research article antiadipogenic effects of aster glehni …luna 4.60 × 250 mm c reversed-phase...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 859624 10 pageshttpdxdoiorg1011552013859624

Research ArticleAntiadipogenic Effects of Aster glehni ExtractIn Vivo and In Vitro Effects

Heon-Myung Lee1 Gabsik Yang1 Tae-Gue Ahn1 Myung-Dong Kim2 Agung Nugroho3

Hee-Juhn Park3 Kyung-Tae Lee4 Wansu Park5 and Hyo-Jin An1

1 Department of Pharmacology College of Oriental Medicine Sangji University Wonju-si Gangwon-do 220-702 Republic of Korea2Department of Physiology College of Oriental Medicine Sangji University Wonju-si Gangwon-do 220-702 Republic of Korea3 Department of Pharmaceutical Engineering Sangji University Wonju-si Gangwon-do 220-702 Republic of Korea4Department of Pharmaceutical Biochemistry College of Pharmacy Kyung Hee University Seoul 130-701 Republic of Korea5 College of Oriental Medicine Kyungwon University Seongnam 461-701 Republic of Korea

Correspondence should be addressed to Hyo-Jin An hjansjackr

Received 29 January 2013 Revised 2 May 2013 Accepted 27 May 2013

Academic Editor Yoshiyuki Kimura

Copyright copy 2013 Heon-Myung Lee et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Aster glehni (AG) is a Korean traditional herb that grows in Ulleungdo Island Republic of Korea None of the several reports onAG include a determination of the effect of AG on adipogenesis The primary aim of this study was to determine whether AGattenuates adipogenesis in mouse 3T3-L1 cells and epididymal fat tissue AG blocked the differentiation of 3T3-L1 preadipocytes ina concentration-dependent manner and suppressed the expression of adipogenesis-related genes such as PPAR120574 CEBP120572 andSREBP1c the master regulators of adipogenesis Male C57BL6J mice were divided randomly and equally into 4 diet groupscontrol diet (CON) high-fat diet (HFD) HFD with 1 AG extract added (AG1) and HFD with 5 AG extract added (AG5)The experimental animals were fed HFD and the 2 combinations for 10 weeks Mice fed HFD with AG gained less body weight andvisceral fat-pad weight than did the mice fed HFD alone Moreover AG inhibited the expression of important adipogenic genessuch as PPAR120574 CEBP120572 SREBP1c LXR and leptin in the epididymal adipose tissue of the mice treated with AG1 and AG5 Thesefindings indicate antiadipogenic and antiobesity effects of AG and suggest its therapeutic potential in obesity and obesity-relateddiseases

1 Introduction

Overweight and obesity are defined as abnormal or excessivefat accumulation that may impair health globally excessivebody weight is the fifth leading risk factor for death Atleast 28 million adults die each year as a result of obe-sity Furthermore 44 of the diabetes burden 23 of theischemic heart disease burden and 7 to 41of the burden ofspecific cancers are attributable to obesity [1]Obesity is now aworldwide health problem regardless of age sex or ethnicityand is associated with fatal diseases [2]

The process by which undifferentiated preadipocytesbecome adipocytes is termed adipogenesis The differen-tiation to adipocyte is a complex developmental proce-dure that involves the coordinated interplay of numeroustranscription factors [3] Peroxisome proliferator-activated

receptor 120574 (PPAR120574) and CCAATenhancer binding pro-teins (CEBP120572) which are crucial transcription factors inadipogenesis activate the expression of other adipocytemarkers [4] Sterol regulatory element binding protein 1c(SREBP1c) is also important in the regulation of obesity andadipocyte differentiation [5] Lipoprotein lipase (LPL) is atranscription gene of the lipogenic enzyme [6] and liver Xreceptor (LXR) activation enhances lipogenesis [7] leptin isa hormone secreted by adipose tissues that plays a key role inregulating energy intake and energy expenditure includingappetitehunger and metabolism [8]

The common side effects of conventional pharmacolog-ical obesity treatmentsmdashhypertension cardiac arrhythmiaconstipation headache steatorrhea and deficiencies of lipid-soluble vitamins and essential fatty acids [9 10]mdashhavecontributed to a focus on herbal medicine as a healthcare

2 Evidence-Based Complementary and Alternative Medicine

modality Several natural products extracted from herbalplants have been shown to be effective for specific conditionsSome natural extracts as well as the principal componentsof the extracts such as berberine and resveratrol haveantiobesity effects [11 12] Studies on the efficacy of herbalmedicines are actively going on in all parts of the world

Aster glehni Franchet et Sckmidt (AG) which is com-mon to Ulleungdo Island Republic of Korea is officiallyrecognized as a regional specialty that grows only in thisregion It has recently been named Aster pseudoglehni LimHyun amp Shin In the republic of Korea the leaves of AGare used in a dish called chwinamul and AG has alsobeen used to treat diabetes mellitus hypercholesterolemiainsomnia and cardiovascular disease [13] According to arecent study AG extract has both sedative and anticonvulsanteffects [14] Previously we reported that the AG rich in CQcompound lowers the total lipid triglyceride phospholipidand cholesterol levels in animal model [15] Although AGrsquoseffect on body weight in rats has been reported [15] nostudy has focused on the antiadipogenic effect of AG in vitroSpecific adipogenic signal genes were examined to elucidatethe specific mechanism underlying the effect of AG Thepresent study considered the antiobesity and antiadipogeniceffects of AG in 3T3-L1 preadipocytes and mice

2 Materials and Methods

21 Plant Material and Preparation of the Extract AG leavesfrom Ulleungdo Island Republic of Korea were dried andcrushed for extraction The dried material was refluxedwith 70 EtOH for 6 h at 60∘C The extract was filteredconcentrated under reduced pressure and then freeze-driedto obtain a solid extract powder

22 Cell Culture The 3T3-L1 mouse preadipocytes werecultured in Dulbeccorsquos Modified Eaglersquos Medium (DMEMLife Technologies Inc Grand Island NY USA) supple-mentedwith 10bovine calf serum 100120583gmL streptomycinand 100 unitsmL penicillin at 37∘C in 10 CO

2 The cells

were grown to confluency in 10 calf serumDMEM Twodays after the cells had reached confluency (day 0) theywere stimulated with a methylisobutylxanthine dexametha-sone and insulin (MDI) induction medium (DMEM con-taining 10 fetal bovine serum (FBS) 05mM 3-isobutyl-1-methylxanthine (IBMX) 1 120583mmL dexamethasone and1 120583gmL of insulin) Two days after stimulation with MDI(day 2) the medium was changed to an insulin medium(DMEM containing 10 fetal bovine serum and 1120583gmL ofinsulin) Two days later (day 4) the medium was changed to10 FBSDMEM The cells were fed with 10 FBSDMEMevery 2 days Full differentiation was achieved by day 8

23 Oil Red O Staining To determine both adipogenicpotential and fat accumulation we stained the cells withoil red O (Sigma Chemical Co St Louis MO) On day8 the cultured cells were washed thrice with phosphate-buffered saline (PBS) and then fixed with 4 formaldehydefor 30min at room temperature The cells were washed 3

times more with PBS and stained with 05 120583gmL oil red Ofor 15min After rinsing with PBS we captured representativephotomicrographs using a microscope and measured fataccumulation

24 AdipoRed Assay 3T3-L1 cells were cultured in 6-wellplates at a density of 3 times 105 cells per well and set up todifferentiate in the presence and absence of AG On day 2 andday 8 of differentiation the cells were exposedwithAdipoRed(Lonza Houston TX USA) and intracellular triglyceride(TG) level was analyzed

25 Leptin Assay For the periods indicated 3T3-L1 cellswere treated with AG or not Conditioned media from thefinal 24 h of culture were collected Leptin concentrationswere determined using an enzyme-linked immunosorbentassay (ELISA Mouse Leptin Assay Kit Immuno-BiologicalLaboratories Co Tokyo Japan)

26 Real-Time PCR Analysis Total RNA was isolated fromthe differentiated 3T3-L1 cells and epididymal adipose tissueby using Easy-Blue Reagent (Intron Biotechnology IncGyoenggi-do Republic of Korea) according to the manu-facturerrsquos instructions Then total RNA qualification wasperformed with an Epoch micro-volume spectrophotometersystem (BioTek Instruments Inc Winooski VT USA) Anequal amount of total RNA was used to synthesize cDNAwith a high-capacity cDNA reverse transcription kit (AppliedBiosystems Foster City CA USA) The program was set for10min of initiation at 25∘C followed by 90min of incubationat 50∘C and 5min at 85∘C Primers were obtained fromBioneer (Daejeon Republic of Korea) All primer sequencesand annealing temperatures are shown in Table 1 A StepOne Plus Real-time PCR system (Applied Biosystems FosterCity CA USA) with an SYBR Green Master Mix (AppliedBiosystems Warrington UK) and primers (Bioneer SeoulRepublic of Korea) was used to carry out a real-time PCRThe steps were as follows 10min at 95∘C 40 cycles of 5 sat 95∘C 45 s at 60∘C a final melting curve of 15 s at 95∘C1min at 60∘C 15 s at 95∘C for tissues or for cells and 5minfollowed by 35 repetitive thermal cycles (94∘C for 30 s 60∘Cfor 30 s and 72∘C for 30 s) Fold changes of gene expressionwere calculated using the comparative threshold cycle (Ct)method (Applied Biosystems) The data were normalized forthe initial control GAPDH

27 Western Blot Analysis The 3T3-L1 cells were lysed ina lysis buffer (25mM HEPES pH 74 100mM NaCl 1mMEDTA 5mM MgCl

2 01mM dithiothreitol and protease

inhibitor mixture) Determination of the protein was carriedout on Tris-glycine SDS-polyacrylamide gels followed by atransfer to PVDF membranes (EMD Millipore CorporationBillerica MA USA) Then the membranes were blockedwith 5 skim milk in Tris-buffered saline containing 01tween 20 (TBST) at room temperature for 1 h and incubatedat 4∘C overnight with 1 1000 dilutions of primary antibodiesThe following day the membranes were washed with TBST3 times for 10min each and made to react with 1 2500

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences and PCR conditions

Gene name 119879119898

(∘C) Size (bp) Sequence 51015840-31015840

Peroxisome proliferator-activated receptorgamma (PPAR120574) 55 176 F TCGGAATCAGCTCTGTGGA

R CCATTGGGTCAGCTCTTGTGCCAATenhancer binding protein alpha(CEBP120572) 55 151 F ACAACGCAACGTGGAGAC

R ACCAAGGAGCTCTCAGGCAGSterol regulatory element binding transcriptionfactor 1 (SREBP1c) 55 198 F ATCGCAAACAAGCTGACCTG

R AGATCCAGGTTTGAGGTGGG

Liver X receptor (LXR) 55 119 F TCCTACACGAGGATCAAGCGR AGTCGCAATGCAAAGACCTG

Lipoprotein lipase (LPL) 55 158 F AGGACCCCTGAAGACACAGCR TTGGGCACCCAACTCTCATA

Leptin 55 143 F CTCCAAGGTTGTCCAGGGTTR AAAACTCCCCACAGAATGGG

dilutions of horseradish peroxidase-conjugated secondaryantibody for 2 h at room temperature After the reaction thecomplexes were visualized with ECL plus detection reagents(GenDEPOT Barker TX USA)

28 Animal Experiments Male C57BL6J mice weighing 15ndash17 g at an age of 3-4 weeks were purchased from DaehanBiolink (DaeJeon Republic of Korea) They were housed(5micecage) under a 12 h light-dark cycle at 23 plusmn 2∘C anda relative humidity of 55 plusmn 10 under conditions thatfollowed the NIH Guide for the Care and Use of LaboratoryAnimals they were allowed diet and water ad libitum After1 week of acclimation 40 mice were divided randomly andequally into 4 groups of 10 each Mice in group 1 (CON) werefed standard chow and those in group 2 (HFD) a HFD (40fat) group 3 (AG1) consisted of mice fed with the same HFDwhich included 1AG extract (1ww inHFD) and group 4(AG5) consisted of mice fed HFD with 5 added AG extract(5 ww in HFD) Mice were fed experimental diet for 10weeks Body weight was recorded weekly After an overnightfast at the end of the experimental period tissue collectionwas performedThe animals were sacrificed by cervical spinedislocation and the livers epididymal retroperitoneal andtotal fat pads were dissected weighed immediately andfrozen at minus80∘C

29 Histological Analysis The liver and the epididymaladipose tissues were fixed with 4 paraformaldehyde andembedded in paraffin Sections of 4 and 8120583m were cut andstained with hematoxylin and eosin (HampE) for analysis ofadipocyte diameter surface area and number The stainedslides were observed with a SZX10 microscope (OlympusSeoul Republic of Korea) and photographed

210 HPLC Analysis TheHPLC apparatus was a Gilson Sys-tem equippedwith a 234 autosampler aUVVIS-155 detectorand a 321 HPLC Pump (Gilson Seoul Republic of Korea) ALuna 460 times 250mm C18 reversed-phase column with 5 120583mparticles (Phenomenex CA USA) was used The columneluent was monitored at UV 246 nm following which allsolvents were degassed with a micromembrane filter (PTFEAdvantec Tokyo Japan) Chromatography was performed at

room temperature at a flow rate of 05mLmin and 10 120583L wasanalyzed for 50min In brief seven CQs and the four extractsdissolved in 80 MeOH were filtered using a syringe filterand injected for HPLC analysis The two mobile phases of005 phosphoric acid (solvent A) and MeOH (solvent B)were used for gradient elution at the rate of 100mLmin0ndash10min 60 A 40 B 10ndash20min 50 A 50 B 20ndash30min 40 A 60 B 30ndash35min 60 A 40 B The twoflavonoids astragalin and kaempferol were also used as thestandard compounds for the analysis Under these HPLCidentical conditions the retention times of astragalin andkaempferol were 144min and 243min respectively Plottingthe peak area (119910 counts) versus concentration (119909 120583gmL)regression equations of 119910 = 2710119909 + 5050 (1198772 = 0998)and 119910 = 5485119909 + 28701 (1198772 = 0999) for astragalin andkaempferol respectively Sample solutions were injected intothe HPLC system at 1000mgmL and the contents weredetermined from the regression equation

211 Statistical Analysis The results are presented as themean plusmn SEM of the 3 independent experiments The datawere analyzed by one-way analysis of variance (ANOVA)statistical analyses were performed using SPSS (Version 190)A 119875 value of lt005 was considered statistically significant

3 Results

31 AG-Mediated Inhibition of Adipogenesis in PreadipocytesTo determine whether AG has an antiobesity effect wecarried out adipocyte differentiation using 3T3-L1 cells bothin the presence and the absence of AG After 8 days oil redO was added to the cultured cells and fat accumulation andintracellular triglyceride levels were analyzed Troglitazonean antidiabetic and anti-inflammatory drug [16] was usedas a positive control for the inhibition of fat accumulationAs shown in Figure 1 the number and size of cytosoliclipid droplets markedly decreased (Figure 1(a)) and reduc-tion in fat accumulation dependent on concentration wasobserved (Figure 1(b)) Intracellular TG was analyzed withthe AdipoRed assay On day 2 the accumulation of TG inthe differentiated cells was similar to that in the AG-treatedcells (Figure 1(c)) After 8 days the AG-treated differentiated


ND CON AG 100120583gmL AG 200120583gmL AG 400120583gmL

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Figure 1 Effects of AG on adipocyte differentiation (a) Fat droplets in adipocyte differentiated for 8 days with or without AG were stainedwith oil red dye (b) Relative lipid accumulation was calculated Troglitazone was used as a positive control Accumulation of intracellulartriglycerides was calculated by AdipoRed assay at (c) day 2 and (d) day 8 in differentiated adipocyte (e) Leptin levels in adipocyte with orwithout AG 119875 lt 005 versus CON group lowast119875 lt 005 versus HF group lowastlowast119875 lt 001 versus HF group and lowastlowastlowast119875 lt 0001 versus HF group

cells showed significantly lower TG levels exhibiting a dose-dependent change than those in the untreated differentiatedcells (Figure 1(d)) To assess cellular capacity to produceadipocyte-derived hormones wemonitored the expression ofleptin a well-documented hormone with anti-diabetic prop-erties in cells of adipocyte lineage Significant suppression ofleptin protein secretion was observed in 3T3-L1 cells by thetreatment with AG (Figure 1(e))

32 AG Regulation of the Expression of Adipogenic Genesin Adipocytes The differentiation of adipocytes frompreadipocytes is correlated with the expression of adipogenicgenes PPAR120574 CEBP120572 and SREBP1c are key regulatorsof adipogenesis and LXR is also involved After celldifferentiation for 8 days in the presence or absence of100 200 and 400 120583gmL of AG the expression levels ofadipogenic markers were determined by real-time PCR The


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ive m

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lowastlowastlowast

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(d)

Figure 2 Effects ofAGon expression of adipogenicmarkersDifferentiated adipocyteswere treatedwith 100 200 and 400 120583gmL respectivelyCells treated with AG were harvested at day 8 and the expressions of genes were analyzed by real-time PCR Each bar represents the mean plusmnSEM of triplicate PCR reactions andmRNA samples were obtained from experimental adipocyte cells 119875 lt 005 versus ND group lowast119875 lt 005versus CON group lowastlowast119875 lt 001 versus CON group and lowastlowastlowast119875 lt 0001 versus CON group

8-day AG treatment of cells reduced the mRNA expressionof PPAR120574 CEBP120572 SREBP1c and LXR (Figure 2)

33 AG-Mediated Reduction of BodyWeight Visceral Epididy-mal Retroperitoneal and Total Fat-Pad Weights and LiverWeight in HFD-Induced Obese Mice Figure 3 indicates thechanges in total body as well as epididymal retroperitonealand total fat pads and liver weight all of which weresignificantly greater in the HFD group than in the controlgroup The increases in body weight fat weights and liverweight in the AG1 group were lesser than those in the HFDgroup (Figures 3(a) 3(b) 3(e) and 3(f)) The correspondingweight increases in the AG5 group were significantly lesserthan those in the HFD and CON groups (Figures 3(a) and3(b)) The food intake amount was not different among eachgroup (Figure 3(c)) so the effect of AG is not an impactof reduced food intake However the HFD groups showedhigher caloric food intake (kcaldaymouse) than the CONgroup (Figure 3(d)) The caloric food intake was also notdifferent among each groups

34 AG-Mediated Amelioration of HFD-Induced Fat Accumu-lation in Liver and Adipose Tissue To determine whether ornot the decreased body visceral and liver weights were due toreduced accumulation of fat we stained representative liverand adipose tissues with HampE As shown in Figure 4 liversections of the HFD group unlike those of the CON groupexhibited lipid deposition in the hepatocytes particularlyaround the central vein Considering mild intracytoplasmichepatocyte vacuolation the histological liver images of theAG1 and AG5 groups indicate that AG ameliorated the accu-mulation of lipids (Figure 4(a)) Because fatty degeneration inadipose tissues is easy to identify we compared the diameterand number of lipid droplets in the adipose tissues As shownin Figure 4(b) AG1 and AG5 decreased the diameter and thenumber of lipid droplets markedly (Figure 4(b))

35 In Vivo AG-Mediated Modulation of Adipogenic-RelatedSignaling Molecules in Adipose Tissue To analyze theinhibitory effects of AG on the adipogenic mRNA expressioninduced by HFD in epididymal white adipose tissue we


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Figure 3 Effects of AG on body weight gain fat-pads weights and liver weights of N HF AG1 and AG5 group mice Mice were fed normaldiet or HFD for 10 weeks in the presence or absence of AG (a) Changes in total body (g) (b) changes in body weight gain (g) (c) food intake(d) caloric food intake (e) visceral fat-pad weight and (f) liver weights in each group are shown 119875 lt 005 versus CON group lowast119875 lt 005versus HF group lowastlowast119875 lt 001 versus HF group and lowastlowastlowast119875 lt 0001 versus HF group

conducted a quantitative real-time PCR analysis As itaffected adipogenesis in vitro AG attenuated the expressionof mRNA related to adipogenesis The expression of PPAR120574was evidently reduced in the epididymal tissue takenfrom AG-treated HFD mice The expression of CEBP120572 adownstream target of PPAR120574 also decreased significantlyOther adipogenic markers such as SREBP1c LXR LPLand leptin were reduced markedly in the epididymal tissueof AG-treated mice (Figure 5(a)) In parallel with mRNA

expressions smaller amounts of PPAR120574 CEBP120572 andSREBP1c proteins were produced in epididymal adiposetissue obtained from AG-treated HFD mice than from micegiven HFD alone (Figure 5(b))

36 Characterization of AG Using HPLC Systems The CQswere analyzed by HPLC using seven standard compoundsTheCQs identified byHPLCusing standard compoundswere3-pCQ 34-DQ 5-CQ and 35-DQ (Figure 6) When the


times100

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Figure 4 Effects of AG on fat accumulation Representative histological images of the (a) liver (b) epididymal adipose tissue as assessed byHampE staining and examined using a light microscope magnification times100 times400 (c) Adipocyte number and diameter

005

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PPAR120574 CEBP120572 SREBP LPLLXR Leptin

lowast

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Figure 5 Effects of AG on expression of adipogenic signal genes (a) Real-time PCR analysis of epididymal adipose tissue from experimentalmice shows the expression of adipogenesis-related transcription Data were normalized to the GAPDH mRNA levels and then comparedto CON group measurements which were assigned a value of 10 (b) Protein levels of PPAR120574 CEBP120572 and SREBP1c were determined byWestern blotting Each bar represents the mean plusmn SEM of 3 independent experiments of the RNA and protein samples obtained from 5 miceper each group 119875 lt 005 versus CON group lowast119875 lt 005 versus HF group lowastlowast119875 lt 001 versus HF group and lowastlowastlowast119875 lt 0001 versus HF group


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Figure 6 HPLC chromatograms of the extract from AG

Table 2 Content of caffeoylquinic acids and flavonoids in AG

Compounds Contents (mgg)35-DQ 4435-CQ 13693-CQ 3253-pCQ 3406Astragalin 132Kaempferol 023

contents of the two flavonoids were quantitatively comparedastragalin and kaempferol were 132mgg and 023mgg(Table 2)

4 Discussion

The number of people with obesity and obesity-relateddiseases such as diabetes mellitus hypertension coronaryartery disease and cancers [17] has increased at an alarmingrate all over the world [18] Consequently the idea of devel-oping antiobesity drugs with no undesirable side effect hasbecome a hot topic [19] Herbal medicine has been looked atas a complementary treatment [20] To keep pace with globalresearch trends we investigated the antiobesity effects of AGa plant found inmountainous terrainThe results of this studyindicated the antiadipogenic effects of AG both in vitro andin vivo In vitro the AG blocked adipocyte differentiation aswell as the accumulation of fat and intracellular TG in 3T3-L1adipocytes AGalso decreased leptin levels dose-dependentlywhich are a marker of adipocyte differentiation In vivo AGreduced body weight fat-pad weight and liver weight Inaddition AG decreased the expression of adipogenic signalgenes in experimental obese mice

Several reports have indicated PPAR120574 CEBP120572 andSREBP1c as the major transcriptional genes involved inadipogenesis [3 21 22] PPAR120574 is a ligand-activated tran-scription factor that plays an important role in the regulation

of obesity It exists as a heterodimer with another nuclearhormone receptor retinoid X receptor or RXR to bindto DNA and it is actively involved in adipogenesis [23]Adipocyte differentiation provokes PPAR120574 activity and ittransfers hormonal stimulation to its target genes such asCEBP120572 and LPL [19 24] Although CEBP120572 is induced byPPAR120574 CEBP120573 and CEBP120575 members of CEBP familyare detected at a very early stage of adipocyte differentiationfollowed by PPAR120574 and they accelerate the process ofadipogenesis acting with PPAR120574 [25] Despite its role as atarget gene of PPAR120574 CEBP120572 acts in a positive-feedbackloop to express PPAR120574 and other adipogenic-related genesIt maintains an enhanced differentiated state [26] SREBP1calso has a profound effect on adipogenesis it stimulatesthe expression of many lipogenic genes and regulates fattyacid and glucose metabolism [27] Furthermore it has beensuggested that SREBP1c is correlated with the production ofan endogenous PPAR120574 ligand that reinforces PPAR120574 activityand for this reason PPAR120574 is a target gene of SREBP1c[28 29] Considering the significance of these factors thattake a leading position in adipogenesis AG seems to suppressadipocyte differentiation by blocking PPAR120574 CEBP120572 andSREBP1c activation

LXR is known to regulate cholesterol and fatty acidmetabolism in liver tissue and inmacrophages [30] RecentlyLXRmdashconsidered one of the major genes for regulation oflipid metabolismmdashhas also been found to be involved in theprocess of adipocyte differentiation It affects the adipocyte-specific gene expression and adipogenesis LXR activationmeditates lipogenesis and adipogenesis by inducing SREBP1cand PPAR120574 [7] Because PPAR120574 directly regulates LXR120572which is one of the LXR families PPAR120574 and LXR form apositive-feedback relation in adipocyte differentiation [31] Inthis study AG decreased LXR levels in 3T3-L1 cells and inepididymal adipose tissues

Leptin a hormone secreted largely by adipose tissueto maintain body fat regulates food intake and energyexpenditure through its action on the hypothalamus Whenthe level of body fat mass decreases plasma leptin levelsfall stimulating appetite and suppressing energy expenditureuntil fatmass is restored Conversely when fatmass increaseslevels of leptin increase and suppress appetite until weight islost Thus adipose tissue mass seems to be under the controlof leptin [32] In brief an activated leptin gene links to astate of obesity In our results the expression of the leptingene was decreased by AG apparently signifying that AGattenuates weight gain and fat accumulation by inhibitingleptin release Leptin resistance the hallmark of high-Fatdiet induced obesity model is a phenomenon in whichcirculating leptin levels decrease in response to target tissuessuch as adipose tissue muscles liver and hypothalamus Inmost cases the leptin resistance could be either primary oracquired In human adiposity although there exit substantialindividual differences at some specific levels of body fatleptin concentrations in the circulation are associated withadipose tissue [33] Therefore the close correlation betweenhyperleptinemia and body weight has resulted in recognitionof leptin resistance as a cause of obesity In our present study


leptin resistance is developed by the administration of a high-fat diet Subsequently treatment with AG decreased leptingene level and this means that AG may ameliorate leptinresistance

In our experiment we used a small amount of the AGextract at doses of 1 and 5 AG (ww in HFD) We usuallyuse 10ndash20 doses for evaluating the antiobesity effect inanimal models [34] However our previous data showed thatAG has a good lipid lowering effect in lower drug doses [15]Thus in this study we reduced AG doses (1 and 5) AGis able to effective in spite of the low doses (1 and 5)Thus mice were fed HFD mixed with AG extract and themethod of feeding mice meant that their intake of AG wasnot equal among them If they were given larger doses of AGextract or if the AG ingestion modality was changed to oraladministration or intraperitoneal injection an antiobesityeffect could be more evident

Adipose tissue is considered a crucial factor in theregulation of many pathological processes and it is linked toboth the immune and the metabolic systems For that reasonobesity is associated with both a chronic inflammatoryresponse and immune functions [35] Further studies areneeded to identify the effects of AG on inflammation and onthe immune system

Caffeoylquinic acids (CQs) are esters of caffeic and quinicacids and are phenolic compounds They exist naturally aschlorogenic acid (3-CQ) cryptochlorogenic acid (4-CQ)neochlorogenic acid (5-CQ) cynarin (15-diCQ) isochloro-genic acid A (35-diCQ) isochlorogenic acid B (34-diCQ)and isochlorogenic acid C (45-diCQ) [36]Their various bio-logical functions include antioxidant antiinflammatory anti-microbial anti-skin-aging antihypercholesterolemia andantihyperglycemia activities Previously we reported that AGhad high quantity of CQs [14] In this study we used CQ-richextract fromAG and downregulation of leptin gene could berelated with leptin sensitivity by CQ compound This effectcan be induced by reduction of triglyceride and insulin levels

5 Conclusion

Wehave demonstrated that AG exerts antiobesity effects bothin vitro and in vivo Its effects on obesity are based on thedownregulation of adipogenic-related transcription factorssuggesting that AG has a potential as a therapeutic agent forobesity and obesity-related diseases

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Heon-Myung Lee and Gabsik Yang contributed equally tothis work

Acknowledgment

This work was supported by a Grant from National ResearchFoundation of Korea (2012-0004407)

References

[1] WorldHealthOrganizatrionWhat Are Overweight andObesityFacts About Overweight and Obesity Report of a WHO consul-tation on overweight and obesity Geneva Switzerland 2012

[2] T C Rosini A S Silva and C D Moraes ldquoDiet-inducedobesity rodentmodel for the study of obesity-related disordersrdquoRevista Da Associacao Medica Brasileira vol 58 pp 383ndash3872012

[3] M I Lefterova and M A Lazar ldquoNew developments inadipogenesisrdquo Trends in Endocrinology andMetabolism vol 20no 3 pp 107ndash114 2009

[4] P Tontonoz E Hu and B M Spiegelman ldquoRegulation ofadipocyte gene expression and differentiation by peroxisomeproliferator activated receptor 120574rdquo Current Opinion in Geneticsand Development vol 5 no 5 pp 571ndash576 1995

[5] M S Brown and J L Goldstein ldquoThe SREBP pathway regu-lation of cholesterol metabolism by proteolysis of a membrane-bound transcription factorrdquoCell vol 89 no 3 pp 331ndash340 1997

[6] C Linehan S Gupta A Samali and L OrsquoConnor ldquoBisphenola-mediated suppression of lpl gene expression inhibits triglyc-eride accumulation during adipogenic differentiation of humanadult stem cellsrdquo PLoSONE vol 7 no 5 Article ID e36109 2012

[7] J B Seo H M Moon W S Kim et al ldquoActivated liver xreceptors stimulate adipocyte differentiation through inductionof peroxisome proliferator-activated receptor 120574 expressionrdquoMolecular and Cellular Biology vol 24 no 8 pp 3430ndash34442004

[8] J M Friedman and J L Halaas ldquoLeptin and the regulation ofbody weight in mammalsrdquo Nature vol 395 no 6704 pp 763ndash770 1998

[9] A M Heck J A Yanovski and K A Calis ldquoOrlistat a newlipase inhibitor for the management of obesityrdquo Pharmacother-apy vol 20 no 3 pp 270ndash279 2000

[10] C A Luque and J A Rey ldquoSibutramine a serotonin-norepinephrine reuptake-inhibitor for the treatment of obesityrdquoAnnals of Pharmacotherapy vol 33 no 9 pp 968ndash978 1999

[11] S Kim Y Jin Y Choi and T Park ldquoResveratrol exerts anti-obesity effects via mechanisms involving down-regulation ofadipogenic and inflammatory processes in micerdquo BiochemicalPharmacology vol 81 no 11 pp 1343ndash1351 2011

[12] Y S Lee W S Kim K H Kim et al ldquoBerberine a naturalplant product activates AMP-activated protein kinase withbeneficial metabolic effects in diabetic and insulin-resistantstatesrdquo Diabetes vol 55 no 8 pp 2256ndash2264 2006

[13] T Kim Korean Plant Resources Publishing Center of SeoulNational University 1996

[14] A NugrohoMH Kim J Choi et al ldquoPhytochemical studies ofthe phenolic substances in Aster glehni extract and its sedativeand anticonvulsant activityrdquo Archives of Pharmacal Researchvol 35 pp 423ndash430 2012

[15] M-H Kim A Nugroho J Choi and H-J Park ldquoThe extractof Aster glehni leaves rich in caffeoylquinic acids preventsatherogenic index oxidative stress and body weight increase inhigh-fat diet-induced ratsrdquo Korean Journal of Pharmacognosyvol 42 no 1 pp 54ndash60 2011

[16] L J H van Tits E Arioglu-Oral C G J Sweep P SmitsA F H Stalenhoef and C J Tack ldquoAnti-inflammatory effectsof troglitazone in nondiabetic obese subjects independent ofchanges in insulin sensitivityrdquo Netherlands Journal of Medicinevol 63 no 7 pp 250ndash255 2005


[17] T Pischon U Nothlings and H Boeing ldquoObesity and cancerrdquoProceedings of the Nutrition Society vol 67 no 2 pp 128ndash1452008

[18] Y Ono E Hattori Y Fukaya S Imai and Y Ohizumi ldquoAnti-obesity effect of Nelumbo nucifera leaves extract in mice andratsrdquo Journal of Ethnopharmacology vol 106 no 2 pp 238ndash2442006

[19] J B Seo S S Choe H W Jeong et al ldquoAnti-obesity effectsof Lysimachia foenum-graecum characterized by decreased adi-pogenesis and regulated lipid metabolismrdquo Experimental andMolecular Medicine vol 43 no 4 pp 205ndash215 2011

[20] P A Sharpe H M Blanck J E Williams B E Ainsworth andJ M Conway ldquoUse of complementary and alternative medicinefor weight control in the United Statesrdquo Journal of Alternativeand Complementary Medicine vol 13 no 2 pp 217ndash222 2007

[21] E D Rosen C J Walkey P Puigserver and B M SpiegelmanldquoTranscriptional regulation of adipogenesisrdquo Genes and Devel-opment vol 14 no 11 pp 1293ndash1307 2000

[22] Z Wu P Puigserver and B M Spiegelman ldquoTranscriptionalactivation of adipogenesisrdquoCurrent Opinion in Cell Biology vol11 no 6 pp 689ndash694 1999

[23] P Tontonoz and BM Spiegelman ldquoFat and beyond the diversebiology of PPAR120574rdquo Annual Review of Biochemistry vol 77 pp289ndash312 2008

[24] K Schoonjans J Peinado-Onsurbe A-M Lefebvre et alldquoPPAR120572 and PPAR120574 activators direct a distinct tissue-specifictranscriptional response via a PPRE in the lipoprotein lipasegenerdquo EMBO Journal vol 15 no 19 pp 5336ndash5348 1996

[25] W-C Yeh Z Cao M Classon and S L McKnight ldquoCascaderegulation of terminal adipocyte differentiation by three mem-bers of the CEBP family of leucine zipper proteinsrdquo Genes andDevelopment vol 9 no 2 pp 168ndash181 1995

[26] R P Brun J B Kim E Hu S Altiok and B M Spiegel-man ldquoAdipocyte differentiation a transcriptional regulatorycascaderdquo Current Opinion in Cell Biology vol 8 no 6 pp 826ndash832 1996

[27] J B Kim and B M Spiegelman ldquoADD1SREBP1 promotesadipocyte differentiation and gene expression linked to fattyacid metabolismrdquo Genes and Development vol 10 no 9 pp1096ndash1107 1996

[28] L Fajas K Schoonjans L Gelman et al ldquoRegulation ofperoxisome proliferator-activated receptor 120574 expression byadipocyte differentiation and determination factor 1sterol reg-ulatory element binding protein 1 implications for adipocytedifferentiation andmetabolismrdquoMolecular andCellular Biologyvol 19 no 8 pp 5495ndash5503 1999

[29] J B Kim H M Wright M Wright and B M SpiegelmanldquoADD1SREBP1 activates PPAR120574 through the production ofendogenous ligandrdquo Proceedings of the National Academy ofSciences of the United States of America vol 95 no 8 pp 4333ndash4337 1998

[30] T T Lu J J Repa and D J Mangelsdorf ldquoOrphan nuclearreceptors as eLiXiRs and FiXeRs of sterol metabolismrdquo TheJournal of Biological Chemistry vol 276 no 41 pp 37735ndash37738 2001

[31] A Chawla W A Boisvert C-H Lee et al ldquoA PPAR120574-LXR-ABCA1 pathway inmacrophages is involved in cholesterol effluxand atherogenesisrdquoMolecular Cell vol 7 no 1 pp 161ndash171 2001

[32] J M Friedman ldquoLeptin and the Regulation of Body WeightrdquoThe Keio Journal of Medicine vol 60 no 1 pp 1ndash9 2011

[33] C J Zhou S Huang J Q Liu et al ldquoSweet tea leaves extractimproves leptin resistance in diet-induced obese ratsrdquo Journalof Ethnopharmacology vol 145 no 1 pp 386ndash392 2013

[34] H-J AnH-K Rim S-E Suh et al ldquoGamiwalbitang composedof four herbs controls body weight increase and lipid levelelevation induced by a high-fat diet in micerdquo Immunopharma-cology and Immunotoxicology vol 32 no 2 pp 307ndash312 2010

[35] H Tilg and A R Moschen ldquoAdipocytokines mediators linkingadipose tissue inflammation and immunityrdquo Nature ReviewsImmunology vol 6 no 10 pp 772ndash783 2006

[36] H F Harrison Jr T R Mitchell J K Peterson W P WechterG F Majetich and M E Snook ldquoContents of caffeoylquinicacid compounds in the storage roots of sixteen sweetpotatogenotypes and their potential biological activityrdquo Journal of theAmerican Society for Horticultural Science vol 133 no 4 pp492ndash500 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


modality Several natural products extracted from herbalplants have been shown to be effective for specific conditionsSome natural extracts as well as the principal componentsof the extracts such as berberine and resveratrol haveantiobesity effects [11 12] Studies on the efficacy of herbalmedicines are actively going on in all parts of the world

Aster glehni Franchet et Sckmidt (AG) which is com-mon to Ulleungdo Island Republic of Korea is officiallyrecognized as a regional specialty that grows only in thisregion It has recently been named Aster pseudoglehni LimHyun amp Shin In the republic of Korea the leaves of AGare used in a dish called chwinamul and AG has alsobeen used to treat diabetes mellitus hypercholesterolemiainsomnia and cardiovascular disease [13] According to arecent study AG extract has both sedative and anticonvulsanteffects [14] Previously we reported that the AG rich in CQcompound lowers the total lipid triglyceride phospholipidand cholesterol levels in animal model [15] Although AGrsquoseffect on body weight in rats has been reported [15] nostudy has focused on the antiadipogenic effect of AG in vitroSpecific adipogenic signal genes were examined to elucidatethe specific mechanism underlying the effect of AG Thepresent study considered the antiobesity and antiadipogeniceffects of AG in 3T3-L1 preadipocytes and mice

2 Materials and Methods

21 Plant Material and Preparation of the Extract AG leavesfrom Ulleungdo Island Republic of Korea were dried andcrushed for extraction The dried material was refluxedwith 70 EtOH for 6 h at 60∘C The extract was filteredconcentrated under reduced pressure and then freeze-driedto obtain a solid extract powder

22 Cell Culture The 3T3-L1 mouse preadipocytes werecultured in Dulbeccorsquos Modified Eaglersquos Medium (DMEMLife Technologies Inc Grand Island NY USA) supple-mentedwith 10bovine calf serum 100120583gmL streptomycinand 100 unitsmL penicillin at 37∘C in 10 CO

2 The cells

were grown to confluency in 10 calf serumDMEM Twodays after the cells had reached confluency (day 0) theywere stimulated with a methylisobutylxanthine dexametha-sone and insulin (MDI) induction medium (DMEM con-taining 10 fetal bovine serum (FBS) 05mM 3-isobutyl-1-methylxanthine (IBMX) 1 120583mmL dexamethasone and1 120583gmL of insulin) Two days after stimulation with MDI(day 2) the medium was changed to an insulin medium(DMEM containing 10 fetal bovine serum and 1120583gmL ofinsulin) Two days later (day 4) the medium was changed to10 FBSDMEM The cells were fed with 10 FBSDMEMevery 2 days Full differentiation was achieved by day 8

23 Oil Red O Staining To determine both adipogenicpotential and fat accumulation we stained the cells withoil red O (Sigma Chemical Co St Louis MO) On day8 the cultured cells were washed thrice with phosphate-buffered saline (PBS) and then fixed with 4 formaldehydefor 30min at room temperature The cells were washed 3

times more with PBS and stained with 05 120583gmL oil red Ofor 15min After rinsing with PBS we captured representativephotomicrographs using a microscope and measured fataccumulation

24 AdipoRed Assay 3T3-L1 cells were cultured in 6-wellplates at a density of 3 times 105 cells per well and set up todifferentiate in the presence and absence of AG On day 2 andday 8 of differentiation the cells were exposedwithAdipoRed(Lonza Houston TX USA) and intracellular triglyceride(TG) level was analyzed

25 Leptin Assay For the periods indicated 3T3-L1 cellswere treated with AG or not Conditioned media from thefinal 24 h of culture were collected Leptin concentrationswere determined using an enzyme-linked immunosorbentassay (ELISA Mouse Leptin Assay Kit Immuno-BiologicalLaboratories Co Tokyo Japan)

26 Real-Time PCR Analysis Total RNA was isolated fromthe differentiated 3T3-L1 cells and epididymal adipose tissueby using Easy-Blue Reagent (Intron Biotechnology IncGyoenggi-do Republic of Korea) according to the manu-facturerrsquos instructions Then total RNA qualification wasperformed with an Epoch micro-volume spectrophotometersystem (BioTek Instruments Inc Winooski VT USA) Anequal amount of total RNA was used to synthesize cDNAwith a high-capacity cDNA reverse transcription kit (AppliedBiosystems Foster City CA USA) The program was set for10min of initiation at 25∘C followed by 90min of incubationat 50∘C and 5min at 85∘C Primers were obtained fromBioneer (Daejeon Republic of Korea) All primer sequencesand annealing temperatures are shown in Table 1 A StepOne Plus Real-time PCR system (Applied Biosystems FosterCity CA USA) with an SYBR Green Master Mix (AppliedBiosystems Warrington UK) and primers (Bioneer SeoulRepublic of Korea) was used to carry out a real-time PCRThe steps were as follows 10min at 95∘C 40 cycles of 5 sat 95∘C 45 s at 60∘C a final melting curve of 15 s at 95∘C1min at 60∘C 15 s at 95∘C for tissues or for cells and 5minfollowed by 35 repetitive thermal cycles (94∘C for 30 s 60∘Cfor 30 s and 72∘C for 30 s) Fold changes of gene expressionwere calculated using the comparative threshold cycle (Ct)method (Applied Biosystems) The data were normalized forthe initial control GAPDH

27 Western Blot Analysis The 3T3-L1 cells were lysed ina lysis buffer (25mM HEPES pH 74 100mM NaCl 1mMEDTA 5mM MgCl

2 01mM dithiothreitol and protease

inhibitor mixture) Determination of the protein was carriedout on Tris-glycine SDS-polyacrylamide gels followed by atransfer to PVDF membranes (EMD Millipore CorporationBillerica MA USA) Then the membranes were blockedwith 5 skim milk in Tris-buffered saline containing 01tween 20 (TBST) at room temperature for 1 h and incubatedat 4∘C overnight with 1 1000 dilutions of primary antibodiesThe following day the membranes were washed with TBST3 times for 10min each and made to react with 1 2500



Gene name 119879119898















3 Results





(a)

ND CON Tro 50 100 200 4000

20

40

60

80

100

120

Differentiated

Relat

ive f

at ac

cum

ulat

ion

()


AG (120583gmL)

(b)

ND CON 100 200 400

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

AG (120583gmL)


(c)

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

ND CON 100 200 400AG (120583gmL)


Intr

a cel

lula

r TG

()

lowastlowast

lowast lowast

(d)

0

2000

4000

6000

8000

10000

12000

Lept

in le

vel i

n 3T

3-L1

adip

ocyt

es (p

gm

L)

lowastlowast

lowastlowast

ND CON 100 200 400AG (120583gmL)


(e)





ND CON 100 200 4000

5

10

15

20

25

Differentiated

Rela

tive m

RNA

PPAR120574

AG (120583gmL)

lowastlowastlowast


(a)

ND CON 100 200 4000

2

4

6

8

Differentiated

Rela

tive m

RNA

CEBP120572

AG (120583gmL)


lowastlowastlowast

(b)

ND CON 100 200 4000

05

1

15

2

25

Differentiated

SREBP1c

Relat

ive m

RNA

AG (120583gmL)


(c)

ND CON 100 200 4000

2

4

6

Differentiated

LXR

Relat

ive m

RNA

AG (120583gmL)

lowastlowastlowast


(d)







20

25

30

35

15

40

0 1 2 3 4 5 6 7 8 9 10Week

lowast


CONHF

AG1AG5

Tota

l bod

y w

eigh

t (g)

(a)

0 1 2 3 4 5 6 7 8 9 10Week

0

5

10

15

20

Wei

ght g

ain

(g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


CONHF

AG1AG5

(b)

0 1 2 3 4 5 6 7 8 9 101

152

253

354

45

CONHF

AG1AG5

Food

inta

ke (g

day

)

Week

(c)

10

12

14

16

18

20

22

0 1 2 3 4 5 6 7 8 9 10Calo

ric in

take

(kca

lmou

sed

ay)

CONHF

AG1AG5

Week

(d)


20

40

60

80

CONHF

AG1AG5

Fat-p

ad w

eigh

t(m

glowast10

00g

bod

y w

eigh

t)

lowastlowastlowast


lowast

lowastlowast

(e)

CON HF AG1 AG50

05

1

15

2

25

Live

r wei

ght (


(f)






times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Gene name 119879119898















3 Results





(a)

ND CON Tro 50 100 200 4000

20

40

60

80

100

120

Differentiated

Relat

ive f

at ac

cum

ulat

ion

()


AG (120583gmL)

(b)

ND CON 100 200 400

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

AG (120583gmL)


(c)

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

ND CON 100 200 400AG (120583gmL)


Intr

a cel

lula

r TG

()

lowastlowast

lowast lowast

(d)

0

2000

4000

6000

8000

10000

12000

Lept

in le

vel i

n 3T

3-L1

adip

ocyt

es (p

gm

L)

lowastlowast

lowastlowast

ND CON 100 200 400AG (120583gmL)


(e)





ND CON 100 200 4000

5

10

15

20

25

Differentiated

Rela

tive m

RNA

PPAR120574

AG (120583gmL)

lowastlowastlowast


(a)

ND CON 100 200 4000

2

4

6

8

Differentiated

Rela

tive m

RNA

CEBP120572

AG (120583gmL)


lowastlowastlowast

(b)

ND CON 100 200 4000

05

1

15

2

25

Differentiated

SREBP1c

Relat

ive m

RNA

AG (120583gmL)


(c)

ND CON 100 200 4000

2

4

6

Differentiated

LXR

Relat

ive m

RNA

AG (120583gmL)

lowastlowastlowast


(d)







20

25

30

35

15

40

0 1 2 3 4 5 6 7 8 9 10Week

lowast


CONHF

AG1AG5

Tota

l bod

y w

eigh

t (g)

(a)

0 1 2 3 4 5 6 7 8 9 10Week

0

5

10

15

20

Wei

ght g

ain

(g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


CONHF

AG1AG5

(b)

0 1 2 3 4 5 6 7 8 9 101

152

253

354

45

CONHF

AG1AG5

Food

inta

ke (g

day

)

Week

(c)

10

12

14

16

18

20

22

0 1 2 3 4 5 6 7 8 9 10Calo

ric in

take

(kca

lmou

sed

ay)

CONHF

AG1AG5

Week

(d)


20

40

60

80

CONHF

AG1AG5

Fat-p

ad w

eigh

t(m

glowast10

00g

bod

y w

eigh

t)

lowastlowastlowast


lowast

lowastlowast

(e)

CON HF AG1 AG50

05

1

15

2

25

Live

r wei

ght (


(f)






times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(a)

ND CON Tro 50 100 200 4000

20

40

60

80

100

120

Differentiated

Relat

ive f

at ac

cum

ulat

ion

()


AG (120583gmL)

(b)

ND CON 100 200 400

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

AG (120583gmL)


(c)

Intr

a cel

lula

r TG

()

0

20

40

60

80

100

120

ND CON 100 200 400AG (120583gmL)


Intr

a cel

lula

r TG

()

lowastlowast

lowast lowast

(d)

0

2000

4000

6000

8000

10000

12000

Lept

in le

vel i

n 3T

3-L1

adip

ocyt

es (p

gm

L)

lowastlowast

lowastlowast

ND CON 100 200 400AG (120583gmL)


(e)





ND CON 100 200 4000

5

10

15

20

25

Differentiated

Rela

tive m

RNA

PPAR120574

AG (120583gmL)

lowastlowastlowast


(a)

ND CON 100 200 4000

2

4

6

8

Differentiated

Rela

tive m

RNA

CEBP120572

AG (120583gmL)


lowastlowastlowast

(b)

ND CON 100 200 4000

05

1

15

2

25

Differentiated

SREBP1c

Relat

ive m

RNA

AG (120583gmL)


(c)

ND CON 100 200 4000

2

4

6

Differentiated

LXR

Relat

ive m

RNA

AG (120583gmL)

lowastlowastlowast


(d)







20

25

30

35

15

40

0 1 2 3 4 5 6 7 8 9 10Week

lowast


CONHF

AG1AG5

Tota

l bod

y w

eigh

t (g)

(a)

0 1 2 3 4 5 6 7 8 9 10Week

0

5

10

15

20

Wei

ght g

ain

(g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


CONHF

AG1AG5

(b)

0 1 2 3 4 5 6 7 8 9 101

152

253

354

45

CONHF

AG1AG5

Food

inta

ke (g

day

)

Week

(c)

10

12

14

16

18

20

22

0 1 2 3 4 5 6 7 8 9 10Calo

ric in

take

(kca

lmou

sed

ay)

CONHF

AG1AG5

Week

(d)


20

40

60

80

CONHF

AG1AG5

Fat-p

ad w

eigh

t(m

glowast10

00g

bod

y w

eigh

t)

lowastlowastlowast


lowast

lowastlowast

(e)

CON HF AG1 AG50

05

1

15

2

25

Live

r wei

ght (


(f)






times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ND CON 100 200 4000

5

10

15

20

25

Differentiated

Rela

tive m

RNA

PPAR120574

AG (120583gmL)

lowastlowastlowast


(a)

ND CON 100 200 4000

2

4

6

8

Differentiated

Rela

tive m

RNA

CEBP120572

AG (120583gmL)


lowastlowastlowast

(b)

ND CON 100 200 4000

05

1

15

2

25

Differentiated

SREBP1c

Relat

ive m

RNA

AG (120583gmL)


(c)

ND CON 100 200 4000

2

4

6

Differentiated

LXR

Relat

ive m

RNA

AG (120583gmL)

lowastlowastlowast


(d)







20

25

30

35

15

40

0 1 2 3 4 5 6 7 8 9 10Week

lowast


CONHF

AG1AG5

Tota

l bod

y w

eigh

t (g)

(a)

0 1 2 3 4 5 6 7 8 9 10Week

0

5

10

15

20

Wei

ght g

ain

(g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


CONHF

AG1AG5

(b)

0 1 2 3 4 5 6 7 8 9 101

152

253

354

45

CONHF

AG1AG5

Food

inta

ke (g

day

)

Week

(c)

10

12

14

16

18

20

22

0 1 2 3 4 5 6 7 8 9 10Calo

ric in

take

(kca

lmou

sed

ay)

CONHF

AG1AG5

Week

(d)


20

40

60

80

CONHF

AG1AG5

Fat-p

ad w

eigh

t(m

glowast10

00g

bod

y w

eigh

t)

lowastlowastlowast


lowast

lowastlowast

(e)

CON HF AG1 AG50

05

1

15

2

25

Live

r wei

ght (


(f)






times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20

25

30

35

15

40

0 1 2 3 4 5 6 7 8 9 10Week

lowast


CONHF

AG1AG5

Tota

l bod

y w

eigh

t (g)

(a)

0 1 2 3 4 5 6 7 8 9 10Week

0

5

10

15

20

Wei

ght g

ain

(g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


CONHF

AG1AG5

(b)

0 1 2 3 4 5 6 7 8 9 101

152

253

354

45

CONHF

AG1AG5

Food

inta

ke (g

day

)

Week

(c)

10

12

14

16

18

20

22

0 1 2 3 4 5 6 7 8 9 10Calo

ric in

take

(kca

lmou

sed

ay)

CONHF

AG1AG5

Week

(d)


20

40

60

80

CONHF

AG1AG5

Fat-p

ad w

eigh

t(m

glowast10

00g

bod

y w

eigh

t)

lowastlowastlowast


lowast

lowastlowast

(e)

CON HF AG1 AG50

05

1

15

2

25

Live

r wei

ght (


(f)






times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















times100

times400

CON HF AG1 AG5

(a)

times100

times400

CON HF AG1 AG5

(b)

11010 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 100 1100

4

8

12


Num

ber o

f cel

ls (

of t

otal

)

10 20 30 40 50 60 70 80 90 1000

4

8

12


umbe

r of c

ells

( o

f tot

al)

(c)


005

115

225

3

47

Relat

ive g

ene e

xpre

ssio

n


lowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CONHF

AG1AG5

(a)

PPAR120574

CEBP120572

SREBP1c

120573-Actin

CON HF AG1 AG5

(b)



3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3-CQ

3-CQ5-CQ

5-CQ

3-pCQ

3-pCQ

minus10

0

10

20

30

40

50

0

25

50

75

5 10 15 20 25

Standard compounds


45-DQ

34-DQ

35-DmQ

35-DQ

35-DQ


Inte

nsity

(mAU

)





4 Discussion











5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion






Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article antiadipogenic effects of aster glehni …luna 4.60 × 250 mm c reversed-phase...

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