Research Article 2-Heptyl-Formononetin Increases...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 926942 13 pageshttpdxdoiorg1011552013926942

Research Article2-Heptyl-Formononetin Increases Cholesterol andInduces Hepatic Steatosis in Mice

Charlotte Andersen1 Janne G Schjoldager1 Christian G Tortzen2

Andreas Vegge1 Majbritt R Hufeldt1 Mette T Skaanild1 Finn K Vogensen3

Karsten Kristiansen4 Axel K Hansen1 and John Nielsen5

1 Department of Veterinary Disease Biology Faculty of Health and Medical Sciences University of Copenhagen1870 Frederiksberg C Denmark

2Department of Chemistry Faculty of Science University of Copenhagen 1870 Frederiksberg C Denmark3 Department of Food Science Faculty of Science University of Copenhagen 1870 Frederiksberg C Denmark4Department of Biology Faculty of Science University of Copenhagen 2200 Copenhagen N Denmark5 Department of Drug Design and Pharmacology Faculty of Health and Medical Sciences University of Copenhagen2100 Copenhagen Oslash Denmark

Correspondence should be addressed to Axel K Hansen akhlifekudk

Received 2 January 2013 Revised 15 March 2013 Accepted 26 March 2013

Academic Editor Kazim Husain

Copyright copy 2013 Charlotte Andersen 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

Consumption of isoflavones may prevent adiposity hepatic steatosis and dyslipidaemia However studies in the area are few andprimarily with genistein This study investigated the effects of formononetin and its synthetic analogue 2-heptyl-formononetin(C7F) on lipid and cholesterol metabolism in C57BL6J mice The mice were fed a cholesterol-enriched diet for five weeks toinduce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplementedformononetin or C7F for three weeks Body weight and composition glucose homeostasis and plasma lipids were comparedIn another experiment mice were fed the above diets for five weeks and hepatic triglyceride accumulation and gene expressionand histology of adipose tissue and liver were examined Supplementation with C7F increased plasma HDL-cholesterol therebyincreasing the plasma level of total cholesterol Supplementation with formononetin did not affect plasma cholesterol but increasedplasma triglycerides levels Supplementation with formononetin and C7F induced hepatic steatosis However formononetindecreased markers of inflammation and liver injury The development of hepatic steatosis was associated with deregulatedexpression of hepatic genes involved in lipid and lipoprotein metabolism In conclusion supplementation with formononetin andC7F to a cholesterol-enriched diet adversely affected lipid and lipoprotein metabolism in C57BL6J mice

1 Introduction

Over the last decade there has been a pronounced increasein the interest of the physiologic and pharmacologic effectsof bioactive compounds Of particular interest in relation tohuman health is a group of naturally occurring compoundscalled isoflavones which exhibit both hormonal and nonhor-monal properties These compounds are found in variouslegumes including soybean green bean and alfalfa sprout[1] Originally isoflavones were studied for their effects onhormone-sensitive cancers osteoporosis menopause and

heart diseases [2] However recently the focus has beendirected towards their effects on lipid metabolism Dataobtained from animal experiments as well as clinical and epi-demiological studies suggest that consumption of isoflavonesmay prevent obesity [3 4] type 2 diabetes [4] atherosclerosis[5] nonalcoholic fatty liver disease [6] and dyslipidaemia [78]Thus isoflavonesmight be useful in targeting themetabolicsyndrome

The most frequently studied isoflavone is genistein fol-lowed by daidzein Supplementationwith high doses of genis-tein or daidzein to rodents fed high-fat diets substantially

2 BioMed Research International

decreases body weight and fat mass [9ndash11] lowers the plasmalevels of total cholesterol triglyceride and LDL-cholesterol[9 10 12 13] and protects against the development of hep-atic steatosis [9 10 14 15] Thus supplementation withisoflavones or synthetic analogues could potentially be usedin prevention andor treatment of obesity dyslipidaemia andhepatic steatosis

Formononetin is an O-methylated isoflavone present indifferent bean types at various levels [16] Previously extractscontaining formononetin have been found to have an influ-ence on fat metabolism [17 18] However as extract alsocontains a range of other compounds it is difficult tomake solid conclusions on the effects of formononetin basedon these studies One study also shows a cardioprotectiveeffect of a derivative of formononetin [19] suggesting thatformononetin and derivatives of formononetin could have apositive influence on obesity and related disorders Howeverthe effects of formononetin remain putative owing to theexistence of a range of other compounds found in extracts

A newly synthesised analogue of formononetin 2-heptylformononetin (C7F) affected lipid accumulation lipolysisand peroxisome proliferator-activated receptor 120574 (PPAR120574)activation in vitromore potently than synthetic formononetin(manuscript in preparation) Therefore we aimed to inves-tigate if formononetin and C7F positively affected lipid andcholesterol metabolism in C57BL6 mice fed a cholesterol-enriched diet by assessing the effect of formononetin andC7F on body weight and composition glucose toleranceplasma lipid composition hepatic steatosis and expressionof genes involved in lipid metabolism and phase I and IImetabolism

2 Materials and Methods

21 Synthesis of Compounds

211 General Experimental Information Commercially avai-lable reagents (Sigma-Aldrich Germany) were used withoutfurther purification unless otherwise noted Solvents used forthe synthesis were of analytical grade dried over activated4A molecular sieves when necessary (all solvents used underdry conditions had a water content of lt25 ppm measuredby coulometric Karl Fischer titration) Analytical thin layerchromatography was performed using precoated silica gel 60F254 plates (MerckGermany) and visualized using eitherUVlight or potassium permanganate stain

Column chromatographywas performed onMerckKisel-gel 60 (0015ndash0040mm) using the dry column vacuumchromatography (DCVC) technique [20] High-performanceliquid chromatography was performed on a Waters 2525 sys-tem equipped with a Waters 2996 photodiode array detectorand aWaters 2767 Sample Manager using a 100mm times 19mmid XTerra prep MS C18 column (Waters Corp MA USA)with a gradient of acetonitrile in Milli-Q water with a flowof 15mLmin (Waters) Melting points were measured on aReichert melting point microscope model N254-1R (Aus-tria) 1H and 13C NMR spectroscopic data were recorded ona Bruker Avance 300 (Bruker BioSpin MRI Germany) using

deuterated solvents as a lock Chemical shifts are reportedin parts per million relative to the residual solvent peak(1H NMR) or the solvent peak (13C NMR) as the internalstandard Accurate mass determinations were performed ona Micromass LCT apparatus (UK) equipped with an AP-ESI probe calibrated with Leu-Enkephalin (5562771 gmol)All spectrophotometric measurements were performed on aShimadzu UV-2101PC UV-vis scanning spectrophotometerwith automatic cell changer and a temperature-controlledwater-jacket-regulated cell holder (Shimadzu Corp Japan)

212 Synthesis of C7F 24-Dihydroxy-41015840-methoxy-deoxybe-nzoin (25 g 97mmol) was dissolved in 50 mL of dry THFThe stirred solution was cooled to 0∘C and triethylamine(41 mL 3 eq) was added After 5min octanoyl chloride(37mL 22 eq) was added and the cooling was stoppedAfter 15min the reaction mixture was acidified with 50 mLof 1M HCl The yellow solution was extracted twice withEtOAc and the combined organic phases were washed withwater sat NaHCO

3aq and brine After removal of the

solvent the resulting residue was dried in vacuo Purificationby dry column vacuum chromatography (EtOAcHeptane onsilicamdash5 gradient) yielded the diester (47 g 95)13C NMR (75MHz CDCl

3) 120575 = 19703 17202 17145

15875 15398 15020 13098 13069 12617 11909 1175011425 5537 4743 3450 3441 3178 2918 2915 2908 29022491 2465 2274 1420 ppm1H NMR (300MHz CDCl

3) 120575 = 780 (d J = 86Hz 1H)

726 (s 1H) 712 (d J = 87Hz 2H) 706 (dd J = 86 22Hz1H) 694 (d J = 22Hz 1H) 686 (d J = 87Hz 2H) 411 (s2H) 379 (s 3H) 382ndash375 (m 4H) 180ndash162 (m 4H) 147ndash118 (m 16H) 094ndash079 (m 6H) ppm

24-Dihydroxy-41015840-methoxy-deoxybenzoin (47 g 92mmol)was dissolved into 60 mL of anhydrous THF and DBU(16mL 11 eq) was added The reaction mixture was heatedto reflux for 1 hour at which point 5mL conc HCl was addedand the heating was continued for 1 hour The reaction wasmonitored by TLC and after complete conversion NaOH(6mL 10M) was added and the solution was refluxed foranother 30min The reaction was again monitored by TLCuntil complete conversion was observed At this point 1MHCl was added until the reaction mixture was neutralisedThe yellow solution was cooled and extracted twice withEtOAc The combined organic phases were washed withwater sat NaHCO

3aq and brine After removal of solvent

the resulting residue was dried in vacuo The residue waspurified by dry column vacuum chromatography (EtOAcheptane on silica 5 gradient) yielding C7F (263 g 78) asa white foam13C NMR (75MHz CDCl

3) 120575 = 17782 16802 16283

15937 15817 13172 12768 12502 12237 11573 11413 102777758 7716 7674 5536 3273 3173 2924 2895 2762 22721418 ppm1H NMR (300MHz CDCl

3) 120575 = 802 (d J = 87Hz 1H)

789ndash740 (bs) 715 (d J = 87Hz 2H) 691 (d J = 87Hz 2H)688ndash683 (m 2H) 378 (s 3H) 258ndash249 (m 2H) 175ndash155(m 2H) 136ndash110 (m 8H) 094ndash076 (m 3H) ppm

BioMed Research International 3

22 Mouse Study

221 Preparation of Experimental Diets The basic diet usedfor preparation of the experimental diets was C1000 (Altro-min Germany) a standard maintenance rodent diet free ofphytoestrogens and with all polysaccharides derived fromcorn starch Three experimental diets were prepared a dietenriched with 2 cholesterol a diet enriched with 2cholesterol and 1000mgkg formononetin (37mmolkg)and a diet enriched with 2 cholesterol and 1300mgkg C7F(36mmolkg) For pelleting 2 gelatin 05 magnesium-stearate and 5 talcumwere added to the diets For composi-tion of the diet See Supplementary Table 1 in Supplementarymaterial available online at httpdxdoiorg1011552013926942

222 Animals and Study Design All animal studies wereperformed in accordance with the Council of Europe Con-vention ETS 123 in which the principles are equivalent tothe PHS Policy on Humane Care and Use of LaboratoryAnimals The study was approved by the Danish AnimalExperimentation Inspectorate (License no 2007-561-1434)All mice were housed in type III makrolon cages (TecniplastItaly) with aspen bedding and environmental enrichment(Tapvei Oy Finland) All mice were housed under environ-mentally controlled conditions with an alternating 12-hourlight dark cycle with access to food and water ad libitumexcept when food was withheld for the experimental proto-cols described in the following Food andwater were changedseveral timesweekly C57BL6JBomTacmice purchased fromTaconic (Denmark) were selected for the study as it is oneof the mostly used mouse strains within this field due to itssusceptibility to diet-induced obesity type 2 diabetes andlow-grade inflammation [21] The mice were approximatelyfour weeks old when recruited to the study as we wanted tostudy the effects in early life as obesity problems are oftenestablished when growing up

Experiment 1 126 male mice were housed in groups of fivemice The mice were randomly divided into five groups andallowed one week of acclimatisation where they were fedthe cholesterol-free C1000 diet After this period one group(119899 = 30) was euthanized and the remaining mice were fedthe cholesterol diet for five weeks to induce hypercholes-terolemia After this period another group (119899 = 23) waseuthanized and the remaining mice were fed the experimen-tal diets (cholesterol (119899 = 25) formononetin (119899 = 23)or C7F (119899 = 25)) for additionally three weeks before theywere euthanized The group size was calculated based uponserum cholesterol levels from a previous study on isoflavonesin mice [22] in which a difference in relation to isoflavonefeeding was approximately 5 with a standard deviationof approximately 6 Thus a power calculation setting thepower to 09 showed that a difference could be shown with25 mice and 119875 lt 001 Body weight was measured weeklyduring the entire period

Experiment 2 32 male mice were housed in groups of fourmice The mice were randomly divided into four groups

(119899 = 8) and allowed one week of acclimatisation where theywere fed the cholesterol-free C1000 diet The group size wascalculated on the basis of a previous study on gene expressionin relation to daidzein feeding [9] in which the relevant geneexpressions were at least 50 different between the groupsand no standard deviation was more than 25Thus a powercalculation setting the power to 09 showed that a differencecould be shown with 8 mice and 119875 lt 001 One groupwas fed the cholesterol-free C1000 diet (chow) for the wholeexperiment The other three groups were fed the cholesterol-enriched diet for five weeks before initiation of experimentalperiod with feeding of cholesterol formononetin or C7F foradditionally five weeks Body weight and food intake weremeasured weekly during the entire period

For termination of both studies the mice were deprivedof food for 12 hours The mice were anesthetised with amixture of Hypnorm (Vetapharma UK) and Dormicum(Roche Denmark) as previously described [23] After loss ofreflexes blood was collected from the retroorbital sinus intoheparinised tubes and the mice were euthanized by cervicaldislocation In experiment 1 the livers were removed anda slice from each liver was transferred to RNA later andkept at minus20∘C Caecum samples were collected asepticallyand immediately frozen at minus80∘C until use In experiment 2white adipose tissues (WAT) (inguinal WAT (iWAT) epi-didymal WAT (eWAT) intrascapular brown adipose tissue(iBAT) and liver were dissected and half was freeze clampedand frozen at minus80∘C and the other half was fixed in 4paraformaldehyde in phosphate buffer and later dehydratedand embedded in paraffin

23 Oral Glucose Tolerance Test An oral glucose tolerancetest was performed in experiment 1 prior to the initiationof the experimental diets and again at termination of theexperimental period The mice were fasted overnight bloodwas collected from the tail vein as previously described [24] attime points minus30 0 30 60 120 and 180 minutes and glucosewas monitored immediately on a FreeStyle Mini glucometer(Hermedico Denmark) as previously described [25] Afterthe first two blood samples at 119905 = 0 each mouse was dosedpo with 2 gkg glucose (500 gL Glucose SAD infusionsolution Veterinary Pharmacy University of CopenhagenDenmark)

24 DXA Scan Prior to euthanasia in experiment 1 DualEnergy X-ray Absorptiometry (DXA) scan (GE LunarProdigy General Electric WI USA) was performed with thescanner running the small animal software from the samemanufacturer The anesthetised mice were placed at specificmarks on a piece of carton to make sure that all animalswere similarly arranged in the scanner The carton alone wasevaluated prior to the first scan with a satisfying result (nomeasureable values) Body weight body fat percentage fatmass bone mineral content and bone mineral density weredetermined

25 Blood Sampling and Analysis of Plasma Lipids Plasmawas harvested after centrifugation at 3000 g at room


temperature for 10min and stored at minus80∘C until anal-ysed From experiment 1 total plasma cholesterol HDL-cholesterol LDL-cholesterol and triglycerides were mea-sured enzymatically and photometrically on ABXPentra400(Horiba Group France) using 120120583L of plasma From exper-iment 2 plasma levels of alanine transaminase (ALT) andaspartate transaminase (AST) were analysed using Biovisionkits (AH Diagnostics Denmark) Samples were run in dupli-cates for all analyses

26 Gene Expression

Samples fromExperiment 1 Total RNAwas isolated from liverslices using the Nucleospin Kit (Macherey Nagel Germany)according tomanufacturerrsquos protocolThe quality of the RNAwas assessed (all 260280 gt 2 and all 260230 gt 17) Thereverse transcription and PCR were set up using RT2 First-strand Kit and RT2 SYBR Green qPCR Master Mix fromSABiosciences (Tebu-bio Denmark) The reactions were setup according to the KitmanualsThe gene expression analysiswas carried out using Mouse Drug Metabolism array platesfrom SA Biosciences Three array plates were set up foreach group of animals (seven animals per plate) For eacharray 15 120583g total RNA was used 021 120583g of total RNA fromeach animal In the mouse drug metabolism array geneexpression of 84 genes involved mainly in phase I and phaseII metabolism can be analysed (Supplementary Table 2)The array also includes negative and positive control andthe following 5 housekeeping genes 120573-glucuronidase hypox-anthine guanine phosphoribosyl transferase 1 heat shockprotein 90 120572 glyceraldehyde-3-phosphate dehydrogenaseand 120573-actin in order to calculate expression changes

Samples from Experiment 2 Total RNA was purified fromeWAT iWAT iBAT and liver using Trizol (Invitrogen Den-mark) and RNA concentration was measured on a Nanodrop(Thermo Scientific Denmark) cDNA was synthesised withRevertAid (Fermentas Germany) according to manufac-turerrsquos instructions Reactions were diluted with 120 120583L ofwater and frozen at minus80∘C until analysed on Roche Light-Cycler 480 (Roche) cDNA was analysed in duplicates in20120583L reactions containing SYBR Green Mastermix (Roche)3 120583L of diluted cDNA and 300 nM of each primer Reactionmixtures were denaturated at 95∘C for 2min followed by 40cycles of 95∘C15 s 60∘C15 s 72∘C20 s Data was analysedusing Roche Lightcycler software and the ΔΔCt methodand normalised to 18S ribosomal RNA Primers for RT-PCR were purchased from TAG Copenhagen (Denmark)(Supplementary Table 3)

27 Gut Microbiota Composition Analysis DNA was extract-ed from the ceacum samples from Experiment 1 using theQIAamp DNA Stool Mini Kit (Qiagen Germany) accordingto the manufacturerrsquos instructions and stored at minus40∘C untilanalysis during which the V3 region of the 16S rRNA genewas amplified by PCR using the following universal primerset PRBA338f and PRUN518r (51015840-C GCC CGC CGC GCGCGG CGG GCG GGG CGG GGG CAC GGG GGG ACTCCT ACG GGA GGC AGC AG-31015840 and 51015840-ATT ACC GCG

GCT GCT GG-31015840) [26] (Eurofins MWG Operon Germany)All reactions were carried out in a 50 120583L volume containing125UHotMaster Taq DNA Polymerase (5 Prime Germany)5 120583L 10times HotMaster Taq Buffer with 25mM MgCl

2(5

Prime)100 ng DNA 10 pmol of each primer 03mM dNTP

(Bioline Germany) and 1 120583g BSA (Sigma-Aldrich) ThePCR reaction was performed on a Robocycler Thermoblock(Stratagene Denmark) Initial denaturation was done at 95∘Cfor 5 minutes and amplification was carried out using 30cycles each including denaturation at 95∘C for 30 secondsannealing at 60∘C for 30 seconds and extension at 72∘C for40 seconds followed by a final elongation step at 72∘C for10 minutes A final product length of approximately 230 bpwas checked by electrophoresis on a 2 agarose gel stainedwith EthidiumBromide (Bio-Rad CAUSA) PCR ampliconswere analysed by DGGE using the INGENYphorU-2 systemaccording to the manufacturerrsquos instructions (INGENY TheNetherlands) The acrylamide concentration in the gel was9 and the linear denaturation gradient was 30 to 65(100 denaturant corresponds to 7M urea and 40 deion-ized formamide) Before loading 35 120583L PCR product wasmixed with 6120583L 6times loading dye In addition to the samplesanalysed an in-house standard PCR product was loadedallowing accurate alignment of lanes and bands within andbetween gels Electrophoresis was performed in 05timesTAE (1timesTAE corresponds to 40mM Tris-acetate 1mM EDTA pH80) at 60∘C for 16 hours at 120 Volt Staining was performedwith a 1 10000 SYBR Gold staining solution (InvitrogenOR USA) in 1times TAE for 1 hour and photographed with UVtransillumination (302 nm) using a Kodak EDAS 290 system(Eastman Kodak)

28 Histology From Experiment 2 sections of paraffin-embedded adipose tissue and liver were cut into 3 120583m thickslices and stained with haematoxylin and eosin according tostandard procedures

29 Triglyceride Measurements From Experiment 2 totallipids were extracted from the liver using a modified versionof the Bligh and Dyer protocol In brief 25mg tissueswas homogenised in potassium phosphate buffer and lipidswere extracted with chloroformmethanol (1 2) HCl wasadded and the chloroform phase transferred to new tubesand evaporated under nitrogen The extract was dissolvedin LPL buffer (2875mM PIPES 5741mM MgCl

2sdot6H2O

0569mgmL BSA-FFA 01 SDS) and analysed with atriglyceride kit (Zen-Bio NC USA)

210 Statistics Area under curve (AUC) was calculated fromweighing and data from the oral glucose tolerance testThree-dimensional principal component analysis (3D-PCA)based on DGGE data was carried out (Applied Maths) Allquantitative data were tested for normality by Anderson-Darling test compared in a general linear model with thesettings group cage (group) and finally significant differ-ences between groups were further evaluated comparingindividual groups by an unpaired two-sample 119905-test Softwaredeveloped by SABiosciences (Tebu-bio) specifically for gene


Table 1 Body composition as shown by DXA scans of C57BL6 mice before cholesterol feeding after five weeks of initial cholesterol feedingand after three additional weeks where the mice were fed the cholesterol-enriched diet supplemented with either formononetin or 2-heptyl-formonetin (C7F) (Experiment 1) Data show mean plusmn SEM Different letters (a b c) denote significant difference between the groups

Fat percentage Body weight(g)

Fat mass(g)

Bone mineralconcentration

(g)

Bone mineral density(mgcm3)

Before test periodAfter acclimatisation (119899 = 30) 193 plusmn 78a 167 plusmn 20a 32 plusmn 18 03 plusmn 01ab 640 plusmn 9a

After initial cholesterol feeding (119899 = 23) 137 plusmn 68b 243 plusmn 13b 32 plusmn 17 03 plusmn 01a 802 plusmn 10b

After test periodCholesterol (119899 = 25) 148 plusmn 82b 280 plusmn 21c 42 plusmn 26 04 plusmn 007b 864 plusmn 6c

Cholesterol + formononetin (119899 = 23) 131 plusmn 96b 278 plusmn 23abc 36 plusmn 28 04 plusmn 011ab 839 plusmn 9bc

Cholesterol + C7F (119899 = 24) 145 plusmn 55b 261 plusmn 22d 32 plusmn 17 04 plusmn 008ab 825 plusmn 10bc

Experimental days0 5 10 15 20 25

Delt

a bod

y w

eigh

t

0

1

2

3

4

5

CholesterolFormononetinC7F

lowastlowastlowast

Figure 1 Body weight of cholesterol fed C57BL6 mice (119899 = 23ndash25) supplemented with either formononetin or 2-heptyl-formonetin(C7F) for three weeks (Experiment 1) Graphs show mean plusmn SEMlowast119875 le 005

expression arrays was used to calculate the fold changesin gene expression for gene expression and 119875 values inExperiment 1 In Experiment 1 all ANOVAs were performedby the software Minitab ver 14 (Minitab PA USA) InExperiment 2 differences between the groups were analysedusing the GLM procedure in SAS (SAS 93 SAS Institute)Data were considered statistically significant when 119875 le 005

3 Results

31 Supplementation with C7F Decreased Body Weight GainAt the end of Experiment 1 mice fed cholesterol plus C7Fweighed significantly less than the mice fed only cholesterol(119875 lt 005) There were no weight differences betweenthe mice fed only cholesterol and those fed cholesterol plusformononetin (Figure 1) The fat percentage was decreasedin all three experimental groups after cholesterol feeding andafter the experimental period compared to the initial accli-matisation period However there were no differences among

the three experimental groups (Table 1) Bone mineral con-centration and bone mineral density increased significantlyfrom the initial acclimatisation period over the cholesterolinduction period till the experimental period but there wereno differences among the experimental groups (Table 1)

Difference in weight development can be due to differ-ences in gut microbiota [27] However even though therewas a significant clustering in gut microbiota composition inrelation to feeding on the 119910- and 119911-axis of the PCA-plot thiswas mainly due to caging (Supplementary Figure 1)

32 The Cholesterol-Enriched Diet Increased Plasma Levels ofTotal Cholesterol and HDL-Cholesterol and Decreased PlasmaLevels of Triglycerides The cholesterol-enriched diet wasexpected to elevate the plasma level of cholesterol Howeveralthough total plasma cholesterol increased after the firstfive weeks of cholesterol feeding the difference was notsignificant Yet after the experimental period the plasmalevel of total cholesterol was significantly higher for micefed cholesterol compared to the mice euthanized beforeinitiation of cholesterol feeding Surprisingly the plasmalevel of HDL-cholesterol was significantly increased andthe plasma level of triglycerides was decreased in mice fedcholesterol compared to mice euthanized before initiation ofcholesterol feeding Cholesterol feeding did not affect LDL-cholesterol (Table 2)

33 Supplementation with C7F Increased Plasma CholesterolMice fed C7F had significantly higher plasma levels of totalcholesterol and HDL-cholesterol than mice fed cholesterolor formononetin Furthermore mice fed formononetin hadincreased plasma level of triglycerides compared to mice fedcholesterol There were no differences between the experi-mental groups with respect to LDL-cholesterol (Table 2)

34 Formononetin and C7F Did Not Affect Glucose ToleranceIsoflavones have been reported to improve glucose uptake invitro and glucose tolerance in vivo [28 29] However micefed cholesterol diet supplemented with either formononetinor C7F did not differ in glucose tolerance as monitored by anoral glucose tolerance test (Figure 2(a)) or in fasting glucose


Table 2 Plasma lipid profiles of C57BL6 mice before cholesterol feeding after five weeks of initial cholesterol feeding and after threeadditional weeks where the mice were fed the cholesterol-enriched diet supplemented with either formononetin or 2-heptyl-formonetin(C7F) (Experiment 1) Data show mean plusmn SEM Different letters (a b c d) denote significant difference (P le 005) between the groups

Total cholesterol (mmolL) HDL (mmolL) LDL (mmolL) Triglycerides (mmolL)Before test period

After acclimatisation (119899 = 30) 312 plusmn 020a 139 plusmn 010a 032 plusmn 005 195 plusmn 055a

After initial cholesterol feeding (119899 = 23) 322 plusmn 026ab 140 plusmn 011ab 035 plusmn 007 113 plusmn 030ab

After test periodCholesterol (119899 = 25) 332 plusmn 025bc 146 plusmn 015b 029 plusmn 006 095 plusmn 027c

Cholesterol + formononetin (119899 = 23) 343 plusmn 027c 144 plusmn 016ab 034 plusmn 014 116 plusmn 029b

Cholesterol + C7F (119899 = 25) 383 plusmn 054d 178 plusmn 018c 028 plusmn 010 103 plusmn 039bc

Time after gavage of glucose (min)0 50 100 150 200

Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)

Figure 2 Glucose homeostasis of cholesterol fed C57BL6 mice (119899 = 23ndash25) supplemented with either formononetin or 2-heptyl-formononetin (C7F) for three weeks (Experiment 1) (a) Glucose clearance assessed by oral glucose tolerance test (2 gkg glucose) (b) Fastingplasma glucose concentration Graphs show mean plusmn SEM

levels (Figure 2(b)) (Experiment 1) compared to control micefed only the cholesterol diet

35 C7F Upregulated the Expression of Gstm1 Isoflavoneshave been shown to affect phase I and II metabolism of drugsin the liver [30] Scatter plots of the liver gene expression inExperiment 1 showed that Gstm1 (glutathione S-transferaseMu 1) was significantly upregulated 24 times in mice fedcholesterol plus C7F compared to those fed only cholesterolFurthermore the expression of Cyp11b2 (aldosterone syn-thase) was 129 times upregulated in mice fed C7F comparedto formononetin (Supplementary Figure 2)

36 Formononetin and C7F Induced Hepatic Steatosis Tofurther asses the effects of formononetin and C7F on lipidmetabolism in liver and adipose tissues a second experimentwas carried out There were no significant differences inweight development in this study perhaps because of thelower number of mice in each group (Supplementary Figure3) Feed intake was measured weekly but showed no differ-ences between the groups (Supplementary Figure 4)

It is well-documented that genistein and daidzein protectagainst the development of hepatic steatosis in rodents fedhigh-fat diets [9 10 14 15] At termination of Experiment 2weight of the liver was significantly increased in the micefed cholesterol compared to the other groups The liverweight was similar for chow and C7F fed mice but increasedfor mice fed formononetin (Figure 3(A)) Quantification oftriglycerides in the liver showed no difference between micefed chow and cholesterol In contrast there was a largeincrease in hepatic accumulation of triglycerides in the micefed formononetin and C7F (Figure 3(B)) The developmentof hepatic steatosis was confirmed by visual examination ofHampE stained sections of the livers (Experiment 2) revealingclear microvesicular structures presumably from fat vacuolesin mice fed C7F and formononetin (Figure 3(C))

37 Formononetin Protected against Hepatic Inflammationand Dysfunction The development of hepatic steatosis isoften associated with hepatic inflammation andor liverinjury Surprisingly the expression of Tnf (tumour necrosisfactors 120572) was similar in mice fed chow cholesterol andC7F but decreased in mice fed formononetin (Figure 3(D))


Wei

ght o

f liv

er (g

)

0

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05

075

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125

15

175

a

b

ac

Chol

este

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Chow

Form

onon

etin

C7F

(A)

Live

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(Mm

g tis

sue)

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20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)

C7FCholesterolChow Formononetin

(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

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este

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etin

C7F

(D)

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activ

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activ

ity (U

L)

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Chol

este

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Chow

Form

onon

etin

C7F

Chol

este

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Chow

Form

onon

etin

C7F

(E)

Figure 3 Development of hepatic steatosis in C57BL6 mice fed chow cholesterol or cholesterol supplemented with formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment 2) (A)Weight of liver (119899 = 8) (B) Triglyceride content in liver (119899 = 6) Total lipids wereextracted from liver using a modified version of the Bligh and Dyer protocol and the content of triglyceride were analysed with a commercialkit (C) Liver sections stained with hematoxylin and eosin (D) Hepatic gene expression of Tnf (tumour necrosis factor 120572) measured by RT-PCR Data is normalised to 18S ribosomal RNA and presented relatively to the expression in chow (119899 = 6) (E) Plasma level of aspartateaminotransferase (AST) and alanine aminotransferase (ALT) (119899 = 6) Graphs show mean plusmn SEM Different letters (a b) denote significantdifference (119875 le 005) between the groups


suggesting less hepatic inflammation in formononetin fedmice Similarly the plasma level of ALT was increased inmice fed cholesterol and C7F compared to mice fed chowwhereas there was no increase for mice fed formononetin(Figure 3(E)) This indicates increased damage to the hepa-tocytes in mice fed cholesterol and C7F but not in mice fedformononetin There was no difference in the plasma level ofAST (Figure 3(E))

38 Formononetin and C7F Decreased Lipogenesis 120573-Oxida-tion and Lipoprotein Metabolism To investigate possibleroutes by which formononetin and C7Fmight induce hepaticsteatosis we measured hepatic expression of genes involvedin lipogenesis 120573-oxidation and lipoprotein metabolism(Experiment 2)

Surprisingly the expressions ofAcaca (acyl-CoA carboxy-lase-1) and Fasn (fatty acid synthase) the rate-limiting genesin lipogenesis were significantly upregulated in cholesterolfed mice compared to chow but similar to chow for micefed formononetin and C7F The pattern was the same forScd1 (stearoyl-CoA desaturase) the rate-limiting gene inthe synthesis of monounsaturated fatty acids although theexpression was increased in formononetin compared to chowbut not as much as in cholesterol-fed mice There were nodifferences in the expressions of the lipogenic transcriptionfactors Srebf1 (sterol regulatory element-binding protein-1c)and Mlxipl (MLX interacting protein-like or carbohydrateresponse element binding protein)The genesGpam (glycerolphosphate acyltransferase) and Dgat2 (diglyceride acyltrans-ferase 2) are both central to the synthesis of triglyceridesCompared to chow-fed mice the expression of Gpam wasupregulated in mice fed cholesterol but similar in mice fedformononetin and C7F There was no difference between thegroups for the expression of Dpat2 (Figure 4(A))

Compared to mice fed chow the expression of the lipoly-tic gene Atgl (adipose triglyceride lipase) was upregulatedin mice fed cholesterol and C7F but not affected in micefed formononetin There was no difference in the expressionof Ppara (peroxisome proliferator-activated receptor 120572) atranscription factor involved in catabolism of fatty acidsThe expression of Acox1 (acyl-CoA oxidase) involved inperoxisomal 120573-oxidation was upregulated in cholesterolfed mice compared to the three other groups whereas theexpression ofCpt1a (carnitine palmitoyl-CoA transferase-1a)involved in mitochondrial 120573-oxidation was the same in allthree groups compared to chow although the expression wasdecreased in mice fed formononetin compared to cholesterol(Figure 4(B))

The expression of Acat2 (acetyl-CoA acetyltransferase 2)responsible for synthesis of cholesteryl esters was similar formice fed cholesterol and chow but downregulated in micefed formononetin and C7F The expressions of Mttp (micro-somal triglyceride transfer protein) which controls theassembly of lipoproteins and Ldlr (low-density lipoproteinreceptor) which mediates endocytosis of ApoB-containinglipoproteins were both increased in mice fed cholesterolcompared to chow but similar chow-fed mice and mice fedformononetin and C7F (Figure 4(C))

As the mice were fed a cholesterol-enriched diet itseemed likely that the metabolism of cholesterol could beaffectedHowever therewas no difference in the level of genescentral in cholesterol metabolism (Hmgcr (3-hydroxy-3-methylglutaryl-Coenzyme A reductase) Cyp7a1 (cholesterol7 alpha-hydroxylase) Nr1h3 (liver X receptor 120572) and Nr1h4(farnesoid X receptor)) (Figure 4(D))

39 C7F Increased Lipogenic and Lipolytic Gene Expressionin iWAT We also examined gene expression in the adiposetissues (Experiment 2) Of interest the expressions of Srebf1and Pparg (PPAR 120574) master regulators of lipogenesis wereupregulated in eWAT in mice fed cholesterol and C7Fcompared to mice on chow (Figure 5(A)) Furthermore theexpressions of Srebf1 Acaca Fasn and Scd1 as well as Atglwere upregulated in iWAT from mice fed C7F comparedto the three other groups (Figure 5(B)) The expressionof Ucp1 (uncoupling protein-1) essential for nonshiveringthermogenesis was upregulated in iBAT from cholesterol-fedmice compared to the three other groups and in iWAT frommice fed C7F compared to the three other groups (Figures5(B) and 5(C)) Also of interest the expression ofEmr1 (EGF-like module containing mucin-like hormone receptor-likesequence 1 or F480) a macrophage marker was increasedin eWAT from mice fed cholesterol and C7F but comparedto cholesterol-fed mice the expression was down-regulatedin both eWAT and iWAT in mice fed formononetin (Figures5(A) and 5(B))

Visual examination of HampE stained sections of eWATiWAT and iBAT showed no differences in size of theadipocytes between the groups (data not shown) (Experi-ment 2)

4 Discussion

Supplementation with formononetin or C7F to C57BL6Jmice fed a cholesterol-enriched diet had limited effectson body weight body composition and glucose toleranceHowever C7F increased the serum level of total cholesteroland HDL-cholesterol More importantly formononetin andC7F induced hepatic steatosis by affecting adipocyte andhepatic gene expression although hepatic gene expression ofTnf was decreased by formononetin

Studies with genistein and daidzein using doses compa-rable to this study show a substantial decrease in body weightand fat mass [9ndash11] and improved glucose tolerance [28]However genistein and daidzein have been supplemented tomice fed high-fat diets and thus getting considerably obesewhich could explain contradictory results in the presentstudy

Surprisingly formononetin and C7F induced hepaticsteatosis Increased lipogenesis andor decreased 120573-oxidationpromote the development of hepatic steatosis [31] Hep-atic gene expression suggested decreased peroxisomal 120573-oxidation but also decreased lipogenesis and decreasedtriglyceride assembly in mice fed formononetin and C7Fcompared to cholesterol suggesting overall decreased hepaticlipid metabolism In mice fed formononetin the expressionof Acox1 was slightly decreased correlating with decreased


Lipogenesis and TG synthesis

Srebf1 Mlxipl Acaca Fasn Scd1 Gpat1 Dgat2

Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F

Atgl Ppara Cpt1a Acox1

Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F

Lipoprotein metabolism

Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F

Cholesterol metabolism

Hmgcr Cyp7a1 Nr1h3 Nr1h4

Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)

Hydrolysis and 120573-oxidation of fatty acids

Figure 4 Hepatic gene expression measured by RT-PCR in C57BL6 mice fed chow cholesterol or cholesterol supplemented formononetinor 2-heptyl-formonetin (C7F) for five weeks (Experiment 2) (A) Genes involved in lipogenesis (Srebf1 (sterol regulatory element-bindingprotein-1c) Mlxipl (carbohydrate response element binding protein) Acaca (acyl-CoA carboxylase 1) Fasn (fatty acid synthase) and Scd1(stearoyl-CoA desaturase 1)) and synthesis of triglycerides (Gpam (glycerol phosphate acyltransferase) andDgat2 (diglyceride acyltransferase2)) (B) Genes involved in hydrolysis and beta-oxidation of fatty acids Atgl (adipose triglyceride lipase) Ppara (peroxisome proliferator-activated receptor 120572)Cpt1a (carnitine palmitoyltransferase 1a) and Acox1 (acyl CoA oxidase) (C) Genes involved in lipoprotein metabolismAcat2 (acetyl-CoA acetyltransferase)Mttp (microsomal triglyceride transfer protein) and Ldlr (low-density lipoprotein receptor) (D) Genesinvolved in cholesterol metabolism Hmgcr (3-hydroxy-3-methyl-glutaryl-CoA reductase) Cyp7a1 (cholesterol 7 alpha-hydroxylase) Nr1 h3(liver X receptor) and Nr1 h3 (farnesoid X receptor) Data is normalised to 18S ribosomal RNA and presented relative to the expression inchow (119899 = 6) Graphs show mean plusmn SEM Different letters (a b c) denote significant difference (119875 le 05) between the groups

expression of Atgl Mice with liver-specific deletion of Atglhave severe hepatic steatosis but normal plasma levels ofglucose triglycerides and cholesterol [32] Thus deceasedlipolysis and 120573-oxidation could partly explain the devel-opment of hepatic steatosis especially for mice fed for-mononetin although decreased lipogenic gene expressioncould counteract this effect In agreement with our resultsgenistein and daidzein decrease lipogenic gene expression[14 33] whereas the expression of genes involved in 120573-oxidation has been decreased in some studies [34 35] but notaffected in others [14 15] This suggests that other factors areinvolved in the increase in hepatic steatosis in this study

The development of hepatic steatosis can also be causedby decreased export of fatty acids from the liver due toderegulated lipoproteinmetabolismMicrosomal triglyceridetransfer protein (MTTP) deficient mice have reduced plasma

triglycerides levels but develop hepatic steatosis withoutinsulin resistance and inflammation [36] Similarly low-density lipoprotein receptor (LDLR) deficient mice alsodevelop hepatic steatosis [37] Thus although we did notobserve a decrease in plasma triglycerides decreased expres-sion of Mttp and Ldlr could be a major cause of the devel-opment of hepatic steatosis in mice fed formononetin andC7F The effects on lipoprotein metabolism by formononetinand C7F in this study are to a large extent supported by astudy in HepG2 cells by Borradaile et al [38] In their studygenistein and daidzein decreased apolipoprotein B secretionthrough decreased MTTP activity and mRNA expressionand decreased acetyl-Coenzyme A acetyltransferase activityHowever they report increased expression of Ldlr Interest-ingly in this study genistein also increased triglyceride massin the cells


eWAT

Srebf1 Pparg Cebpa Acaca Fasn Scd1 Atgl Ucp1 Emr1

Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT

Pparg Cebpa Ucp1 Emr1

Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)

Figure 5 Adipocyte gene expression in C57BL6 mice fed chow cholesterol or cholesterol supplemented formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment 2) Gene expressionmeasured by RT-PCR of Srebf1 (sterol regulatory element-binding protein-1c) Pparg (peroxisome proliferator-activated receptor 120574)Cebpa (CCAATenhancer-binding protein120572)Acaca (acyl-CoA carboxylase 1) Fasn(fatty acid synthase) Scd1 (stearoyl-CoA desaturase 1) Atgl (adipose triglyceride lipase) Ucp1 (uncoupling protein 1) and Emr1 (F480) in(A) eWAT (B) iWAT and (C) iBATmeasured by RT-PCR Data is normalised to 18S ribosomal RNA and presented relative to the expressionin chow (119899 = 6) Graphs show mean plusmn SEM Different letters (a b) denote significant difference (119875 le 05) between the groups

Other isoflavones have been shown to decrease lipogenicgene expression in adipocytes both in vitro [39ndash41] andin vivo [10 14] In 3T3-L1 preadipocytes lower concen-trations of C7F increase lipid accumulation whereas highconcentrations decrease lipid accumulation (manuscript in

preparation) This response is similar to genistein both invitro (unpublished results) and in vivo [11] Based on thisstudy it is not possible to conclude why C7F in contrastto other isoflavones increased lipogenic gene expressionin vivo Both genistein and daidzein have been shown to


induce lipolysis [40ndash42] Increased expression of Atgl by C7Fsuggests increased lipolysis in iWATwhich could explainwhythere is no increase in fat mass despite increased lipogenicgene expression Moreover this could imply a flux of fattyacids from iWAT to the liver

Despite increased accumulation of triglycerides in theliver formononetin decreased level of plasma ALT andhepatic expression of Tnf indicating diminished liver dam-age and lower hepatic inflammation Furthermore thedecreased expression of Emr1 in iWAT and eWAT suggestslower infiltration of macrophages in mice fed formononetinIsoflavones are known to be anti-inflammatory compoundsand other studies also report decreased plasma levels ofAST ALT and tumour necrosis factor 120572 [6 13 43] anddecreased adipocyte and hepatic expression of Tnf [10 43]Accumulation of lipids in hepatocytes impairs the oxidativecapacity of the mitochondria thereby increasing the gen-eration of reactive oxygen species Reactive oxygen speciestrigger lipid peroxidation release of inflammatory cytokinesand cell death and thereby induce hepatic inflammation andfibrosis [44] Some of the effects of isoflavones have beenattributed to the antioxidative capacity Yet formononetin hasa lower antioxidative capacity than genistein and daidzein[45] This could partly explain why formononetin and C7Fdid not prevent hepatic steatosis Still the lower levels ofplasma ALT and hepatic expression of Tnf in formononetinfed mice compared to C7F fed mice could be due to a higherantioxidative capacity of formononetin than C7F

In contrast to our results a range of studies show thatplasma total cholesterol LDL-cholesterol and triglyceridesare decreased by genistein [10 12 13] daidzein [9] andformononetin [46] However LDL is a difficult parameter inmice as the levels are normally very low and the variation stillsubstantial [9] Conversely the effects on HDL-cholesterolvary some studies show upregulation [10 13 28] one studyshows downregulation [12] and two studies show no effect[14 46] Still based on the development of hepatic steatosisand dysregulated lipid and lipoprotein metabolism it seemsplausible that plasma lipid composition was dysregulated inmice fed formononetin and C7F The increased plasma levelof total cholesterol in mice fed C7F seemed to be caused by arise in HDL-cholesterolWhen LDL circulates in the blood itcan slowly build up in the inner walls of the arteries formingplaques leading to atherosclerosis In contrast HDL tends tocarry cholesterol away from the arteries and back to the liverThus the increase in HDL-cholesterol could protect againstcardiovascular diseases However in contrast to humansHDL is the essential cholesterol fraction of mice whereas thelevel of LDL-cholesterol is minimal [47] Therefore it can bedifficult to affect the level of LDL-cholesterol in mice and toextrapolate data on lipid profiles from mice to humans

Our study suggests that even though bioactive com-pounds have very similar structures the biological actionscan be very different It is a possibility that the differentactions of formononetin and C7F reported in this studyare specifically due to the use of a cholesterol-enriched dietinstead of chow and high-fat diets used in other studies Itwould therefore be interesting to assess the metabolic effects

of genistein and daidzein using other diets like a cholesterol-enriched diet to see if this affects the health benefits associatedwith these compounds

5 Conclusions

In conclusion we showed that supplementation with for-mononetin and C7F to C57BL6J mice fed a cholesterol-enriched diet induced hepatic steatosis affecting adipocyteand hepatic gene expression Of note in spite of the hepa-tosteatotic phenotype formononetin but not C7F decreasedmarkers of inflammation and liver injury

Conflict of Interests

All authors declare no conflict of interests

Acknowledgments

This study was carried out as part of the research program ofthe UNIK Food Fitness amp Pharma for Health and Disease(see httpwwwfoodfitnesspharmakudk) supported by theDanish Ministry of Science Technology and Innovationand the BEST strategic initiative supported by the RoyalVeterinary and Agricultural University The authors wish tothank Helene Farlov for excellent technical assistance

References

[1] C R Cederroth and S Nef ldquoSoy phytoestrogens and metabo-lism a reviewrdquo Molecular and Cellular Endocrinology vol 304no 1-2 pp 30ndash42 2009

[2] M S Kurzer and X Xu ldquoDietary phytoestrogensrdquo AnnualReview of Nutrition vol 17 pp 353ndash381 1997

[3] A Oslashrgaard and L Jensen ldquoThe effects of soy isoflavones onobesityrdquo Experimental Biology and Medicine vol 233 no 9 pp1066ndash1080 2008

[4] T Usui ldquoPharmaceutical prospects of phytoestrogensrdquo Endo-crine Journal vol 53 no 1 pp 7ndash20 2006

[5] R P Patel and S Barnes ldquoIsoflavones andPPAR signaling a crit-ical target in cardiovascular metastatic and metabolic diseaserdquoPPAR Research Article ID 153252 2010

[6] M Yalniz I H Bahcecioglu N Kuzu et al ldquoPreventive roleof genistein in an experimental non-alcoholic steatohepatitismodelrdquo Journal of Gastroenterology and Hepatology vol 22 no11 pp 2009ndash2014 2007

[7] K Taku K Umegaki Y Sato Y Taki K Endoh and S Watan-abe ldquoSoy isoflavones lower serum total and LDL cholesterol inhumans ameta-analysis of 11 randomized controlled trialsrdquoTheAmerican Journal of Clinical Nutrition vol 85 no 4 pp 1148ndash1156 2007

[8] X G Zhuo M K Melby and S Watanabe ldquoSoy isoflavoneintake lowers serum LDL cholesterol a meta-analysis of 8 ran-domized controlled trials in humansrdquoThe Journal of Nutritionvol 134 no 9 pp 2395ndash2400 2004

[9] M H Kim J S Park J W Jung K W Byun K S Kang andY S Lee ldquoDaidzein supplementation prevents non-alcoholicfatty liver disease through alternation of hepatic gene expressionprofiles and adipocyte metabolismrdquo International Journal ofObesity vol 35 pp 1019ndash1030 2011


[10] M H Kim K S Kang and Y S Lee ldquoThe inhibitory effectof genistein on hepatic steatosis is linked to visceral adipocytemetabolism in mice with diet-induced non-alcoholic fatty liverdiseaserdquo British Journal of Nutrition vol 104 no 9 pp 1333ndash1342 2010

[11] M Penza C Montani A Romani et al ldquoGenistein affects adi-pose tissue deposition in a dose-dependent and gender-specificmannerrdquo Endocrinology vol 147 no 12 pp 5740ndash5751 2006

[12] J Y Yang S J Lee HW Park and Y S Cha ldquoEffect of genisteinwith carnitine administration on lipid parameters and obesityin C57B16J mice fed a high-fat dietrdquo Journal of Medicinal Foodvol 9 no 4 pp 459ndash467 2006

[13] S S Mohamed P Nallasamy P Muniyandi V Periyasami andA CaraniVenkatraman ldquoGenistein improves liver function andattenuates non-alcoholic fatty liver disease in a rat model ofinsulin resistancerdquo Journal of diabetes vol 1 no 4 pp 278ndash2872009

[14] A Crespillo M Alonso M Vida et al ldquoReduction of bodyweight liver steatosis and expression of stearoyl-CoA desat-urase 1 by the isoflavone daidzein in diet-induced obesityrdquo Bri-tish Journal of Pharmacology vol 164 pp 1899ndash1915 2011

[15] Y M Lee J S Choi M H Kim M H Jung Y S Lee and JSong ldquoEffects of dietary genistein on hepatic lipid metabolismand mitochondrial function in mice fed high-fat dietsrdquo Nutri-tion vol 22 no 9 pp 956ndash964 2006

[16] N Yumiko K Akiko T Yukari I Susumu and T YasuhideldquoContent and composition of isoflavonoids in mature or imma-ture beans and bean sprouts consumed in Japanrdquo Journal ofHealth Science vol 47 pp 394ndash406 2001

[17] G Pakalapati L Li N Gretz E Koch andMWink ldquoInfluenceof red clover (Trifolium pratense) isoflavones on gene andprotein expression profiles in liver of ovariectomized ratsrdquoPhytomedicine vol 16 no 9 pp 845ndash855 2009

[18] P Shen M H Liu T Y Ng Y H Chan and E L Yong ldquoDif-ferential effects of isoflavones from Astragalus Membranaceusand Pueraria Thomsonii on the activation of PPAR120572 PPAR120574and adipocyte differentiation in vitrordquoThe Journal of Nutritionvol 136 no 4 pp 899ndash905 2006

[19] S Zhang X Tang J Tian et al ldquoCardioprotective effect of sul-phonated formononetin on acutemyocardial infarction in ratsrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 6pp 390ndash395 2011

[20] D S Pedersen and C Rosenbohm ldquoDry column vacuumchromatographyrdquo Synthesis no 16 pp 2431ndash2434 2001

[21] S A Schreyer D L Wilson and R C Leboeuf ldquoC57BL6 micefed high fat diets as models for diabetes-accelerated atheroscle-rosisrdquo Atherosclerosis vol 136 no 1 pp 17ndash24 1998

[22] J S Kang W K Lee C W Lee et al ldquoImprovement of high-fat diet-induced obesity by a mixture of red grape extractsoy isoflavone and l-carnitine implications in cardiovascularand non-alcoholic fatty liver diseasesrdquo Food and ChemicalToxicology vol 49 no 9 pp 2453ndash2458 2011

[23] P A Flecknell Laboratory Animal Anaesthesia Academic PressLondon UK 1996

[24] S D Christensen L F Mikkelsen J J Fels T B Bodvarsdottirand A K Hansen ldquoQuality of plasma sampled by differentmethods for multiple blood sampling in micerdquo LaboratoryAnimals vol 43 no 1 pp 65ndash71 2009

[25] K Dahl K Buschard D X Gram A J F DrsquoApice and A KHansen ldquoGlucose intolerance in a xenotransplantation modelstudies in alpha-gal knockout micerdquo APMIS vol 114 no 11 pp805ndash811 2006

[26] L Ovreas L Forney F L Daae and V Torsvik ldquoDistribu-tion of bacterioplankton in meromictic lake saelenvannet asdetermined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNArdquo Applied andEnvironmental Microbiology vol 63 no 9 pp 3367ndash3373 1997

[27] H J Flint ldquoObesity and the gut microbiotardquo Journal of ClinicalGastroenterology vol 45 pp S128ndashS132 2011

[28] S Ae Park M S Choi S Y Cho et al ldquoGenistein and daidzeinmodulate hepatic glucose and lipid regulating enzyme activitiesinC57BLKsJ-dbdbmicerdquoLife Sciences vol 79 no 12 pp 1207ndash1213 2006

[29] M S ChoiU J Jung J YeoM J Kim andMK Lee ldquoGenisteinand daidzein prevent diabetes onset by elevating insulin leveland altering hepatic gluconeogenic and lipogenic enzyme activ-ities in non-obese diabetic (NOD) micerdquo DiabetesMetabolismResearch and Reviews vol 24 no 1 pp 74ndash81 2008

[30] Y J Moon X Wang and M E Morris ldquoDietary flavonoidseffects on xenobiotic and carcinogen metabolismrdquo Toxicologyin Vitro vol 20 no 2 pp 187ndash210 2006

[31] E Fabbrini S Sullivan and S Klein ldquoObesity and nonalcoholicfatty liver disease biochemical metabolic and clinical implica-tionsrdquo Hepatology vol 51 no 2 pp 679ndash689 2010

[32] JWWu S PWang F Alvarez et al ldquoDeficiency of liver adiposetriglyceride lipase in mice causes progressive hepatic steatosisrdquoHepatology vol 54 no 1 pp 122ndash132 2011

[33] E S ShinHH Lee S Y ChoHW Park S J Lee andT R LeeldquoGenistein downregulates SREBP-1 regulated gene expressionby inhibiting site-1 protease expression in HepG2 cellsrdquo TheJournal of Nutrition vol 137 no 5 pp 1127ndash1131 2007

[34] S Kim I Sohn Y S Lee and Y S Lee ldquoHepatic gene expressionprofiles are altered by genistein supplementation in mice withdiet-induced obesityrdquo The Journal of Nutrition vol 135 no 1pp 33ndash41 2005

[35] M J Ronis Y Chen J Badeaux and T M Badger ldquoDietary soyprotein isolate attenuates metabolic syndrome in rats via effectson PPAR LXR and SREBP signalingrdquoThe Journal of Nutritionvol 139 no 8 pp 1431ndash1438 2009

[36] KMinehira S G Young C J Villanueva et al ldquoBlockingVLDLsecretion causes hepatic steatosis but does not affect peripherallipid stores or insulin sensitivity in micerdquo Journal of LipidResearch vol 49 no 9 pp 2038ndash2044 2008

[37] F Rodrıguez-Sanabria A Rull G Aragones et al ldquoDifferentialresponse of two models of genetically modified mice fed withhigh fat and cholesterol diets relationship to the study of non-alcoholic steatohepatitisrdquo Molecular and Cellular Biochemistryvol 343 no 1-2 pp 59ndash66 2010

[38] N M Borradaile L E De Dreu L J Wilcox J Y Edwardsand M W Huff ldquoSoya phytoestrogens genistein and daidzeindecrease apolipoprotein B secretion from HepG2 cells throughmultiple mechanismsrdquo Biochemical Journal vol 366 no 2 pp531ndash539 2002

[39] H J Park M A Della-Fera D B Hausman S Rayalam SAmbati and C A Baile ldquoGenistein inhibits differentiation ofprimary human adipocytesrdquo Journal of Nutritional Biochem-istry vol 20 no 2 pp 140ndash148 2009

[40] K Szkudelska L Nogowski and T Szkudelski ldquoGenisteinaffects lipogenesis and lipolysis in isolated rat adipocytesrdquoJournal of Steroid Biochemistry and Molecular Biology vol 75no 4-5 pp 265ndash271 2000

[41] A W Harmon and J B Harp ldquoDifferential effects of flavonoidson 3T3-L1 adipogenesis and lipolysisrdquo American Journal ofPhysiology vol 280 no 4 pp C807ndashC813 2001


[42] K Kandulska L Nogowski and T Szkudelski ldquoEffect of somephytoestrogens on metabolism of rat adipocytesrdquo ReproductionNutrition Development vol 39 no 4 pp 497ndash501 1999

[43] G Ji Q Yang J Hao et al ldquoAnti-inflammatory effect of genis-tein on non-alcoholic steatohepatitis rats induced by high fatdiet and its potential mechanismsrdquo International Immunophar-macology vol 11 no 6 pp 762ndash768 2011

[44] A P Rolo J S Teodoro and C M Palmeira ldquoRole of oxidativestress in the pathogenesis of nonalcoholic steatohepatitisrdquo FreeRadical Biology and Medicine vol 52 no 1 pp 59ndash69 2012

[45] C E Rufer and S E Kulling ldquoAntioxidant activity of isoflavonesand their major metabolites using different in vitro assaysrdquoJournal of Agricultural and Food Chemistry vol 54 no 8 pp2926ndash2931 2006

[46] L Qiu H Ye L Chen Y Hong F Zhong and T Zhang ldquoRedclover extract ameliorates dyslipidemia in streptozotocin-induced diabetic C57BL6 mice by activating hepatic PPARal-phardquo Phytotherapy Research vol 26 no 6 pp 860ndash864 2011

[47] A A Pendse J M Arbones-Mainar L A Johnson M KAltenburg and N Maeda ldquoApolipoprotein E knock-out andknock-in mice atherosclerosis metabolic syndrome andbeyondrdquo Journal of lipid research vol 50 pp S178ndash182 2009

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


decreases body weight and fat mass [9ndash11] lowers the plasmalevels of total cholesterol triglyceride and LDL-cholesterol[9 10 12 13] and protects against the development of hep-atic steatosis [9 10 14 15] Thus supplementation withisoflavones or synthetic analogues could potentially be usedin prevention andor treatment of obesity dyslipidaemia andhepatic steatosis

Formononetin is an O-methylated isoflavone present indifferent bean types at various levels [16] Previously extractscontaining formononetin have been found to have an influ-ence on fat metabolism [17 18] However as extract alsocontains a range of other compounds it is difficult tomake solid conclusions on the effects of formononetin basedon these studies One study also shows a cardioprotectiveeffect of a derivative of formononetin [19] suggesting thatformononetin and derivatives of formononetin could have apositive influence on obesity and related disorders Howeverthe effects of formononetin remain putative owing to theexistence of a range of other compounds found in extracts

A newly synthesised analogue of formononetin 2-heptylformononetin (C7F) affected lipid accumulation lipolysisand peroxisome proliferator-activated receptor 120574 (PPAR120574)activation in vitromore potently than synthetic formononetin(manuscript in preparation) Therefore we aimed to inves-tigate if formononetin and C7F positively affected lipid andcholesterol metabolism in C57BL6 mice fed a cholesterol-enriched diet by assessing the effect of formononetin andC7F on body weight and composition glucose toleranceplasma lipid composition hepatic steatosis and expressionof genes involved in lipid metabolism and phase I and IImetabolism

2 Materials and Methods

21 Synthesis of Compounds

211 General Experimental Information Commercially avai-lable reagents (Sigma-Aldrich Germany) were used withoutfurther purification unless otherwise noted Solvents used forthe synthesis were of analytical grade dried over activated4A molecular sieves when necessary (all solvents used underdry conditions had a water content of lt25 ppm measuredby coulometric Karl Fischer titration) Analytical thin layerchromatography was performed using precoated silica gel 60F254 plates (MerckGermany) and visualized using eitherUVlight or potassium permanganate stain

Column chromatographywas performed onMerckKisel-gel 60 (0015ndash0040mm) using the dry column vacuumchromatography (DCVC) technique [20] High-performanceliquid chromatography was performed on a Waters 2525 sys-tem equipped with a Waters 2996 photodiode array detectorand aWaters 2767 Sample Manager using a 100mm times 19mmid XTerra prep MS C18 column (Waters Corp MA USA)with a gradient of acetonitrile in Milli-Q water with a flowof 15mLmin (Waters) Melting points were measured on aReichert melting point microscope model N254-1R (Aus-tria) 1H and 13C NMR spectroscopic data were recorded ona Bruker Avance 300 (Bruker BioSpin MRI Germany) using

deuterated solvents as a lock Chemical shifts are reportedin parts per million relative to the residual solvent peak(1H NMR) or the solvent peak (13C NMR) as the internalstandard Accurate mass determinations were performed ona Micromass LCT apparatus (UK) equipped with an AP-ESI probe calibrated with Leu-Enkephalin (5562771 gmol)All spectrophotometric measurements were performed on aShimadzu UV-2101PC UV-vis scanning spectrophotometerwith automatic cell changer and a temperature-controlledwater-jacket-regulated cell holder (Shimadzu Corp Japan)

212 Synthesis of C7F 24-Dihydroxy-41015840-methoxy-deoxybe-nzoin (25 g 97mmol) was dissolved in 50 mL of dry THFThe stirred solution was cooled to 0∘C and triethylamine(41 mL 3 eq) was added After 5min octanoyl chloride(37mL 22 eq) was added and the cooling was stoppedAfter 15min the reaction mixture was acidified with 50 mLof 1M HCl The yellow solution was extracted twice withEtOAc and the combined organic phases were washed withwater sat NaHCO

3aq and brine After removal of the

solvent the resulting residue was dried in vacuo Purificationby dry column vacuum chromatography (EtOAcHeptane onsilicamdash5 gradient) yielded the diester (47 g 95)13C NMR (75MHz CDCl

3) 120575 = 19703 17202 17145

15875 15398 15020 13098 13069 12617 11909 1175011425 5537 4743 3450 3441 3178 2918 2915 2908 29022491 2465 2274 1420 ppm1H NMR (300MHz CDCl

3) 120575 = 780 (d J = 86Hz 1H)

726 (s 1H) 712 (d J = 87Hz 2H) 706 (dd J = 86 22Hz1H) 694 (d J = 22Hz 1H) 686 (d J = 87Hz 2H) 411 (s2H) 379 (s 3H) 382ndash375 (m 4H) 180ndash162 (m 4H) 147ndash118 (m 16H) 094ndash079 (m 6H) ppm

24-Dihydroxy-41015840-methoxy-deoxybenzoin (47 g 92mmol)was dissolved into 60 mL of anhydrous THF and DBU(16mL 11 eq) was added The reaction mixture was heatedto reflux for 1 hour at which point 5mL conc HCl was addedand the heating was continued for 1 hour The reaction wasmonitored by TLC and after complete conversion NaOH(6mL 10M) was added and the solution was refluxed foranother 30min The reaction was again monitored by TLCuntil complete conversion was observed At this point 1MHCl was added until the reaction mixture was neutralisedThe yellow solution was cooled and extracted twice withEtOAc The combined organic phases were washed withwater sat NaHCO

3aq and brine After removal of solvent

the resulting residue was dried in vacuo The residue waspurified by dry column vacuum chromatography (EtOAcheptane on silica 5 gradient) yielding C7F (263 g 78) asa white foam13C NMR (75MHz CDCl

3) 120575 = 17782 16802 16283

15937 15817 13172 12768 12502 12237 11573 11413 102777758 7716 7674 5536 3273 3173 2924 2895 2762 22721418 ppm1H NMR (300MHz CDCl

3) 120575 = 802 (d J = 87Hz 1H)

789ndash740 (bs) 715 (d J = 87Hz 2H) 691 (d J = 87Hz 2H)688ndash683 (m 2H) 378 (s 3H) 258ndash249 (m 2H) 175ndash155(m 2H) 136ndash110 (m 8H) 094ndash076 (m 3H) ppm


22 Mouse Study












26 Gene Expression





2(5





2sdot6H2O






Fat mass(g)


(g)








Delt

a bod

y w

eigh

t

0

1

2

3

4

5


lowastlowastlowast



3 Results
















Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)








Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















22 Mouse Study












26 Gene Expression





2(5





2sdot6H2O






Fat mass(g)


(g)








Delt

a bod

y w

eigh

t

0

1

2

3

4

5


lowastlowastlowast



3 Results
















Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)








Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















26 Gene Expression





2(5





2sdot6H2O






Fat mass(g)


(g)








Delt

a bod

y w

eigh

t

0

1

2

3

4

5


lowastlowastlowast



3 Results
















Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)








Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Fat mass(g)


(g)








Delt

a bod

y w

eigh

t

0

1

2

3

4

5


lowastlowastlowast



3 Results
















Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)








Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Glu

cose

(mm

olL

)

0

5

10

15

20


minus50

(a)

Chol

este

rol

Form

onon

etin

C7F

0

1

2

3

4

5

Fasti

ng g

luco

se (m

mol

L)

(b)








Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Wei

ght o

f liv

er (g

)

0

025

05

075

1

125

15

175

a

b

ac

Chol

este

rol

Chow

Form

onon

etin

C7F

(A)

Live

r TG

(Mm

g tis

sue)

0

20

40

60

80

100

a

b

Chol

este

rol

Chow

Form

onon

etin

C7F

(B)


(C)

Relat

ive e

xpre

ssio

n of

Tnf

0

02

04

06

08

1

12a

b

a

Chol

este

rol

Chow

Form

onon

etin

C7F

(D)

AST

activ

ity (U

L)

0

2

4

6

8

10

12

14

ALT

activ

ity (U

L)

0

2

4

6

8

10

12

14

a

b

ab

b

Chol

este

rol

Chow

Form

onon

etin

C7F

Chol

este

rol

Chow

Form

onon

etin

C7F

(E)











4 Discussion







Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

FormononetinC7F


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

FormononetinC7F


Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4 Discussion







Relat

ive e

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n

005

115

225

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4

ChowCholesterol

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(A)

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Relat

ive e

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n

0

05

1

15

2

25

a

b

aaba

baba

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ac

bc

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(B)

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Acat2 Mttp Ldlr

Relat

ive e

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n

002040608

1121416

a

b

cb

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a

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(C)

ChowCholesterol

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Relat

ive e

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n

0

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(D)







eWAT


Relat

ive e

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n

0

05

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15

2

25

a

bb

ab ab

a

abb

a

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ive e

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ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Relat

ive e

xpre

ssio

n

005

115

225

335

4

ChowCholesterol

FormononetinC7F

a

b

a abb

aa

b

cac

a

b

a

(A)

ChowCholesterol

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Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

b

aaba

baba

b

ac

bc

a

(B)

ChowCholesterol

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Acat2 Mttp Ldlr

Relat

ive e

xpre

ssio

n

002040608

1121416

a

b

cb

ab

a

a

b

a

(C)

ChowCholesterol

FormononetinC7F



Relat

ive e

xpre

ssio

n

0

05

1

15

2

(D)







eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















eWAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

25

a

bb

ab ab

a

abb

a

b

ab

b

ChowCholesterol

FormononetinC7F

(A)

iWAT


Relat

ive e

xpre

ssio

n

005

115

225

335

ab ab

ab

ab a a

bab b

a

b

a

b

a

b

a

b b

a a

b

ChowCholesterol

FormononetinC7F

(B)

iBAT


Relat

ive e

xpre

ssio

n

0

05

1

15

2

a

b

a

aab

a

b

ChowCholesterol

FormononetinC7F

(C)










5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions




Acknowledgments


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 2-Heptyl-Formononetin Increases...

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Transcript of Research Article 2-Heptyl-Formononetin Increases...