consumption of quercetin and quercetin- containing apple...

The Journal of Nutrition

Nutrition and Disease

Consumption of Quercetin and Quercetin-Containing Apple and Cherry Extracts AffectsBlood Glucose Concentration, HepaticMetabolism, and Gene Expression Patterns inObese C57BL/6J High Fat–Fed Mice1–4

Sarah M Snyder,5 Bingxin Zhao,5 Ting Luo,4 Clive Kaiser,6 George Cavender,5 Jill Hamilton-Reeves,7

Debra K Sullivan,7 and Neil F Shay5*

5Department of Food Science and Technology, Oregon State University, Corvallis, OR; 6Department of Horticulture, Oregon State

University, Milton-Freewater, OR; and 7Department of Dietetics and Nutrition, University of Kansas Medical Center, Kansas City, KS

Abstract

Background: Intake of polyphenols and polyphenol-rich fruit extracts has been shown to reducemarkers of inflammation,

diabetes, and hepatic complications that result from the consumption of a high-fat (HF) diet.

Objective: The objective of this study was to determine whether mice fed polyphenol-rich apple peel extract (AE), cherry

extract (CE), and quercetin, a phytochemical abundant in fruits including apples and cherries,wouldmodulate the harmful effects

of adiposity on blood glucose regulation, endocrine concentrations, and hepatic metabolism in HF-fed C57BL/6J male mice.

Methods: Groups of 8-wk-old mice (n = 8 each) were fed 5 diets for 10 wk, including low-fat (LF; 10% of total energy)

and HF (60% of total energy) control diets and 3 HF diets containing polyphenol-rich AE, CE, and quercetin (0.2% wt:wt).

Also, an in vitro study used HepG2 cells exposed to quercetin (0–100 mmol/L) to determine whether intracellular lipid

accumulation could be modulated by this phytochemical.

Results:Mice fed the HF control diet consumed 36%more energy, gained 14 g more body weight, and had;50% elevated

blood glucose concentrations (all P< 0.05) than did LF-fedmice.Mice fed HF diets containing AE, CE, or quercetin became as

obese as HF-fed mice, but had significantly lower blood glucose concentrations after food deprivation (236%, 222%,

222%, respectively; P < 0.05). Concentrations of serum C-reactive protein were reduced 29% in quercetin-fed mice

comparedwith HF-fed controls (P < 0.05). A qualitative evaluation of liver tissue sections suggested that fruit phytochemicals

may reduce hepatic lipid accumulation. A quantitative analysis of lipid accumulation in HepG2 cells demonstrated a dose-

dependent decrease in lipid content in cells treated with 0–100 mmol quercetin/L (P < 0.05).

Conclusions: In mice, consumption of AE, CE, or quercetin appears to modulate some of the harmful effects associated

with the consumption of an obesogenic HF diet. Furthermore, in a cell culture model, quercetin was shown to reduce

intracellular lipid accumulation in a dose-dependent fashion. J Nutr 2016;146:1001–7.

Keywords: apples, cherries, C57BL/6J mice, high-fat diet, phytochemicals, polyphenols, PPAR-a, quercetin

Introduction

As of 2010, more than one-third of adults in America wereconsidered to be obese (1), and the prevalence of metabolic

syndrome, a collection of clinical risk factors for cardiovasculardisease, stroke, kidney disease, and type 2 diabetes mellitus, hadrisen to 23% of US adults (2). Clinical risk factors for metabolicsyndrome include hyperglycemia, hypertriglyceridemia, hyper-lipidemia, greater waist circumference, high blood pressure, andhigh cholesterol concentrations. Beltran-Sanchez et al. (2) reportedthat the prevalence of metabolic syndrome decreased by 2.6% in

4 Supplemental Text and Supplemental Tables 1–5 are available from the ‘‘Online

Supporting Material’’ link in the online posting of the article and from the same

link in the online table of contents at http://jn.nutrition.org.

*To whom correspondence should be addressed. E-mail: neil.shay@oregonstate.

edu.

1 Funding for NFS was provided by the Oregon State University College of

Agricultural Sciences and the Blue Mountain Horticultural Society. Support for JH-R

was provided by amentored research training award, the KL2 TR000119-04, a Clinical

and Translational Science Award grant from the National Center for Advancing

Translational Sciences (NCATS) awarded to the University of Kansas Medical Center

for Frontiers: The Heartland Institute for Clinical and Translational Research.2 Author disclosures: SM Snyder, B Zhao, T Luo, C Kaiser, G Cavender,

J Hamilton-Reeves, DK Sullivan, and NF Shay, no conflicts of interest.3 The contents are solely the responsibility of the authors and do not necessarily

represent the official views of the NIH or NCATS.

ã 2016 American Society for Nutrition.

Manuscript received December 18, 2015. Initial review completed January 22, 2016. Revision accepted February 22, 2016. 1001First published online April 6, 2016; doi:10.3945/jn.115.228817.

Downloaded from https://academic.oup.com/jn/article-abstract/146/5/1001/4589914by gueston 20 May 2018

the last decade (2000–2010), along with hypertriglyceridemia(29.2%) and elevated blood pressure (28.3%). Despite thisdecrease in overall metabolic syndrome, the study found thatthe prevalence of hyperglycemia and elevated waist circumfer-ence, or abdominal obesity, rose 7% and 10.7%, respectively,during the same time period. It can be postulated that thedecrease in hypertriglyceridemia and elevation in blood pres-sure and LDL cholesterol concentrations is a result of anincreased use of antihypertensive and lipid-modifying drugs tolower cardiovascular disease risk. However, few improvementshave been observed for the prevalence of hyperglycemia orabdominal obesity.

Previous studies have shown that fruits including apples andcherries and their phytochemical extracts are effective fordecreasing the risk factors associated with metabolic diseasessuch as abdominal fat accumulation, type 2 diabetes, heartdisease, and inflammation (3–11). Apple consumption corre-lated with a decreased risk of developing type 2 diabetes andcardiovascular disease in the Women�s Health Study (12, 13).Two other studies (6, 7) demonstrated the ability of apples toinhibit lipid oxidation, reduce cholesterol, and improve glucosetolerance. Cherries also are able to help decrease the risk ofdeveloping metabolic diseases by reducing fat accumulation,body weight, cholesterol, and TG concentrations, improvingglucose and insulin regulation, and promoting an anti-inflammatory state (8, 10, 11, 14). One phytochemical in these2 commonly consumed fruits is quercetin, a compound shownpreviously to produce an anti-inflammatory effect (15). TheC57BL/6J mouse strain was used to model the consumption of ahigh-calorie, high-fat (HF)8 diet by humans. When fed a normallow-fat (LF) diet, these mice remain normal weighted andhealthy; however, when fed an HF diet, hyperphagia and obesityresults. Hypothesizing that intake of apple peel extract (AE),cherry extract (CE), and quercetin brings metabolic benefits, wefirst observed their effect on hepatic metabolic markers andglucose control. Second, we compared observed physiologiceffects to the molecular level by observing nuclear hormonereceptor (NHR)–regulated hepatic gene expression. NHRs aretranscription factors that potently activate or repress sets ofgenes related to specific metabolic pathways. Because someNHRs exhibit promiscuity in that they may bind many differentmolecules as ligands, we hypothesized that one metabolic actionof phytochemicals is mediated by these promiscuous NHRs.Three receptors of particular interest to us were PPARa, which isinvolved in lipid oxidation; PPARg, which regulates adipogen-esis and glucose control; and Pregnane X Receptor, which isinvolved in xenobiotic and cholesterol metabolism and excretion(16). The PPARa receptor, for example, regulates a set of genesrelated to FA oxidation, including carnitine palmitoyltransferase1a (Cpt1a) and acyl-CoA oxidase 1 (Acox1). This study wasperformed to identify metabolic and gene expression changeswhen commonly consumed polyphenol-rich foods, such asapples and cherries, and their components, such as quercetin,were provided to HF-fed, obese mice.

We hypothesized that mice fed polyphenol-rich extractsderived from apples and cherries and quercetin would modulatethe harmful effects of adiposity produced by an obesogenic HFdiet.

Methods

Preparation of AE and CE. The extraction protocol was modifiedbased on published methods (5, 17, 18), and the detailed method is

described in Supplemental Text. Briefly, fruit purees were subjected to

solid-phase extraction with the use of a hydrophobic polymer (Amberlite

FPX-66; Rohm and Haas). The ethanol eluent was evaporated and

freeze-dried to a powder.

Quantification of phytochemicals by HPLC. Anthocyanins were

analyzed by HPLC. Total anthocyanins were measured by AOAC

method 2005.02 (pH differential method). Polymeric color and

browning were measured by bisulfite bleaching. Total phenolic com-

pounds were measured by the Folin-Ciocalteu spectrophotometric

method. Phenolic compounds were analyzed by HPLC with the use of

the method that has been proposed as an AOAC method for phenolic

compound analysis. Antioxidants were measured by fluorescence recov-

ery after photobleaching and oxygen radical absorbance capacity assays

(19) and provided in Supplemental Table 1. The content of individ-

ual compounds in extracts was determined qualitatively by comparison

of HPLC chromatograms of extracts and mixtures of phytochemical

standards.

Mouse diet study. Forty 6-wk-old male C57BL/6J mice (JacksonLaboratories) were randomly divided into 5 groups (n = 8): an LF group

(10% energy from fat), an HF group (60% energy from fat), an HF plus

0.2% quercetin (HF+QUE) group, an HF plus 0.2% cherry extract (HF

+CE) group, and an HF plus 0.2% apple peel extract (HF+AE) group.

Equal amounts of extracts were used rather than using different amounts

of extracts standardized to a single constituent or total polyphenols. The

same amount of free quercetin was used in a fifth diet. Mice were housed

4/cage under standard conditions and acclimated for 2 wk with access to

standard rodent diet and ad libitum water and a 12-h light/dark cycle.

Experimental diets (Research Diets) and water were consumed ad

libitum for 10 wk (Supplemental Table 2). Body weights, energy intake,

and spillage were measured weekly (Figure 1). At the end of the study,

food was withheld from mice for 6 h before anesthetization with

isofluorane inhalation. Mice were killed by cardiac puncture and cervical

dislocation, blood was collected, and glucose concentrations were

measured with the use of a ReliOn Ultima Blood Glucose Monitoring

System (Abbott). Serum was obtained by centrifugation at 2000 3 g for

15 min at 4�C. Organ weights were measured and liver tissue was stored

in RNAlater (AM7021; Ambion). Liver RNA was isolated by using

Trizol (no. 15596–026; Ambion) and following the suggested product

protocol. The mouse protocol was approved by the Institutional Animal

Care and Use Committee at Oregon State University.

Intraperitoneal glucose tolerance test. A glucose tolerance test was

performed at week 6. Food was withheld for 6 h before initial baseline

glucose measurement. At time zero, a small tail cut was made and the

initial baseline glucose measurement was taken. At this time, 10 mL 20%

glucose in 0.9% saline/g body weight was injected intraperitoneally.

Mice then were returned to their cage and tail blood glucose was

measured every 30 min for 2 h (20–23).

Real time-PCR (RT-PCR). RT-PCR protocol was conducted essentiallyas recommended by the reagent supplier (Real-time PCR Handbook; Life

Technologies) and as modified by Nam and Knutson (24). Reagents used

included Applied Biosystems High Capacity cDNAReverse Transcription

Kits (Life Technologies) and Multiscribe Reverse Transcriptase. Reaction

conditions were 10 min at 25�C, 120 min at 37�C, and 5 min at 85�C.Real-time PCR was completed with the use of an Applied Biosystems

7900HT Fast thermal cycler and SensiMix SYBR Master Mix (Origene)

and following the manufacturer-suggested protocol. Standard curve

samples were measured in triplicate for all primers tested. The DDct

method for PCR and Rpl30 typically was used as the housekeepingmRNA. The PCR reaction cycle was 10 min at 95�C followed by 40

cycles of 5 s at 95�C and 20 s at 60�C. Relative mRNA levels for samples

were calculated with the use of standard curve data, and 1-factorANOVAwas used to determine significance.

8 Abbreviations used:Acox1, acyl-CoA oxidase 1; AE, apple peel extract; CE, cherry

extract; Cpt1a, carnitine palmitoyltransferase 1a; CRP, C-reactive protein; HF,

high-fat; HF+AE, HF plus 0.2% apple peel extract; HF+CE, HF plus 0.2% cherry

extract; HF+QUE, HF plus 0.2% quercetin; LF, low-fat; NHR, nuclear hormone

receptor; PAI-1, plasminogen activator inhibitor 1a; Rpl30, ribosomal

protein L30; RT-PCR; real time PCR; Scd1, stearoyl-CoA desaturase 1.

1002 Snyder et al.


Tissue fixation and staining. Tissue fixation and staining was doneaccording to standard histologic protocols. Trichrome staining was used

and images were captured digitally.

Serum lipid analyses. Serum was analyzed for TGs; total, HDL, LDL,

and VLDL cholesterol; and liver cytosolic enzymes (alanine aminotrans-

ferase and aspartate aminotransferase) with the use of an automatedVitros

250 (Ortho Clinical Diagnostics). Directly measured values included totaland HDL cholesterol, TGs, and alanine and aspartate aminotransferase.

VLDL cholesterol was calculated with the formula TGs/2.2 and LDL

cholesterol was calculated as a difference as follows: calculated LDL

cholesterol = (total cholesterol – HDL cholesterol – VLDL cholesterol).

Serum cytokines and adipokines. Cytokine concentrations were

measured with the use of MILLIPLEX MAP kits (MAP2MAG-76K,MCYTMAG-70K-PX32, and MADKMAG-71K; Millipore) by follow-

ing the manufacturer�s instructions. Plates were read on a Luminex 200

instrument with the use of xPONENT software. Data were analyzed by

ANOVAwith the use of Graph Pad software.

Cell culture. HepG2 is a hepatocellular carcinoma cell line (no. HB-8065;

ATCC); cells were maintained in 10% FBS in DMEM supplemented with

0.2% gentamicin and 0.2% fungizone. Reagents were from Gibco; cellswere maintained in a humidified incubator at 37�C with 5% CO2.

To evaluate the effect of one purified compound, HepG2 cells were

seeded with 1.4 3 106 cells/mL (5 3 106 cells/well) in 6-well plates for

24 h. Cell medium was then replaced with 1% FBS DMEM and

incubated for another 24 h. Cells were incubated with 500 mmol oleic

acid/L to induce intracellular hyperlipidemia, and cotreated with control

(vehicle alone) or quercetin (10–100 mg/mL), concentrations that weredetermined by MTT assay (Life Technologies). Quercetin was dissolved

in DMSO at 100 mmol/L. After 24 h of incubation, the medium was

removed and Oil Red O (AMRESCO) staining was performed on cells

with the use of standard protocols. Oil Red O was collected from stainedcells and absorbance was measured at 500 nm.

Statistical analysis. All data are means 6 SEMs. One-factor and

repeated-measures ANOVA was performed with the use of GraphPadPrism 6. Post hoc testing was performed with the use of Tukey�s multiple

comparison testing after ANOVA indicated significance. P values of

#0.05 were considered to be significant, and P values between 0.05 and0.10 were referred to as trending toward significance. For glucose

tolerance testing, a second ANOVA was completed only for all HF-fed

groups to identify whether differences existed within the HF-fed

groups (Figure 2B). This approach also was used in measurements ofgene expression, in which only HF-fed groups were compared.

Results

Extract composition. Phytochemical extracts are highly puri-fied ethanol- and sugar-free powders rich in anthocyanins and

FIGURE 1 Final body weight (A) and weekly energy intake (B) of male

C57BL/6J mice fed an LF diet or HF diet alone or an HF diet containing

apple peel extract, cherry extract, or quercetin for 10 wk. Energy intake

was measured as a total for each group over the course of the study.

Values are means6 SEMs, n = 8. Means without a common letter differ,

P , 0.05. HF, high-fat; HF+AE, HF plus 0.2% apple peel extract; HF+CE,

HF plus 0.2% cherry extract; HF+QUE, HF plus 0.2% quercetin; LF, low-fat.

FIGURE 2 Glucose concentration (A) and glucose tolerance (B) after

food deprivation of male C57BL/6J mice fed an LF diet or HF

diet alone or an HF diet containing apple peel extract, cherry extract, or

quercetin for 6 wk. Mice were food-deprived 6 h before baseline blood

glucose measurement. Values are means 6 SEMs, n = 8. Means

without a common letter differ, P , 0.05. *Tended to differ from HF

when only HF-fed groups were compared, 0.05 , P , 0.01. HF, high-

fat; HF+AE, HF plus 0.2% apple peel extract; HF+CE, HF plus 0.2%

cherry extract; HF+QUE, HF plus 0.2% quercetin; LF, low-fat.

Metabolic improvement with cherry, apple, and quercetin intake 1003


polyphenols. The compositions are shown in Supplemental Table 1.Cherry extract polyphenols were predominantly chlorogenic acid,epicatechin, and rutin (quercetin-3-O-rutinoside). AEs were high inunconjugated quercetin and the quercetin conjugates quercetin3-rhamnoside and rutin, along with lower amounts of chlorogenicacid and epicatechin. The AUCs indicated that the quercetincontent of the CE was <5% of total polyphenols; for AE, all formsof quercetin combinedwere;30%of the total polyphenol content.

Energy intake and weight gain. The body weight of the miceand their energy intake was not different between the HF-fedtreatment groups; however, all HF-fed groups were different fromthe LF-fed control group (P # 0.0001). In terms of grams of dietconsumed per day, the LF-fed andHF-fed groupswere not different;however, this comparison does not consider the difference in energydensity of the LF and HF diets. At week 10, HF-fed mice weighed;14 g more than the LF-fed mice (P < 0.01). No difference wasobserved in energy consumed between the groups of HF-fed mice.

Glucose concentrations and glucose tolerance. Baselineglucose measurements were taken at week 6 and at the end of thestudy on every mouse with the use of glucose test strips and ahandheld glucometer. At week 6, all groups fed HF plus AE, CE,or quercetin had significantly lower baseline glucose concentra-tions than did the mice fed the HF diet alone (P < 0.05) and all3 were also not different from LF-fed mice (Figure 2). At week 10,the HF+CE, HF+QUE, and HF+EA groups had intermediatebaseline glucose concentrations compared with the HF-fed andLF-fed control groups, and the HF+AE treatment group hadbaseline glucose concentrations that were similar to those of theHF-fed control mice (data not shown). All HF-fed mice hadreduced glucose response compared with LF-fed mice as measuredby AUC in an intraperitoneal glucose tolerance test. In comparisonwith the HF-fed mice, both the HF+AE– and HF+QUE–fed miceshowed improved glucose sensitivity (Figure 2B, P < 0.05).

Serum markers. Serum collected by heart puncture at necropsywas analyzed for lipids and other blood markers (Supplemental

Table 3). LDL, VLDL, and HDL cholesterol and TG concen-trations showed no difference between all HF treatment groups,but HDL cholesterol concentrations were higher in HF-fed micethan in LF-fed mice (P = <0.001). Total cholesterol was lower inLF-fed mice than in HF-fed mice (P < 0.01), and elevation in totalcholesterol in HF-fed compared with LF-fed mice was amelioratedby including AE in the HF diet at a concentration that did not differsignificantly from either the LF-fed orHF-fed group. The remainingHF treatment groups showed no differences from the HF-fedcontrol group for serum cholesterol. Creatinine concentrationswere decreased in the HF+QUE–fed mice compared with allother groups (P # 0.0001). Concentrations of alanine amino-transferase and aspartate aminotransferase showed no differencebetween any diet groups (Supplemental Table 3).

Serum hormone, cytokine, and adipokine concentrations werealso measured (Supplemental Table 4). Several markers indicatedprofound metabolic differences between LF- and HF-fed mice,including C-reactive protein (CRP), insulin, leptin, plasminogenactivator inhibitor 1 (PAI-1), and resistin; other markers werenot different. CRP was reduced in quercetin-fed mice comparedwith HF-fed control and HF+AE–fed mice (P < 0.05) and wasnot distinguishable from that in the LF-fed group. Serum PAI-1 concentrations inHF+AE– andHF+CE–fedmice were lower thanthose in HF-fed control mice (P < 0.05). Leptin concentrationstended to be lower (P # 0.10) in HF+CE–fed mice than in HF-fedcontrol and HF+QUE–fed mice.

Hepatic gene expression. Various hepatic mRNAs wereshown to be upregulated by diet when measured via Realtime-PCR (Figure 3). Compared with in HF-fed controlmice, Cpt1a was higher in HF+AE–fed mice, and Acox1 washigher in HF+AE– and HF+QUE–fed mice (P < 0.05). Stearoyl-CoA desaturase 1 (Scd1) mRNA levels were measured andfound not to be significantly different between groups. OthermRNAs were tested; however, no statistically significant dif-ferences in gene expression were observed (SupplementalTable 5).

FIGURE 3 Cpt1a (A), Acox1 (B), and Scd1 (C) mRNA levels in male

C57BL/6J mice fed an LF diet or HF diet alone or an HF diet containing

apple peel extract, cherry extract, or quercetin for 10 wk. Values are

normalized to Rpl30 gene expression and are expressed as a fold

difference compared with the HF control diet. Values are means 6SEMs, n = 4–8. Means without a common letter differ, P , 0.05.

Acox1, acyl-CoA oxidase 1; Cpt1a, Carnitine palmitoyltransferase 1a;

HF, high-fat; HF+AE, HF plus 0.2% apple peel extract; HF+CE, HF

plus 0.2% cherry extract; HF+QUE, HF plus 0.2% quercetin; Rpl30,

ribosomal protein L30; Scd1, stearoyl-CoA desaturase 1.

1004 Snyder et al.


Histology. Liver tissue sections were stained to identify areas offat accumulation and fibrosis; representative images for eachgroup are in Figure 4. Slides of the livers from the HF-fed miceexhibited moderate to severe zone 3 (centrilobular) steatosis torandom and bridging areas of microvacuolar steatosis withlesser areas of macrovacuolar steatosis. Inflammation was rareand frank fibrosis was not evident. Slides of the livers of theLF-fed mice exhibited mild periportal or random microvacuo-lization. In the livers of the HF-fed control mice, many cell nucleiwere shifted from a location in the center of the hepatocyte tothe periphery, apparently due to interference from normal cell

structure because of the presence of numerous large fat globules. Incontrast, hepatocytes from the AE-, CE-, and quercetin-fed miceappeared to have an intermediate appearance. A qualitative visualinspection of the liver at necropsy confirmed these histologicobservations. The livers of the LF-fedmicewere dark red; the liversof the HF-fed control mice were a pale tan color, suggestive of ahigh fat content; and the various phytochemical-fed groups hadlivers with intermediate coloration.

In vitro hyperlipemia. Finally, within the confines of a simplecell culture trial, we evaluated the hypothesis that quercetincould have an impact on cellular lipid accumulation. HepG2cells were induced to become hyperlipidemic by incubation witholeic acid (cis-9-Octadecenoic). This MUFA has been shownto promote lipid accumulation without cell death in severaldifferent cell culture models. When coincubated with quercetin,a dose-dependent reduction in cellular lipid accumulation wasobserved (Figure 5; P < 0.05).

Discussion

The obese state correlates with the risk of type 2 diabetes,cardiovascular disease, hepatosteatosis, and chronic inflamma-tion. In animal models, phenolic acid– and anthocyanin-richextracts have been shown to ameliorate some of the metabolicdysfunction that results from obesity (5, 8, 25–27). Althoughsome studies have examined the effect of the intake of apples,cherries, and their phytochemical extracts, to our knowledge,the present study is unique in that it examines the relative effectsof these 2 phytochemical extracts side by side, along withthe abundant phytochemical, quercetin, by using the HF-fedC57BL/6J mouse model. At the given level of incorporation intothe diet (0.2% wt:wt), this intake is equivalent to a person�sconsuming approximately two 500-mg quercetin capsules/d,when the FDA-recommended dose conversion factor of 12.3 isused to translate mouse to human (28). Similar calculationsshow that the amount of AE and CE provided to these mice issimilar to a few servings of each given fruit per day for a human.This conversion is necessary when one compares the metabolicrate and energy intake of a mouse, which might consume anamount of food equivalent to ;10% of its own body weight,

FIGURE 4 Liver histology stained with trichrome from male C57BL/

6J mice fed an LF diet or HF diet alone or an HF diet containing apple

peel extract, cherry extract, or quercetin for 10 wk. White globules

within cells identify lipid accumulation. Scale: 0.5 inch = 50 mm. HF,

high-fat; HF+AE, HF plus 0.2% apple peel extract; HF+CE, HF plus

0.2% cherry extract; HF+QUE, HF plus 0.2% quercetin; LF, low-fat.

FIGURE 5 Oil Red O staining of HepG2 cells exposed to oleic acid and

various concentrations (0–100 mmol/L) of quercetin for 24 h. Intracellular

lipid accumulation was measured. Values are means 6 SEMs, n = 8.

Means without a common letter differ, P , 0.05. QUE, quercetin.



with humans, who typically consume a substantially lower amountof energy each day in proportion to their body weight.

In week 6, baseline glucose concentrations were shown to bereduced in all mice fed the HF diet plus a supplement comparedwith HF-fed control mice. This antidiabetic effect might bemediated through regulatory factors, including PPARg or AMP-activated protein kinase.

No difference was observed in the HF-fed groups for LDL,VLDL, and HDL cholesterol and TGs. The absence of sup-plemental cholesterol in our diets may explain these data.Still, serum total cholesterol was reduced (P < 0.05) inHF+AE–fed mice compared with HF-fed mice. Creatinine con-centrations were decreased in HF+QUE–fed mice comparedwith both HF-fed control and LF-fed mice. Because elevatedserum creatinine is indicative of renal failure and is associatedwith high blood pressure and diabetes mellitus (29), it may be ofinterest to further explore this finding.

Several markers of chronic inflammation response andendocrine function were changed in mice fed the HF diets thatwere supplemented. Perhaps most importantly, the additionof quercetin to the HF diet resulted in a reduction in CRPconcentrations to the concentrations observed in LF-fed mice.

The hyperinsulinemia observed in HF-fed mice at week 6was ameliorated in HF+AE– and HF+QUE–fed mice to a levelstatistically equivalent to that observed in LF-fed mice. Thesedata are consistent with the glucose AUC data reported in Figure2 in that AUC values for those 2 groups tended to be lower thanAUC values measured in HF-fed mice (P < 0.10). Changes werealso observed in serum PAI-1 concentrations for both extract-fedgroups. PAI-1 is a marker of metabolic syndrome and an inhib-itor of fibrinolysis, and contributes to the pathology of fibrosis intissues, including the liver and kidney.

Phytochemicals have been shown to have an impact onNHR-mediated regulation, notably for PPARa (11, 26, 30, 31), PPARg(26, 30, 32), and PXR (33, 34), and regulation via thetranscription factor Nuclear factor (erythroid-derived 2)-like 2(35). FAs and fibrates have been found to serve as ligands forPPARa, along with other natural compounds, such as soyisoflavones (30, 31) and compounds in tart cherry powder (11).In 2003, Mezei et al. (30) observed an antidiabetic andhypolipidemic effect of soy isoflavones and attributed the effectsto an increase in PPAR pathway activation. In 2006, Mezei et al.(31) further confirmed the role of soy isoflavones in activatingPPARa by using a PPARa knockout model. The expression ofCpt1awas measured to confirm PPARa activity. The Cpt1a geneis strongly regulated by PPARa, and serves as a good marker forPPARa activation (37). Other genes that are regulated by PPARaare Acox1 and Scd1 (11, 37).

Cpt1a mRNA levels were increased in HF+AE–fed mice.Similar results were observed for Acox1, for which HF+AE– andHF+QUE–fed mice had increased relative expression. Scd1mRNA expression appeared to parallel Cpt1a and Acox1, butno statistical differences were observed (P = 0.11). Recently,Okla et al. (37) demonstrated the potency of ellagic acid(4,4,5,5,6,6-Hexahydroxydiphenic acid 2,6,2,6-dilactone) inreducing hepatic lipid concentrations, perhaps involving PPARaas well.

Our qualitative histologic observations of the liver suggest areduction in intracellular fat, especially in the HF+AE– and HF+CE–fed mice. A cell culture study also confirmed the ability ofquercetin to remediate the dysregulated accumulation of lipid inHepG2, a hepatoma cell line commonly used as a human cellculture model for the hepatocyte. Reductions in hepatic lipidcontent would be entirely consistent with PPARa activation.

It is amply clear that bioactives likely act via a number ofdifferent mechanism and pathways (35). In the present study, itis suggested that NHR activation may explain our data.Remarkably, these physiologic and molecular changes occurredwithout a significant reduction in body weight or calorie con-sumption, suggesting some degree of metabolic improvementuncoupled from obesity.

We do not suggest that quercetin is the single bioactive factorin these foods or food extracts, but rather that the entireassembly of constituents needs to be considered the bioactiveentity. A challenge for future research is not only to describe theimprovements produced by the intake of specific healthful foodsor phytochemicals, but also to determine what beneficial syner-gies may be produced by consuming complementary healthyfoods containing a variety of bioactive compounds, acting onmultiple and molecular-level regulatory pathways.

AcknowledgmentsWe thank Robert Durst at the Linus Pauling Institute foranalysis of phytochemical content of fruit extracts, MichaelPellizzon of Research Diets, Inc. for helpful discussion, andMisty Bechtel at the University of Kansas Medical Center foranalysis of serum cytokines, hormones, and adipokines. SMS,JH-R, DKS, and NFS designed the research; SMS, BZ, TL, CK,GC, and NFS conducted the research; SMS and NFS analyzedthe data and wrote the paper; and NFS had primary respon-sibility for the final content. All authors read and approved thefinal manuscript.

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