Inhibition of adipocyte differentiation in 3T3-L1 cell line by quercetin or isorhamnetin

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1 Louisin Stte University LSU Digitl Commons LSU Mster's Theses Grdute School 2012 Inhibition of dipocyte differentition in 3T3-L1 cell line by quercetin or isorhmnetin Din Gbriel Crvjl-Aldz Louisin Stte University nd Agriculturl nd Mechnicl College, Follow this nd dditionl works t: Prt of the Life Sciences Commons Recommended Cittion Crvjl-Aldz, Din Gbriel, "Inhibition of dipocyte differentition in 3T3-L1 cell line by quercetin or isorhmnetin" (2012). LSU Mster's Theses This Thesis is brought to you for free nd open ccess by the Grdute School t LSU Digitl Commons. It hs been ccepted for inclusion in LSU Mster's Theses by n uthorized grdute school editor of LSU Digitl Commons. For more informtion, plese contct grdetd@lsu.edu.

2 INHIBITION OF ADIPOCYTE DIFFERENTIATION IN 3T3-L1 CELL LINE BY QUERCETIN OR ISORHAMNETIN A Thesis Submitted to the Grdute Fculty of the Louisin Stte University nd Agriculturl nd Mechnicl College in prtil fulfillment for the requirements for the degree of Mster of Science in The Deprtment of Food Science by Din Crvjl-Aldz B.S., Zmorno University, 2007 December 2012

3 Dedicted to my beloved fmily Crvjl-Aldz nd the most importnt person in my life Clr Veg, my grndmother ii

4 ACKNOWLEDGEMENTS I would like to express my hertfelt thnks to Dr. Jck N. Losso who tught, guided nd supported me during my studies t Louisin Stte University. Also, I would like to pprecite the help nd dvice provided by my dvisory committee, Dr. John Finley nd Dr. Frederick Enright. As well, I wnt to express my specil grtitude to Ms. Kren Mcdonough who helped me throughout this reserch. Another importnt person I wnt to sincerely thnk is Chncellor Dr. Willim Richrdson, who mintins nd supports the interntionl exchnge progrm between Zmorno nd Louisin Stte University. I m very thnkful to my lb-mtes Gbriel Crespo, Chenfei Go, Behnm Keshvrz, Dmir Torrico, Pul Mgut nd Alfredo Prudente for their invluble support. I would lso like to thnk my friends. Especilly Ntli Ltorre, Adrin Soto, Nmrt Krki, Sriknth Erpin, Luis Vrgs nd Frnklin Vc for lwys provided me their support, help, encourgement nd dvice. Finlly, my sincerest thnk to the Zmorno Agriculturl Society (ZAS). iii

5 TABLE OF CONTENTS ACKNOWLEDGEMENTS..iii LIST OF FIGURES..vi ABBREAVIATIONS.viii ABSTRACT...x CHAPTER 1: INTRODUCTION.. 1 CHAPTER 2: LITERATURE REVIEW Definition of Obesity Risk Fctors for Obesity Obesity nd Adipogenesis Obesity nd Inflmmtion Adipokines Dietry Approch to Reduce Obesity Quercetin Isorhmnetin T3-L1 Cell Line.. 10 CHAPTER 3: MATERIALS AND METHODS Regents Cell Culture MTS Cell Vibility Assy Oil Red O Stining Intrcellulr Triglycerides Biomrkers during 3T3-L1 Differentition Enzyme Linked Immunosorbent Assy for Pro-inflmmtory Cytokines Western Blot Anlysis Sttisticl Anlysis CHAPTER 4: RESULTS AND DISCUSSION Cell Vibility Oil Red O Stining Effect of Quercetin on Lipid Accumultion Effect of Isorhmnetin on Lipid Accumultion Effects of Quercetin or Isorhmnetin on the Secretion of MCP-1 during Cell Differentition Effects of Quercetin on the Secretion of MCP-1 during Cell Differentition Effects of Isorhmnetin on the Secretion of MCP-1 during Cell Differentition Expression of PPAR-γ, C/EBP-α, nd β-ctenin during 3T3-L1 Cell Differentition Effects of Quercetin on PPAR-γ, C/EBP-α, nd β-ctenin during T3-L1 Cell Differentition Effects of Isorhmnetin on PPAR-γ, C/EBP-α, nd β-ctenin during T3-L1 Cell Differentition...34 CHAPTER 5: CONCLUSIONS.. 38 iv

6 REFERENCES. 39 VITA v

7 LIST OF FIGURES Figure 2.1. Body Mss Index Clssifiction.. 3 Figure 2.2. Stges in dipogenesis...5 Figure 2.3. Contribution of dipokines to obesity nd metbolic syndrome bnormlities..6 Figure 2.4. Domins nd structure of diponectin.7 Figure 2.5. Quercetin structure 10 Figure 2.6. Isorhmnetin structure...10 Figure 3.1. Protocol for chemicl-induction of differentition 13 Figure 3.3. Interction of bioctive compounds with 3t3-l1 cell line.. 15 Figure 4.1. Effects of quercetin on 3t3-l1 cell vibility...18 Figure 4.2. Effects of isorhmnetin on 3t3-l1 cell vibility.18 Figure 4.3. Effect of quercetin on lipid ccumultion in 3t3-l1 cells..20 Figure 4.4. Effect of quercetin on 3t3-l1 cells.22 Figure 4.5. Effect of isorhmnetin on lipid ccumultion on 3t3-l1 cells...23 Figure 4.6. Effect of isorhmnetin on 3t3-l1 cells...25 Figure 4.7. Effect of quercetin on MCP-1 levels (pg/µg protein) during 3T3-L1 dipocyte differentition...26 Figure 4.8. Effect of isorhmnetin on MCP-1 levels (pg/µg protein) during 3T3-L1 dipocyte differentition...27 Figure 4.9. Western blot nlysis results for control cells during dipocyte differentition period of 10 dys..28 Figure β-ctenin expression in control cells during dipocyte differentition...29 vi

8 Figure C/EBP-α expression during dipocyte differentition Figure PPAR-γ expression during dipocyte differentition..30 Figure Western blot nlysis of B-ctenin, PPAR-γ, nd C/ebp-α on dy 10 in response to quercetin concentrtion from 25nM to 25 µm.31 Figure β-ctenin expression in quercetin (25 nm to 25 µm) treted cells.33 Figure C/EBP-α expression in quercetin (25 nm to 25 µm) treted cells.. 33 Figure PPAR-γ expression in quercetin (25 nm to 25 µm) treted cells 34 Figure Western blot nlysis of β-ctenin, PPAR-γ, nd C/EBP-α on dy 10 in response to isorhmnetin concentrtion from 25nM to 25 µm...35 Figure β-ctenin expression in isorhmnetin (25 nm to 25 µm) treted cells...36 Figure C/EBP-α expression in isorhmnetin (25 nm to 25 µm) treted cells Figure PPAR-γ expression in isorhmnetin (25 nm to 25 µm) treted cells..37 vii

9 ABBREVIATIONS AMM ATCC β-ctenin BSA BMI C/EBP-α C/EBP-β C/EBP- δ CHD DM DMEM FBS GRAS hamscs IBMX IL-6 IS LDL Adipocyte Mintennce Medium Americn Type Culture Collection Bet-ctenin Bovine serum lbumin Body Mss Index CCAAT/enhncer-binding protein-lph CCAAT/enhncer-binding protein-bet CCAAT/enhncer-binding protein-delt Coronry hert disese Differentition Medium Dulbecco s modified Egle s medium Fetl bovine serum Generl recognized s sfe Humn dipose tissue-derived stem cells 3-isobutyl-1-methylxnthine Interleukin-6 Isorhmnetin Low-density lipoprotein MCP-1 Mcrophge chemottrctnt protein -1 PAI-1 PBS PEM PPAR-γ PVDF Q RIPA ROS Plsminogen ctivtor inhibitor-1 Phosphte buffered sline Pre-dipocyte Expnsion Medium Peroxidse prolifertor-ctivted regultor-gmm Polyvinylidene difluoride Quercetin Rdioimminoprecipittion ssy buffer Rective oxygen species viii

10 RXR SREBP TNF-α WAT 3T3-L1 Retinoid X receptor Sterol regultory element binding protein Tumor necrosis fctor-lph White dipose tissue Cell line which ws originted from clonl expnsion of murine Swiss 3T3 cells nd contin only single cell type ix

11 ABSTRACT Obesity hs become mjor helth problem worldwide. Obesity increses the risk of hypertension, dibetes, nd certin types of cncer. Quercetin is bioctive compound widely found in vriety of foods tht re consumed dily. Isorhmnetin is bioctive compound found in some foods nd lso is quercetin metbolite. Mny studies hve reported the nti-obesity nd nti-inflmmtion properties of quercetin nd isorhmnetin. The objective of this study ws to test the effect of quercetin or isorhmnetin t physiologicl nd suprphysiologicl concentrtions on the inhibition of the differentition process of 3T3-L1 pre-dipocyte to dipocyte. Cell vibility results demonstrted no significnt difference (P > 0.05) between non differentited cells, control nd quercetin or isorhmnetin treted cells. During dipocyte differentition for 8 dys in the presence of quercetin or isorhmnetin, cell vibility ws bove 94.84% nd 97.63%, respectively. Red oil O stining ssy ws performed in order to evlute the inhibitory effect of quercetin or isorhmnetin on cytoplsmic lipid droplet ccumultion. Significnt differences (P < 0.05) were reported. Isorhmnetin ws more effective thn quercetin in inhibiting cytoplsmic lipid droplet ccumultion. Neither quercetin nor isorhmnetin hd n effect on the expression of mcrophge chemottrctnt protein -1 (MCP-1). CCAAT/enhncer binding protein α (C/EBP-α) ws down-regulted by quercetin or isorhmnetin. Compred to control quercetin decresed PPAR-γ 1 nd 2 expressions by ± 3.17% nd ± 12.39%, while isorhmnetin decresed PPAR-γ 1 nd 2 expressions by ± 9.51% nd 2.01 ± 32.46%, respectively. β-ctenin ws not dose dependent either for quercetin or isorhmnetin nd did not follow specific trend. Tken together, our dt indicte tht isorhmnetin more thn quercetin cn exert potentil nti-obesity effects by inhibiting differentition of pre-dipocytes t physiologicl concentrtions. x

12 CHAPTER 1 INTRODUCTION Obesity is result of n imblnce between intke of energy nd energy expenditure [1].Obesity is serious helth problem tht is implicted in vrious diseses including hypertension, type II dibetes, coronry hert diseses, nd cncer [2]. Obesity is chrcterized by incresed dipose tissue mss tht results from incresed ft cell size (hypertrophy) or number (hyperplsi). Adipose tissue is mjor energy reservoir in the body; it stores the excess energy s lipids nd releses it on demnd. Moreover, dipocytes constitute n endocrine system which secrete hormones known s dipokines [3]. Phytochemicl tretments cn regulte dipose tissue mss by the inhibition of dipogenesis from fibroblstic pre-dipocytes to mture dipocytes s demonstrted in recent studies [4]. Quercetin is one of the mjor flvonol in the western diet; the min sources re pples, onions, red wine nd te [5]. It is bioctive compound widely studied due to its unique bility to ct s n ntioxidnt (low doses) or pro-oxidnt (high doses) depending on its concentrtion [6]. Quercetin hs been studied for its nti-inflmmtory nd lipid-regulting properties [7]. Since flvonoids re modified during bsorption in the smll intestine, severl studies hd been concentrted on plsm metbolites fter quercetin intke. Three of the mjor metbolites of quercetin re quercetin s is, isorhmnetin nd tmrixetin [8]. Isorhmnetin is flvonoid found in sebuckthorn [9] or s quercetin metbolite in plsm fter quercetin intke [8]. Isorhmnetin hs recently been reported to hve ntioxidnt ctivity, the bility to increse the resistnce of humn low-density lipoprotein to oxidtion, nd ntitumor ctivity [10]. Adipose tissue hyperplsi triggers the trnsformtion of pre-dipocytes into dipocytes. Adipogenesis or dipocyte differentition is multifceted process which includes chnges in morphology, hormone sensitivity, nd gene expression. In this process, members of CCAAT/enhncerbinding protein (C/EBP) - trnscription fctor fmily nd peroxidse prolifertor-ctivted regultor-γ (PPAR-γ) ct together in order to regulte dipocyte differentition [9]. During dipogenesis dipokines 1

13 s mcrophge chemottrctnt protein -1 (MCP-1) re relesed. MCP-1 is novel dipokine which hs n importnt role in the development of obesity-ssocited insulin resistnce [11]. The expression of MCP-1 by dipose tissue induces inflmmtory rections nd insulin resistnce, suggesting the suppression of MCP-1 is importnt for the mngement of metbolic syndrome [12]. Severl in vitro studies hve shown tht flvonoids t concentrtions exceeding plsm levels hve nti-inflmmtory ctivity, decresed expression of PPAR-γ, C/EBP-α, nd intrcellulr cytoplsmic lipid droplet ccumultion; but the physiologicl significnce of the results is questionble. Therefore, the min objective of this study ws to evlute nd compre the effect of quercetin or isorhmnetin t physiologicl concentrtions nd suprphysiologicl dosges on 3T3-L1 cell line. Steps tken to chieve the objective included: 1. Determining cytotoxic concentrtions of quercetin or isorhmnetin during dipocyte differentition. 2. Demonstrting the lipid droplet ccumultion fter the dipocyte differentition process nd the inhibition of lipid droplet ccumultion by quercetin or isorhmnetin tretments. 3. Investigting the secretion of MCP-1 during the dipocyte differentition. 4. Studying the expression of β-ctenin, C/EBP-α, nd PPAR-γ in the presence or bsence of quercetin or isorhmnetin. 2

14 CHAPTER 2 LITERATURE REVIEW 2.1. Definition of Obesity Obesity is defined s bnorml or excessive ft ccumultion, for women gluteofemorl or lowerbody wheres for men bdominl or upper-body [2]. Obesity results from n excess in energy intke nd low energy expenditure. There re different methods to mesure body ft: (1) skin fold mesurement, (2) wist circumference, or (3) body mss index. However, Body Mss Index (BMI) is commonly used. BMI is defined s the weight in kilogrms divided by the squre height in meters (kg/m 2 ), nd the resulting index is clssified s underweight, overweight, obese, or over-obese (Figure 2.1). Figure 2.1. Body Mss Index clssifiction. Adpted from: 3

15 2.2. Risk Fctors for Obesity Obesity is product of the imblnce between energy intke nd energy expenditure, which leds to the pthologicl growth of dipocytes [1]. The growth of the dipose tissue cn be result of hypertrophy nd/or hyperplsi of the cells; which leds to increse in cell size nd ft cell number, respectively [13]. Obesity is now recognized s chronic nd systemtic inflmmtory disese; the molecules secreted, known s dipokines, regulte crbohydrte nd lipid metbolism, immune function nd blood cogulbility, nd my serve s blood mrkers of crdiometbolic risk [14]. Obesity increses risk to cquire severl disorders tht re directly linked to high mortlity nd morbidity, including dibetes, hypertension, coronry hert disese (CHD), dyslipidemi, gllbldder disese, nd certin kinds of cncer [2]. Obesity is complex disorder with strong genetic bsis nd multifctoril etiology [15] Obesity nd Adipogenesis In periods of excess energy, the eukryotes store excess energy s tricylglycerol nd mobilize the stored lipids during deprivtion periods; this energy reserve is known s white dipose tissue (WAT). A drmtic increse in the rte of obesity is result of the excessive ccumultion of WAT [16]. This phenomenon is clled dipogenesis or dipocyte differentition. During this, fibroblst-like predipocytes differentite into lipid-lden nd insulin-responsive mture dipocytes [17]. Adipogenesis occurs under the regultion of severl trnscriptionl fctors, such s sterol regultory element binding protein (SREBP), CCAAT/enhncer binding proteins (C/EBPs) nd peroxisome prolifertor-ctivted receptor γ (PPAR- γ)[18]. The role of PPAR isoforms in dipogenesis is not completely understood. The PPARs form heterodimers with the retinoid X receptor (RXR) nd regulte trnscription through this binding. Peroxisome prolifertor-ctivted receptor γ is the most dipose specific nd hs been shown to be enough to induce growth rrest or dipogenesis initition besides plying criticl role in the regultion of dipocyte differentition. Peroxisome prolifertor-ctivted receptor α hs been lso reported to be 4

16 cpble of inducing dipocyte differentition, but is less dipogenic thn PPAR- γ. Finlly, peroxisome prolifertor-ctivted δ is not dipocyte specific, however is highly expressed in dipose tissue [19]. C/EBP fmily ws mong the first trnscription fctors confirmed to perform n importnt role in dipocyte differentition. Isoforms of C/EBP metbolize lipid nd cholesterol-relted compounds nd re expressed in tissues such s liver [20]. Although C/EBP-α is not dipocyte specific, is expressed before the trnscription of most dipocyte-specific genes hs strted, nd in some instnces is sufficient to induce differentition. C/EBP-α, C/EBP-β, nd C/EBP-δ re isoforms; ech one hs distinct sequentil nd sptil expression during dipocyte differentition. C/EBP-α express reltively lte in differentition, menwhile β nd δ re present in pre-dipocytes, nd their levels increse during differentition [21]. The expression of PPAR- γ is directly induced by the coexpression of C/EBP-β nd δ; lthough C/EBP-α nd PPAR- γ ppernce rises intensely during dipocyte differentition. This suggests tht n increse in C/EBP-β bove certin level induces the expression of PPAR- γ. The lignd ctivtion of PPAR- γ with C/EBP-α leds to full dipocyte differentition [21]. An overview of stges in dipogenesis is represented in Figure 2.2. Figure 2.2. Stges in dipogenesis [19] 5

17 2.4 Obesity nd Inflmmtion Adipocyte cells hve n ctive role s endocrine cells relesing vrious bioctive substnces clled dipocytokines or dipokines, which impct directly the regultion of food intke, insulin sensitivity, energy metbolism, nd the vsculr microenviroment [12]. Recently, reserch hs focused on the concept tht obesity s chronic low-grde systemtic inflmmtion stimultes insulin resistnce nd the production of inflmmtory biomrkers [22]. Adipocytes relese dipokines nd bioctive peptides, including leptin, diponectin, tumor necrosis fctor-lph (TNF-α), resistin, interleukin-6 (IL-6), plsminogen ctivtor inhibitor-1 (PAI-1), mcrophge chemottrctnt protein-1 (MCP-1) [23].The sitution worsens when dipocytes relese excessive mounts of cytokines/ dipokines s TNF-α, resistin, IL-6, PAI-1, MCP-1 nd others; which circulte vi the vsculr system to insulin trget tissues such s liver, muscle nd islet cells inducing insulin resistnce [24]. Inflmmtion produced by obesity is prtilly medited by multiple cellulr stresses, such s oxidtive stress, endoplsmic reticulum, nd hypoxi [25]. Figure 2.3. Contribution of dipokines to obesity nd metbolic syndrome bnormlities [26]. 6

18 Adipokines Adiponectin is the most bundnt nd studied dipokine; it is exclusively relesed in dipocytes [14]. Full-length diponectin is constituted of 244 mino cids, found in plsm t levels of 3-30 mg/ml, nd forms three mjor oligomeric complexes with biologicl functions s trimer, hexmer, nd highmoleculr-mss form (Figure 2.4) [27]. Adiponectin is lso found in smller form tht consists of globulr domin existing in plsm in very smll mounts [28]. Adiponectin increses ftty cid oxidtion while decreses glucose production, nd lso plys role s nti-inflmmtory [29]. Figure 2.4. Domins nd structure of diponectin [27] Numerous studies revel the negtive correltion of plsm diponectin nd obese subjects depicting higher concentrtion of diponectin in non-obese subjects [30]. Arit et l. [30] found negtive correltion between plsm concentrtion of diponectin nd BMI in men nd women (r = -0.71, P < ; nd r = -0.51, P < respectivily). Individuls with low concentrtions of plsm diponectin hve reduced insulin sensitivity, nd tend to develop Type II dibetes [31]. Elevted plsm 7

19 diponectin levels were found in low BMI dibetic nd non-dibetic subjects [32]. The physiologicl role of diponectin in humns is not yet clerly elucidted [30]. Leptin is nother dipocytokine product of the obese (ob) gene, tht reduces ppetite by sending stiety signl to the centrl nervous system [27]. It is 16 kd hormone nd minly relesed by dipocytes in order to control body weight [14]. Concentrtion of leptin circultion in plsm is proportionl to totl body diposity nd direct nutritionl stte [33]. It is present in humn serum in rnges of 1-15 ng/ml in non-obese individuls, nd levels more thn 30 ng/ml in individuls with BMI 30 kg/m 2 [34]. Leptin lso stimultes thyroid-medited thermogenesis nd ftty cid oxidtion. Deficiency of leptin is ssocited with incresed ppetite nd mnifest obesity in mice nd humns [35]. Interleukin-6 (IL-6) is n inflmmtory cytokine tht is correlted with hyperglycemi, insulin resistnce, nd type 2 dibetes mellitus [36]. Mice chroniclly exposed to IL-6 develop heptic insulin resistnce [37]. About 25% of circulting IL-6 in humns is relesed by subcutneous dipose tissue [38]. Tumor necrosis fctor-lph (TNF-α) is cytokine whose expression is incresed in dipose tissue nd highly found in circultion of obese nd insulin resistnt individuls [39]. TNF-α hs n importnt effect on whole body lipid nd glucose metbolism [40], it inhibits tyrosine phosphoryltion of IRS-1 which decreses insulin signling [41]. Moreover, TNF- α is directly involved in the ctivtion of pro-inflmmtory subcellulr pthwys nd induces the production of rective oxygen species (ROS) [42]. Plsminogen ctivtor inhibitor-1 (PAI-1) is secreted in high concentrtion by dipose tissue. Plsm nd dipocyte concentrtions of PAI-1 correlte with the levels of viscerl ft nd triglycerides [43]. It hs been suggested tht PAI-1 my probbly destroy dipocytes cusing hypertrophy [26]. Mcrophge chemottrctnt protein -1 (MCP-1) is novel dipokine which hs n importnt role in the development of obesity-ssocited insulin resistnce [11]. The expression of MCP-1 by dipose tissue induces inflmmtory rections nd insulin resistnce, suggesting their suppression of MCP-1 is importnt for the mngement of metbolic syndrome [12]. 8

20 2.5. Dietry Approch to Reduce Obesity Consumption of vegetbles, legumes nd fruits hve been ssocited with lrge number of helth benefits, some of them re prevention of obesity, type 2 dibetes mellitus nd crdiovsculr disese, becuse of their flvonoid nd polyphenolic content [44]. Polyphenols, including flvonoids, re bioctive compounds with nti-oxidtive nd nti-inflmmtory properties [45]. Flvonoids re polyphenolic compounds tht re widely found in fruits nd vegetbles in glycosylted forms. Quercetin is one of the mjor flvonol in the western diet; the min sources re pples, onions, red wine nd te [5]. The estimted verge intke of flvones nd flvonols per dy is pproximted 25 mg [46]. An extensive literture demonstrtes tht phenolic compounds including quercetin hve importnt nti-inflmmtory nd ntiobesity properties [7, 22, 24, 45, 47] Quercetin Quercetin (3, 3, 4, 5-7-penthydroxyflvone) is polyphenol extensively found in vriety of foods tht re consumed dily. Quercetin hs the bsic structure of one or more hydroxylted benzene rings, nd lso contins severl hydroxyl groups ttched to the diphenyl propne (C6-C3-C6) bckbone [48].This bioctive compound hs been studied becuse of its unique bility to ct s n ntioxidnt (low doses) or pro-oxidnt (high doses) depending on its concentrtion [6]. Quercetin hs lso been studied for its nti-inflmmtory nd lipid-regulting properties [7]. Quercetin dosge of up to 100 mg/dy is generl recognized s sfe (GRAS). Sixty percent of orlly ingested quercetin is bsorbed. The dily intke is only 6 to 31 mg. Quercetin cn be bsorbed s glycoside or s n glycone. Few studies hve been done on the evlution of bsorption of quercetin, most studies were performed with low doses tht could be chieved by norml diet [6]. Egert et l. estblished tht quercetin could be bsorbed without difficulty. They found quercetin in blood plsm fter two weeks of dministrtion, in verges of 145 nm, 217 nm, nd 380 nm when subjects received supplements of 50 mg/dy, 100 mg/dy, nd 150 mg/dy respectively. The metbolites, isorhmnetin nd tmrixetin, were between 9 nd 23 nm [8]. 9

21 Figure 2.5. Quercetin structure Isorhmnetin Isorhmnetin (3,4,5,7-tetrhydroxy-3 -methoxyflvone) is n ctive flvonol glycone. It is one of the primry metbolites of quercetin. It is recognized for its ntioxidnt ctivity, cytoprotective cpcity, nti-dipogenesis nd nti-inflmmtory effects [49]. Its ntioxidnt ctivity hd been ttributed to its bility to increse the resistnce of humn low-density lipoprotein (LDL) to oxidtion by Cu 2+ [10]. Isorhmnetin ctivte the p38mapk pthwy preventing endothelil cell injuries cused by oxidized LPL [50]. Anti-dipogenesis properties hd been reported by the down regultion of the expression of C/EBPα nd PPAR-γ in 3T3-L1 murine cells [9], nd lso by the stbiliztion of the β-ctenin protein in humn dipose tissue-derived stem cells (hamscs) [51]. Figure 2.6. Isorhmnetin structure T3-L1 Cell Line Mny models nd techniques re being used in order to evlute nd understnd dipocyte biology [52]. 3T3-L1 is pre-dipose cell line which ws originted from clonl expnsion of murine 10

22 Swiss 3T3 cells nd contin only single cell type [53]. This cell line hs been using widely in more thn 5000 published rticles on dipogenesis nd the biochemistry of dipocytes for the lst 30 yers, becuse of its potentil to differentite from fibroblst to complete dipocytes [54]. Severl investigtions use 3T3-L1 cells becuse it helps in identifying key moleculr mrkers including trnscription fctors nd vrious pthwys during pre-dipocyte differentition [52]. Numerous protocols cn be used to induce differentition from pre-dipocytes to dipocytes, but the most commonly used gents re insulin, dexmethsone, nd 3-isobutyl-1-methylxnthine (IBMX) [55] t concentrtions of 1 μg/ml, 0.25 μm, nd 0.5 μm, respectively. Pre-dipocytes contin less mount of lipid droplets ccumulted, but four dys fter induction they strt to ccumulte lipids tht grow in size nd number over the differentition time [54]. 11

23 CHAPTER 3. MATERIALS AND METHODS 3.1. Regents Dulbecco s modified Egle s medium (DMEM) ws purchsed from Gibco (Life Technologies, Prsley, PA), clf serum nd fetl bovine serum (FBS) were obtined from ATLANTA biologicl (Lwrenceville, GA). Dexmethsone, insulin, isorhmnetin, nd oil red O were purchsed from Sigm- Aldrich (St. Louis, MO). Methylisobutylxnthine (IBMX) ws obtined from Cymn Chemicl Compny (Ann Arbor, MI). Quercetin ws purchsed from LKT Lbs (St. Pul, MN). Anti-β-ctenin, β- ctin, C/EBP-α, nd PPAR-γ monocolonl were obtined from Cell Signling (Dnvers, MA). Alkline phosphte-conjugted secondry ntibody ws from Snt Cruz Biotechnology (Snt Cruz, CA). SuperSignl West Pico Chemiluminescent Substrte ws from Thermo Scientific (Rockford, IL) Cell Culture In this study, 3T3-L1 cell line ws used to model the inhibitory effects of quercetin or isorhmnetin ginst pre-dipocyte differentition to dipocyte cells. The cell line ws obtined from Americn Type Culture Collection (ATCC) (Rockville, MD). 3T3-L1 is continuous substrin of 3T3 (Swiss lbino) developed through clonl isoltion [54]. 3T3-L1 pre-dipocyte differentition ws performed following the ATCC protocol, illustrted in Figure 3.1. Cells were seeded nd mintined with Pre-dipocyte Expnsion Medium (PEM) for 48 h by incubting t 37 C nd 5% CO 2, or fter reching 100% confluence. Next, the identicl volume of medium ws replced with Differentition Medium (DM) (Dy 0) nd incubted for 48 h t 37 C in humidified tmosphere contining 5% CO 2. After tht, the Differentition Medium ws replced with Adipocyte Mintennce Medium (AMM); AMM ws chnged every 72h. Finlly, the cells were fully differentited t dy 10 fter induction. Pre-dipocyte cells could be fully differentited between 8 to 12 dys fter DM ppliction. To exmine the effect of quercetin or isorhmnetin on dipocyte differentition, 3T3-L1 cells were treted with PEM until dy 10 in the bsence or presence of 12

24 concentrtions from 20 nm to 25 µm of quercetin or isorhmnetin. Also, flsk of cells contining PEM (non-differentited cells) ws mintined until dy 10. Medi formultions re described below in Tble 3.1. Tble 3.1. Description of medi formultions MEDIUM FORMULATION Pre-dipocyte Expnsion 90% Dubelcco s Modified Egle s Medium (DMEM), nd 10% Bovine Clf Serum Differentition 90% DMEM, 10% Fetl Bovine Serum (FBS), 1.0 μm Dexmethsone, 0.5 mm Isobutylmethylxnthine (IBMX), nd 1.0 μg/ml Insulin Adipocyte Mintennce 90% DMEM, 10% FBS, nd 1.0 μg/ml Insulin PRE-ADIPOCYTE ADIPOCYTE Dy 0 DM Dy 2 AMM Dy 5 AMM Dy 8 AMM Dy 10 Figure 3.1. Protocol for chemicl-induction of differentition. Differentition Medium (DM), Adipocyte Mintennce Medium (AMM) MTS Cell Vibility Assy 3T3-L1 cells were seeded in 96-well pltes t density of 1.0x10 4 cells per well. The cells were initited for differentition s previously described. Stock solutions of 100 μm nd 10 μm of pure quercetin or isorhmnetin were prepred with PEM, DM, nd AMM. Different concentrtions of quercetin (25 nm to 25 μm), nd isorhmnetin (25 nm to 25 μm) of the totl volume of the medi used were tested during the differentition period of eight dys. The cells were incubted for 1, 3, 5, 7, nd 8 dys t 37 C in n incubtor with 5% CO 2. After incubtion, cell vibility ws determined ccording to 13

25 the protocol provided by the supplier of CellTiter 96 AQ ueous One solution (Promeg, Mdison, WI). Twenty microliters of MTS regent ws dded to every well nd incubted under the sme conditions for 4h. Absorbnce of the pltes ws red t 490 nm in BioRd Model 680 micro plte reder (Hercules, CA). The number of vible cells ws directly proportionl to the bsorbnce of formzn formed due to the reduction of MTS. Cell vibility ws expressed s the percentge of control cells. Tretments were tested in triplictes Oil Red O Stining Intrcellulr Triglycerides 3T3-L1 cells were seeded in 6-well pltes t density of 8x10 4 cells per well. Cells were subjected to differentition following the protocol described before including non-differentited, control, nd different concentrtions of quercetin (25 nm to 25 μm) or isorhmnetin (25 nm to 25 μm) in triplictes. At dy 10 of differentition, 3T3-L1 dipocytes were wshed with PBS nd fixed with 10% formlin for 30 min. Then they were wshed with distilled wter twice, the cells were stined for 45 minutes in 37 C incubtor with diluted oil red O solution. Tretments were photogrphed with microscope OLYMPUS Model DP72 (Center Vlley, PA). Finlly, the dye retined in 3T3-L1 cells ws eluted with isopropnol nd pipetted in 96 well-plte to mesure the bsorbnce by microplte spectrophotometer BIORAD Benchmrk Plus TM (Phildelphi, PA) t 510 nm. Briefly, the oil red O solutions ws prepred from 0.05 g of oil red O dissolved in 100 ml of propnol (stock solution), then six prts of stock solution ws mixed with four prts of distilled wter, nd this mixture ws filtered by vcuum. 3.5 Biomrkers during 3T3-L1 Differentition In order to study the inhibitory effects of quercetin, or isorhmnetin on differentition; the prevention pproch ws tested. Cells were treted in presence or bsence of quercetin or isorhmnetin before nd during the dipocyte differentition process. The 3T3-L1 cells were seeded in T25 flsks t density of 200x10 3 cells per flsk nd subjected to differentition following the protocol described before. Cells were treted with quercetin or isorhmnetin t concentrtions of 0 to 25 μm from the expnsion 14

26 period until the completion of dipocyte differentition on dy 10. Finlly, the superntnt nd the cell lystes were collected for further nlysis. Pre-dipocyte 3T3-L1 cell line Incubte with Q or IS from expnsion period until dy 10 Collect At dy superntnt 10 dd 250 µm/l nd cell of plmitic lystes cid for 24 h Superntnt Cell lystes MCP-1 PPAR s, C/EBP-α, β-ctenin Figure 3.3. Interction of bioctive compounds with 3T3-L1 cell line Enzyme Linked Immunosorbent Assy for Pro-inflmmtory Cytokines To determine the inhibitory effect of quercetin or isorhmnetin on differentition, the superntnts were collected on dy 0, 2, 5, 7, nd 10 nd MCP-1 levels were determined by ELISA using commercil kits from ebioscience (Sn Diego, CA). All nlyzes were performed in triplictes Western Blot Anlysis In order to determine the nti-dipogenesis properties of quercetin or isorhmnetin during 3T3-L1 differentition, PPAR-γ, C/EBP-α, nd β-ctenin were identified on dy 10 fter differentition. 3T3-L1 cells were rinsed once with ice-cold phosphte buffered sline (1X PBS), then lysed in 240 µl of rdioimminoprecipittion ssy buffer (RIPA) supplemented with protese inhibitor, sodium orthovndte nd PMSF for 30 min t 4 C. Cells were hrvested by scrping nd trnsferred into n eppendorf, incubted on ice for 20 min, centrifuged t 12,000 rpm for 30 min, nd the superntnt ws sved in fresh eppendorf. Protein concentrtion in the superntnt ws determined by the BCA method 15

27 using BCA protein Assy Regents (Pierce). Extrcts contining 20 µg protein from control, quercetin or isorhmnetin treted 3T3-L1 cells were mixed with LDS smple buffer (Invitrogen, Crlsbd, CA), wter, boiled for 3 minutes for protein denturtion, nd 20 µl of smples were loded onto 12 well 4-12% Bis-Tris gels (Invitrogen, Crlsbd, CA), rn for 35 min t 200 V nd 125 ma. After protein seprtion, they were trnsferred to polyvinylidene difluoride (PVDF) membrne (Invitrogen, Crlsbd, CA) for 1h 10 min t 30 V nd 170 ma. Five percent bovine serum lbumin (BSA) in 1X PBS contining 0.05% Tween 20 (PBS-Tween) ws used to block the membrne for 1h t room temperture. Then the membrne ws incubted with primry ntibody (dissolved in PBS-Tween contining 5% BSA s mnufcturer s instructions) overnight t 4 C with gentle gittion. The next dy, the membrne ws wshed 3 times for 10 min ech with PBST, secondry ntibody (dissolved in PBS-Tween contining 5% BSA s mnufcturer s instructions) ws incubted for 1h t room temperture, nd wshed gin 3 times for 10 min ech with PBST. Finlly the blots were developed by enhnced chemiluminescence with SuperSignl West Pico Chemiluminescent substrte nd exposed to X-ry film (Kodk X-omt 1000A processor) Sttisticl Anlysis Dt were expressed s mens ± stndrd devition from triplictes of ech experiment. Sttisticl nlysis ws performed using the Sttisticl Anlysis Softwre (SAS) (version 9.2). Differences between control nd tretments were determined by nlysis of vrince (ANOVA) nd followed by Tukey nlysis. A P-vlue of < 0.05 ws considered sttisticlly significnt. 16

28 CHAPTER 4 RESULTS AND DISCUSSION 4.1. Cell Vibility To determine the effects of quercetin or isorhmnetin on cell vibility during dipocyte differentition period of 8 dys, MTS cell vibility ssy ws performed on 3T3-L1 cells treted with 0 to 25 µm quercetin or 0 to 10 µm isorhmnetin, nd non-differentited cells mintined only on PEM. Dt were collected on dys 1, 3, 5, 7, nd 8. The cell vibility results re expressed s the percentge test cells surviving compred to control cells. Tretments were performed in triplictes. The cell vibility results for quercetin re shown in Figure 4.1. There ws no significnt difference (P = ; P > 0.05) between non differentited cells, control, nd 25 nm to 10 µm quercetin tretments. The cell vibility results for isorhmnetin re shown in Figure 4.2. Similrly to quercetin, there ws no significnt difference (P = ; P > 0.05) between non differentited cells, control, nd 25 nm to 10 µm isorhmnetin treted cells. During dipocyte differentition in the presence of quercetin or isorhmnetin, verge cell vibility ws nd 97.63%, respectively. Bsed on the results, quercetin or isorhmnetin t the concentrtions used were not cytotoxic to 3T3-L1 during the differentition from pre-dipocytes to dipocytes. The chnges in lipid ccumultion nd morphology of the cells were clerly observed on dy 4, fter the differentition medi ws dded. The complete dipocyte differentition could be chieved between 8 to 10 dys [54]. Becuse the vibility of control, quercetin or isorhmnetin treted, nd nondifferentited cells ws higher thn 90 %; 3T3-L1 cells were mintined for 10 dys for further observtions in terms of lipid ccumultion, morphology nd biomrker mesurements. Yng et l. performed cell prolifertion ssy where the cell vibility ws mesured on the third dy fter the drug ddition (quercetin-3-o-(6 -Feruloyl)-β-D-Glctopyrnoside or quercetin) nd their results showed no significnt difference in prolifertion [13]. 17

29 CELL VIABILITY (% of control) CELL VIABILITY (% of control) QUERCETIN ND Control 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm Dy 1 Dy 3 Dy 5 Dy 7 Dy 8 Figure 4.1. Effects of quercetin on 3T3-L1 cell vibility. Non-differentited 3T3-L1 cells were treted with quercetin (0 to 25 µm) for 0 to 8 dys. The vlues re expressed s percentge of control cells ISORHAMNETIN ND Control 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm Dy 1 Dy 3 Dy 5 Dy 7 Dy 8 Figure 4.2. Effects of isorhmnetin on 3T3-L1 cell vibility. Non-differentited 3T3-L1 cells were treted with isorhmnetin (0 to 10 µm) for 0 to 8 dys. The vlues re expressed s percentge of control cells. 18

30 4.2. Oil Red O Stining The inhibitory effect of quercetin or isorhmnetin on lipid ccumultion ws evluted on dy 10 of tretment, fter the differentition ws induced by dding the differentition medi contining vrious concentrtions of quercetin or isorhmnetin. The retined dye by the intrcellulr lipids ws eluted with isopropnol nd mesured t 510 nm. Tretments were compred to control or non-differentited cells. There were significnt sttisticl differences (P < ; P < 0.05) between non differentited cells, control nd quercetin or isorhmnetin treted cells. Sttisticl nd Tukey s indicted tht there were differences between control isorhmnetin treted, quercetin treted non-differentited, quercetin treted isorhmnetin treted, nd non-differentited control. The results re expressed s percentge of control. Non-differentited cells showed lipid ccumultion of ± 9.35% less thn control Effect of Quercetin on Lipid Accumultion The oil red O stining results for quercetin re shown in Figure 4.3. There ws significnt difference (P = ; P < 0.05) between non differentited cells, control, nd 25 nm to 25 µm quercetin treted cells. Tukey s test stted tht cells treted with 25 µm quercetin were not significntly different from non-differentited cells. Cells treted with 25 µm quercetin down regulted the lipid droplet ccumultion by 8.64 ± 9.54% compred to control. However, the difference ws significnt between non-differentited cells nd 25 nm to 10 µm quercetin. Similrly, Yng et l. stted in their investigtion tht concentrtion of 25 µm decresed lipid ccumultion round 15.9 ± 2.5%, nd concentrtion of 12.5 µm ws not significntly different from control [4]. Furthermore, they found tht combintions of quercetin with resvertrol down regulted the lipid ccumultion by 68.7 ± 0.7%. On the other hnd, Yng et l. reported tht concentrtions of quercetin from 0.1 to 20 µm hve potentil in demonstrting concentrtion-dependent inhibition of dipocyte differentition [13]. Our results suggest tht quercetin lone might not be effective in inhibiting lipid ccumultion s combintion of quercetin nd resvertrol. 19

31 Lipid cummultion (% of control) QUERCETIN b b ND Control 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm 25 μm QUERCETIN Dy 10 Figure 4.3. Effect of quercetin on lipid ccumultion in 3T3-L1 cells. The vlues were expressed s percentge of control cells. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05) mong groups. Differentition ws induced in 3T3-L1 cells for 10 dys with or without quercetin or isorhmnetin. Oil red O stined dipocytes treted with quercetin (0 25 µm) were photogrphed t dy 10. Microscopic observtion ws conducted with n OLYMPUS Model DP72 (Center Vlley, PA) t 5X mgnifiction. Figure 4.4 shows the pictures obtined for quercetin tretments in comprison with nondifferentited cells or control. The pictures clerly show the difference between non-differentited cells nd control t dy 10. Non-differentited cells (Fig A) mintined the initil fibroblst morphology nd show no lipid ccumultion except for control (Fig B), where the intrcellulr lipid ccumultion is noticeble by oil red O stining. As shown in Figure 4.4. (C-J), the concentrtion of quercetin from 25 nm to 25 µm did not highly ttenute the lipid ccumultion in differentited dipocytes s demonstrted by oil red O stining. 20

32 A. Non Differentited cells dy 10 (5X) B. Control dy 10 (5X) C. Q 25 nm dy 10 (5X) D. Q 50 nm dy 10 (5X) E. Q 100 nm dy 10 (5X) F. Q 250 nm dy 10 (5X) Figure 4.4. Effect of quercetin on 3T3-L1 cells. (A) non-differentited cells, (B) control, nd from (C) to (J) 25 nm to 25 µm quercetin treted, respectively. Pictures were obtined with microscope OLYMPUS Model DP72 nd tken t 5X mgnifiction. 21

33 (Figure 4.4. continued) G. Q 500 nm dy 10 (5X) H. Q1 µm dy 10 (5X) I. Q 10 µm dy 10 (5X) J. Q 25 µm dy 10 (5X) Figure 4.4. Effect of quercetin on 3T3-L1 cells. (A) non-differentited cells, (B) control, nd from (C) to (J) 25 nm to 25 µm quercetin treted, respectively. Pictures were obtined with microscope OLYMPUS Model DP72 nd tken t 5X mgnifiction Effect of Isorhmnetin on Lipid Accumultion The oil red O stining results for isorhmnetin re shown in Figure 4.5. There ws significnt difference (P = 0.001; P < 0.05) between non differentited cells, control nd 25 nm to 25 µm isorhmnetin treted cells. Tukey s test results showed tht cells treted from 50 nm to 25 µm isorhmnetin were not significntly different from non-differentited cells. However, control cells were significntly different from ll other tretments. Even 25 nm isorhmnetin ws significntly different 22

34 Lipid cummultion (% of control) from control with difference of ± 12.62% compred to the control. In contrst Lee, et l. reported tht concentrtions of 25 nd 50 µm isorhmnetin significntly lower the lipid ccumultion [9]. ISORHAMNETIN b c bc bc c c c c c ND Control 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm 25 μm ISORHAMNETIN Dy 10 Figure 4.5. Effect of isorhmnetin on lipid ccumultion on 3T3-L1 cells. The vlues re expressed s percentge of control cells. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05) mong groups. Stined dipocytes treted with isorhmnetin (0 25 µm) were photogrphed on dy 10 fter oil red O stining by microscope OLYMPUS Model DP72 (Center Vlley, PA) t 5X mgnifiction. Figure 4.6 shows the pictures obtined for isorhmnetin tretments in comprison with non-differentited cells nd control. The pictures clerly exhibit the difference between non-differentited cells nd control t dy 10. Non-differentited cells (Fig A) on dy 10 mintined the fibroblst morphology nd show no lipid ccumultion compred to the control (Fig B), where the morphologicl ltertions were evident s ccumultion of lipid droplets in the cytoplsm. As shown in Figure 4.6. (C-J), the concentrtion of isorhmnetin from 25 nm to 25 µm ttenuted the lipid ccumultion in differentited dipocytes s demonstrted by oil red O stining. These observtions mtch with the results obtined in the dye quntifiction t 510 nm by spectrophotometry. 23

35 A. Non Differentited cells dy 10 (5X) B. Control dy 10 (5X) C. IS 25 nm dy 10 (5X) D. IS 50 nm dy 10 (5X) E. IS 100 nm dy 10 (5X) F. IS 250 nm dy 10 (5X) Figure 4.6. Effect of isorhmnetin on 3T3-L1 cells. (A) non-differentited cells, (B) control, nd from (C) to (J) 25 nm to 25 µm quercetin treted, respectively. Pictures were obtined with microscope OLYMPUS Model DP72 nd tken t 5X mgnifiction. 24

36 (Figure 4.6. continued) G. IS 500 nm dy 10 (5X) H. IS 1 µm dy 10 (5X) I. IS 10 µm dy 10 (5X) J. IS 25 µm dy 10 (5X) Figure 4.6. Effect of isorhmnetin on 3T3-L1 cells. (A) non-differentited cells, (B) control, nd from (C) to (J) 25 nm to 25 µm quercetin treted, respectively. Pictures were obtined with microscope OLYMPUS Model DP72 nd tken t 5X mgnifiction Effects of Quercetin or Isorhmnetin on the Secretion of MCP-1 during Cell Differentition To determine the effect of quercetin or isorhmnetin on secretion of cytokine MCP-1, the superntnts were collected on dy 0, 2, 5, 7, nd 10. Only smples from dys 0, 2, nd 10 were mesured by ELISA using commercil kits from ebioscience (Sn Diego, CA). Tretments were compred ginst the control nd non-differentited cells. There were significnt sttisticl differences (P < ; P < 0.05) between non differentited cells, control nd quercetin or isorhmnetin tretments. The mens seprtion by Tukey s suggested tht there re differences between non-differentited control, control 25

37 Concentrtion (pg/µg) isorhmnetin treted, nd quercetin treted non-differentited. Between quercetin treted cells nd isorhmnetin treted cells significnt difference ws not evident. The results were expressed per the protein concentrtion of the superntnt obtined by BCA protein ssy Effects of Quercetin on the Secretion of MCP-1 during Cell Differentition The MCP-1 results for secretion over the dipocyte differentition of quercetin re shown in Figure 4.7. There ws no significnt difference (P = ; P > 0.05) between non differentited cells, control nd 25 nm to 25 µm quercetin treted cells. These dt suggest tht quercetin does not ffect the MCP-1 levels per dy during the dipocyte differentition. Extrcts rich in p-coumric cid, quercetin, nd resvertrol chnge levels of MCP-1 [56] suggesting tht quercetin lone might not hve inhibition properties ginst MCP-1. Studies on 3T3-L1 cells with urptene t concentrtions from 0 to 100 µm hd reported results tht suggest decrese in the levels of MCP-1. Additionlly, the effects of levels of urptene on MCP-1 were dose dependent [12] QUERCETIN Control ND 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm 25 μm Dy 0 Dy 2 Dy 10 Figure 4.7. Effect of quercetin on MCP-1 levels (pg/µg protein) during 3T3-L1 dipocyte differentition. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). 26

38 Concentrtion (pg/µg) Effects of Isorhmnetin on the Secretion of MCP-1 during Cell Differentition The levels of MCP-1 in the superntnts of 3T3-L1cells treted with vehicle, undifferentited, or isorhmnetin re shown in Figure 4.8. There ws no significnt difference (P = ; P > 0.05) between non differentited cells, control nd 25 nm to 25 µm isorhmnetin tretments. These dt suggest tht isorhmnetin does not ffect the MCP-1 levels during dipocyte differentition. Other studies in humn umbilicl rtery smooth muscle cells stimulted with TNF-α for 24 h in order to rouse the MCP-1 secretion showed tht quercetin t 2 µm to 10 µm decresed the secretion of MCP-1 to 18% nd 67%, respectively. However, isorhmnetin which is metbolite of quercetin did not ffect MCP-1 secretion [57] ISORHAMNETIN Control ND 25 nm 50 nm 100 nm 250 nm 500 nm 1 μm 10 μm 25 μm Dy 0 Dy 2 Dy 10 Figure 4.8. Effect of isorhmnetin on MCP-1 levels (pg/µg protein) during 3T3-L1 dipocyte differentition. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05) Expression of PPAR-γ, C/EBP-α, nd β-ctenin during 3T3-L1 Cell Differentition In order to understnd the differentition process of 3T3-L1 cells during the cell culture the expression of β-ctenin, PPAR-γ, nd C/EBP-α ws monitored. Cell lystes were collected fter predipocyte expnsion medium (PEM) ws replced with differentition medium (DM). Tht ws considered dy 0 (D0); next smples were collected fter 5, 16, nd 24 hours. Subsequently fter 48 h, 27

39 DM ws replced by dipocyte mintennce medium (AMM) every 72 h, smples were sved t dy 2 (D2), dy 5 (D5), dy 7 (D7), nd dy 10 (D10). Finlly, smple from non-differentited cells ws collected on dy 10. The totl cell lystes were nlyzed by western blot nlysis s described under the mterils nd methods. Results re shown in Figure 4.9. Figure 4.9, demonstrted tht the β-ctenin level decresed until dy 5, nd incresed slightly t dy 7 nd 10. Non-differentited cells hd similr level of β-ctenin s D0. Peroxisome prolifertorctivted receptor-γ (PPAR-γ) levels incresed by dy 10 (D10). CCAAT/enhncer binding protein α (C/EBP) ws visible on dy 10 (D10) nd β-ctenin level decresed. Lee et l. showed tht β-ctenin level decresed t erly period fter DM ws dded, while differentition ws criticl s reveled by the significnt up-regultion of PPAR-γ nd C/EBP-α [58]. DIFFERENTIATION D0 5h 16h 24h D2 D5 D7 D10 ND MW(kD) β-ctenin 92 PPAR-γ 2 PPAR-γ C/EBP-α β-ctin 45 Figure 4.9. Western blot nlysis results for control cells during the dipocyte differentition period of 10 dys. 28

40 Concentrtion (% of Dy 0) Western blots developed by enhnced chemiluminescence were quntittively nlyzed by Quntity One softwre. Figure shows the results obtined for β-ctenin expression during dipocyte differentition. β-ctenin level ws decresed slowly until D5, nd incresed in dy 7, nd 10. There ws significnt difference for β-ctenin expression (P < ; P < 0.05) during dipocyte differentition. These dt show tht β-ctenin expression decresed to ± 2.43% of control during dipocyte differentition. β - ctenin b bc c d e e e f D0 5h 16h 24h D2 D5 D7 D10 ND Figure β-ctenin expression in control cells during dipocyte differentition. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). C/EBP-α expression through dipocyte differentition is shown in Figure 4.11.; there ws significnt difference (P < ; P < 0.05). C/EBP-α p30 nd p42 were mintined the sme from D0 to D7, nd incresed significntly in ± 0.40% nd ± 1.24% respectively from D7 to D10. Also, C/EBP-α p30 nd p42 level in non-differentited cells ws slightly higher thn the level mintined until D7. 29

41 Concentrtion (% of D0) Concentrtion (% of D10) C/EBP-α b b c c c c c c c c c c c c c c D0 5h 16h 24h D2 D5 D7 D10 ND C/EBP-α p42 C/EBP-α p30 Figure C/EBP-α expression during dipocyte differentition. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). PPAR-γ expression through dipocyte differentition is shown in Figure 4.12.; there ws significnt difference (P < ; P < 0.05). Highest levels of PPAR-γ 1 nd 2 were t D7 nd decresed by ± 1.34% nd ± 4.06% respectively from D7 to D10. Also, PPAR-γ 1 nd 2 levels of nondifferentited cells on D10 were not significntly different from D0.Other importnt observtion ws tht PPAR-γ 1 nd 2 levels were normlly incresing until D2 nd decresed by 3.27 ± 0.91% nd ± 0.34% respectively t D5. PPAR-γ h g g f c b b d b d e e c f h g 0.00 D0 5h 16h 24h D2 D5 D7 D10 ND PPAR-γ 2 PPAR-γ 1 Figure PPAR-γ expression during dipocyte differentition. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). 30

42 4.5. Effects of Quercetin on PPAR-γ, C/EBP-α, nd β-ctenin during 3T3-L1 Cell Differentition To quntify the effect of quercetin - dipocyte differentition by quercetin, we determined the expression of β-ctenin, PPAR-γ, nd C/EBP-α in lystes of quercetin treted cells t dy 10, nd compred with control nd non-differentited cells s described under the mteril nd methods. The results shown in Figure 4.13 clerly show tht β-ctenin level is more intense in non-differentited cells; nd decresed fter quercetin tretment. PPAR-γ decresed with incresing concentrtion of quercetin; t 25 µm quercetin the PPAR-γ 1 nd 2 were t the lowest levels nd lowers even thn non-differentited cells. C/EBP p30 nd p42 levels behved similrly to PPAR-γ 1 nd 2. They decresed criticlly t 25 µm quercetin nd were even lowers thn non-differentited cells. These results suggest tht PPAR-γ 1 nd 2 nd C/EBP-α p30 nd p42 expressions were concentrtion dependent. QUERCETIN DAY 10 nm µm ND CTR MW (kd) β-ctenin 92 PPAR-γ 2 PPAR-γ C/EBP-α β-ctin 45 Figure Western blot of β-ctenin, PPAR-γ, nd C/EBP-α on dy 10 in response to quercetin concentrtion from 25 nm to 25 µm. 31

43 The results of β-ctenin expression in cells treted with vehicle, non-differentited or 25 nm to 25 µm quercetin re shown in Figure The highest expression of β-ctenin level ws in nondifferentited cells, nd the lowest t 25 µm quercetin. Level of β-ctenin did not follow trend, these suggest it expression ws not dose dependent. There ws significnt difference in β-ctenin expression (P < ; P < 0.05) mong incresing concentrtions of quercetin from 25 nm to 25 µm. Nondifferentited cells hd β-ctenin level ± 1.59% higher thn control. However, these dt suggest tht quercetin concentrtions cn ffect β-ctenin expression. C/EBP-α levels in quercetin treted cells re shown in Figure 4.15.; there ws significnt difference (P < ; P < 0.05). C/EBP-α level did not hve specific trend in response to quercetin concentrtions. However, t incresing concentrtions of quercetin the levels of C/EBP-α p30 nd p42 decresed grdully. Overll, C/EBP- α p30 nd p 42 levels decresed by ± 2.66% nd ± 2.60%, respectively compred to control. Recent studies hve shown tht quercetin t 25 µm did not hve ny effect on the expression of C/EBP-α p30 nd p42, wheres quercetin nd resvertrol significntly decresed the expression by 60.2 ± 1.2% nd 45.4 ± 1.2% respectively [4] PPAR-γ levels in cells treted with 25 nm to 25 µm quercetin re shown in Figure 4.16.; there ws significnt difference (P < ; P < 0.05). PPAR-γ level hd decresed s quercetin concentrtion incresed. We lso observed tht level of PPAR-γ decresed grdully, this suggest dependent dosge reltion between PPAR-γ nd quercetin. PPAR-γ 1 nd 2 lowest levels were observed t 25 µm quercetin. Overll the PPAR-γ 1 nd 2 levels decresed by ± 3.17% nd ± 12.39% respectively compred to control. Similr study hd reported no significnt effect on PPAR-γ 1 nd 2 expression t concentrtions of 12.5 or 25 µm quercetin [4]. Other flvonoid compounds s nobiletin t concentrtion from 0 to 100 µm hd reported similr results bout down-regultion of PPAR-γ expression s quercetin [3]. 32

44 Concentrtion (% of control) Conceentrtion (% of control) β - ctenin b d d e f c h f g ND Control 25nM 50nM 100nM 250nM 500nM 1uM 10uM 25uM Figure β-ctenin expression in quercetin (25 nm to 25 µm) treted cells. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). C/EBP-α e d b b d b b b b b e d d c f e ND Control Q25nM Q50nM Q100nM Q250nM Q500nM Q1uM Q10uM Q25uM C/EBP-α p42 C/EBP-α p30 Figure C/EBP-α expression in quercetin (25 nm to 25 µm) treted cells. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). 33

45 Concentrtion (% of control) PPAR-γ bc d d cd cd d cd b e b ND Control 25nM 50nM 100nM 250nM 500nM 1uM 10uM 25uM PPAR-γ 2 PPAR-γ 1 Figure PPAR-γ expression in quercetin (25 nm to 25 µm) treted cells. Results re presented s men ±S.D. (n = 3). Letters with different superscripts re significntly different (P < 0.05). 4.6 Effects of Isorhmnetin on PPAR-γ, C/EBP-α, nd β-ctenin during 3T3-L1 Cell Differentition To quntify inhibition of dipocyte differentition by isorhmnetin, the expression of β-ctenin, PPAR-γ, nd C/EBP-α in cells treted with 25 nm to 25 µm ws exmined on dy 10, nd compred with control nd non-differentited cells s described in the mteril nd methods. The expression of β-ctenin, PPAR-γ nd C/EBP-α on D10 is shown in Figure The β-ctenin level is highly expressed in non-differentited cells; lso β-ctenin is expressed mong isorhmnetin tretments. PPAR-γ 1 nd 2 expressions decresed sensitively with incresing concentrtions of isorhmnetin PPAR-γ 1 nd 2 levels were lowest even in comprison with nondifferentited cells. C/EBP-α p30 nd p42 levels lso decresed. These results suggest tht PPAR-γ nd C/EBP-α expressions re dosge dependent. 34

46 ISORHAMNETIN DAY 10 nm µm ND CTR MW (kd) β-ctenin 92 PPAR-γ 2 PPAR-γ C/EBP-α β-ctin 45 Figure Western blot nlysis of β-ctenin, PPAR-γ, nd C/EBP-α on dy 10 in response to isorhmnetin concentrtion from 25 nm to 25 µm. β-ctenin expression in response of isorhmnetin concentrtion from 25 nm to 25 µm, control nd non-differentited cells is shown in Figure Highest expression of β-ctenin level ws in control cells, nd the lowest in 100 nm isorhmnetin treted cells. Level of β-ctenin does not follow specific trend, these suggest it expression is not dosge dependent. There ws significnt difference for β-ctenin expression (P < ; P < 0.05) mong isorhmnetin tretments. Quercetin mintined levels of β- ctenin over ± 0.30%, while isorhmnetin levels were bove ± 0.95% of control. These results suggested tht quercetin hd better effects to mintin β-ctenin level thn isorhmnetin. The results of C/EBP-α level in cells treted with 25 nm to 25 µm re shown in Figure There ws significnt difference (P < ; P < 0.05) mong t incresing concentrtions of isorhmnetin from 25 nm to 25 µm. C/EBP-α p30 nd p42 levels decresed with incresing concentrtion 35