 (B), IRS3 (C), and IRS4 (4) respectively and then detected with laminin receptor by Western blot analysis.

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Fig. 13. Laminin receptor antibody antagonized the inhibitory effects of EGCG on insulin-stimulated pMEK and pIRS2 levels. The 3T3-L1 preadipocytes were cultured for 2 days and then starved for overnight. The cells were pretreated with 67-kDa laminin receptor antibody (5 ug/ml) for 60 min, and then treated with EGCG (20 μM)for 90 min, and 100 nM insulin for another 5 min. (A) pMEK was measured by Western blot analysis, and was corrected with β-actin. (B) pIRS2 was measured by Western blot analysis.

39

Appendix 1. Medium: DMEM (Dulbecco’s modified Eagle medium), 3.7g NaHCO3, 10% FBS,

100 unit/ml Penicillin, and 100 µg/ml Streptomycin, add ddH2O to 1000 ml and adjust pH to 7.4. The medium was filtered through 0.2 µM top filter and then stored at 4°C.

2. 10× PBS: 8 g NaCl

0.2 g KCl 2.16 g NaHPO4•7H2O 0.2 g KH2PO4 add ddH2O to 1 L adjust pH to 7.4

3. Lysis buffer: 1 mM EDTA 1 mM PMSF 1 mM Na3VO4 (Phosphtase inhibitor) 20 mM Tris-HCl (pH 7.6) 0.2% Triton-X 100

4. 2× sample buffer: 100 mM Tris-Cl(PH 6.8) 4% SDS 20% glycerol 0.2% bromophenol blue 10 ml β-mecaptoethenol

5. 30% Acrylamide/bisacrylamide: 145 g acrylamide

5 g bisacrylamide add ddH2O to 500 ml

6. 4× lower (running) Tris buffer: Tris 36.34g add ddH2O 180 ml adjust pH to 8.8 then add ddH2O to 200 ml

7. 4× upper (stacking) Tris buffer: Tris 12.2 g add ddH2O 80 ml adjust pH to 6.8 then add ddH2O to 100 ml

40

8. Lower SDS-PAGE: 12% Resolving gel 10ml 15ml 20ml

H2O 3.3 4.9 6.6

30% acrylamide mix 4.0 6.0 8.0

1.5 M Tris (pH8.8) 2.5 3.8 5.0

10% SDS 0.1 0.15 0.2

10% ammonium persulfate 0.1 0.15 0.2

TEMED 0.004 0.006 0.008

9. Upper SDS-PAGE:

10. Tank buffer: 6.056 g Tris 28.828 g Glycine 2 g SDS add ddH2O to 2 L in total volume store at RT

11. Transfer buffer: Tris 2.32g Glycine 1.17g add ddH2O 400 ml then add methanol 100ml

12. Stain buffer: 0.125 g Coomassie blue R in 200 ml methanol

35 ml acetic acid add ddH2O to 500 ml in total volume store at RT.

13. Destain buffer: 40% methanol 10% acetic acid

5% stacking gel 2ml 6ml

H2O 1.4 4.2

30% acrylamide mix 0.33 0.99

1.5 M Tris (pH6.8) 0.25 0.75

10% SDS 0.02 0.06

10% ammonium persulfate 0.02 0.06

TEMED 0.002 0.006

41

store at RT

14. PBST: 1X PBS 499.5 ml Tween-20 500 µl

15. 5% non-fat milk: 5 g in 100 ml PBST 16. Ampicillin stock solution: 100 mg ampicillin in 1 ml ddH2O store at -20°C