lable at ScienceDirect

Nutrition 29 (2013) 443–449

Contents lists avai

Nutrition

journal homepage: www.nutr i t ionjrnl .com

Basic nutritional investigation

High-fat diet blunts activation of the nuclear factor-kB signaling pathway inlipopolysaccharide-stimulated peritoneal macrophages of Wistar rats

Maria C. Borges M.Sc. a, Marco A.R. Vinolo Ph.D. b, Amanda R. Crisma Ph.D. b, Ricardo A. Fock Ph.D. c,Primavera Borelli Ph.D. c, Julio Tirapegui Ph.D. d, Rui Curi Ph.D. b, Marcelo M. Rogero Ph.D. a,*aDepartment of Nutrition, School of Public Health, University of Sao Paulo, Sao Paulo, BrazilbDepartment of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, BrazilcDepartment of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, BrazildDepartment of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil

a r t i c l e i n f o

Article history:Received 3 February 2012Accepted 16 June 2012

Keywords:InflammationMacrophagesHigh-fat dietNuclear factor-kBRat

This work was financially supported by the Fundac~aoEstado de S~ao Paulo (FAPESP; 09/50269-0) and thDesenvolvimento Cient�ıfico e Tecnol�ogico (CNPq; 470supported by a fellowship from FAPESP (09/02371-0)* Corresponding author. Tel.: þ55-11-3061-7850; f

E-mail address: mmrogero@usp.br (M. M. Rogero)

0899-9007/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.nut.2012.06.008

a b s t r a c t

Objective: The present study was designed to investigate the effect of a high-fat diet (HFD) on theinflammatory response of peritoneal macrophages.Methods: Male Wistar rats were fed a control diet (n ¼ 12) or an HFD (n ¼ 12) for 12 wk. Aftereuthanasia, peritoneal macrophages were collected and stimulated (or not) with lipopolysaccha-ride (LPS). Results from the assays using peritoneal macrophages were analyzed with one-wayanalysis of variance or an equivalent non-parametric test. The level of significance adopted was0.05.Results: Consumption of the HFD was associated with significant increases in weight gain and fatdepots (P < 0.05). Despite having no influence in systemic markers of inflammation, such as inter-leukin (IL)-6, tumor necrosis factor-a, and plasminogen activator inhibitor-1, the HFD intakesignificantly decreased insulin sensitivity, as evaluated by the homeostasis model assessment index(P < 0.05). A decreased production of IL-1b, IL-6, IL-10, and nitric oxide in response to the LPSstimulationwas observed inperitonealmacrophages from theHFD group (P< 0.05). Also, in HFD-fedanimals, LPS incubation did not increase IL-1b and IL-6 mRNA expression (P < 0.05). These effectswere associatedwith an attenuation of IkB inhibitor kinase-b phosphorylation and nuclear factor-kBactivation in response to LPS and with a failure to decrease IkB inhibitor-a expression (P < 0.05).Conclusion: Chronic consumption of an HFD decreased the LPS-induced inflammatory response ofperitoneal macrophages, which was associated with a downregulation of the nuclear factor-kBsignaling pathway.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Obesity and its associated conditions, such as insulin resis-tance, dyslipidemia, and hypertension, referred to as metabolicsyndrome, are well-known risk factors for most chronic diseasesand represent major challenges for basic and clinical research.Several rodent models have been used to study the pathogenesisof metabolic syndrome and its health impact. Because of the

de Amparo �a Pesquisa doe Conselho Nacional de418/2009-5). M. C. B. was.ax: þ55-11-3061-7705..

ll rights reserved.

polygenic nature of this condition, monogenic models of obesityand diabetes, such as the ob/obmouse or the obese Zucker (fa/fa)rat, do not properly reflect this human disease [1]. For thisreason, researchers have been using high-fat diets (HFDs) inrodent models to promote increased adiposity, hyperglycemia,insulin resistance, and hepatic steatosis [2]. However, thecontribution of these HFD models to the understanding ofmetabolic syndrome has limitations because of the frequent useof unbalanced composition diets resulting from a low nutrientdensity and the addition of unpurified ingredients [3].

It is generally accepted that obesity and the consumption ofHFDs in rodents lead to chronic low-grade inflammation, whichis heavily involved in the pathogenesis of metabolic syndromeand chronic diseases. This chronic inflammatory response seemsto be triggered by and to occur predominantly in adipose tissue,

Table 1Composition of experimental diets

CON* HFD

Ingredients (g/1000 kJ)Cornstarch 37.14 7.09Sucrose 5.98 5.98Casein 8.38 8.38Soybean oil 2.39 2.39Lard 0.00 13.25Cellulose 2.99 2.99Mineral mix 2.09 2.09Vitamin mix 0.60 0.60L-cysteine 0.11 0.11Choline bitartrate 0.15 0.15Tert-butyl-hydroquinone 0.0005 0.0017

Fatty acids (% total fat)C14:0 0.81 � 0.01 1.29 � 0.03C16:0 14.06 � 0.03 23.25 � 0.13C18:0 4.42 � 0.01 12.01 � 0.22P

SFA 19.29 � 0.03 36.56 � 0.20C18:1 u9 27.09 � 0.03 40.07 � 0.06P

MUFA 27.09 � 0.03 40.07 � 0.06C18:2 u6 48.37 � 0.07 21.88 � 0.22C18:3 u6 0.23 � 0.00 –

C18:3 u3 4.96 � 0.01 1.48 � 0.03C20:5 u3 0.06 � 0.00 –P

PUFA 53.62 � 0.06 23.37 � 0.25P

u6 48.59 � 0.07 21.88 � 0.22P

u3 5.03 � 0.01 1.48 � 0.03u6:u3 ratio 9.7:1 14.8:1

CON, control diet; HFD, high-fat diet; MUFA, monounsaturated fatty acid; PUFA,polyunsaturated fatty acid; SFA, saturated fatty acid

* According to AIN-93M [11].

M. C. Borges et al. / Nutrition 29 (2013) 443–449444

although other sites may be involved, such as the liver, pancreas,hypothalamus, and skeletal muscle [4]. A hallmark of obesity-/HFD-induced low-grade chronic inflammation is the infiltrationof macrophages intowhite adipose tissue, which is believed to becentral to the development of inflammation, insulin resistance,and other comorbidities [5]. The inflammatory response inmacrophages is highly influenced by the nuclear factor (NF)-kBsignaling pathway. The enzymatic complex IkB kinase (IKK),which is composed of two catalytic subunits (IKK-a and IKK-b)and a regulatory subunit (IKK-g), is known to be crucial to thispathway. IKK-a/b promotes the phosphorylation of the inhibitorof kB (IkB), which leads to IkB degradation and the release ofNF-kB. This nuclear factor (NF-kB) translocates to the nucleusand promotes the transcription of targeted genes, includingtumor necrosis factor (TNF)-a, interleukin (IL)-1b, and induciblenitric oxide (NO) synthase [6,7].

Several studies have focused on unraveling the role of adiposetissue-infiltrated macrophages in the inflammatory and meta-bolic abnormalities observed in HFD-fed animals [8]. Neverthe-less, little is known about the response of macrophages residingin other tissues to HFD intake. Much of the current fundamentalknowledge about macrophage biology comes from the analysisof cells drawn from the peritoneal cavity [9]; however, littleattention has been paid to the impact of HFD on peritonealmacrophages. Amar et al. [10] observed that peritoneal macro-phages from HFD-fed animals showed increased TNF-a mRNAexpression. However, under ex vivo stimulation with lipopoly-saccharide (LPS), these cells decreased the production of proin-flammatory cytokines (e.g., IL-1b and TNF-a) and decreased thebinding of NF-kB to the promoters of TNF-a and IL-10. Thesefindings suggest that an HFD may influence the peritonealmacrophage inflammatory response in a complex manner andthat NF-kB signaling might be involved. Thus, the present studywas designed to investigate the effect of a nutrient-adjusted,semi-purified HFD on the inflammatory response and on theNF-kB signaling pathway in peritoneal macrophages.

Materials and methods

Animals and treatments

Two-month-old male Wistar rats (initial weight 298 � 27 g) were obtainedfrom the animal laboratory of the Faculty of Medicine at the University of SaoPaulo. Rats were housed in plastic cages (two to three rats per cage) in anatmosphere of 55 � 10% relative humidity, at 22 � 2�C, with a 12-h light/12-hdark cycle (lights on at 07:00 h). Rats were given free access to food and water.Body mass and diet intake were recorded three times per week. The study wasapproved by the ethics committee on animal experimentation of the Institute ofTropical Medicine, University of Sao Paulo according to the guidelines of theBrazilian College on Animal Experimentation (protocol no. 029/2008).

After acclimatization to a semi-purified diet based on American Institute ofNutrition recommendations for the adult rodent (AIN-93M) for 10 d [11], ratswere randomly assigned to the HFD group or the control (CON) group. For 12 wk,the CON group (n ¼ 12) received the AIN-93M diet (total energy, 75.8% carbo-hydrates, 9.3% fat, and 14.9% protein), whereas the HFD group (n ¼ 12) receivedan AIN-93M–based diet enriched with lard (total energy, 24.2% carbohydrates,60.9% fat, and 14.9% protein). In a pilot study, it was observed that the rats in theHFD group consumed approximately 30% less diet than the rats in the CON group.However, given the higher energy density of the HFD (23.2 kJ/g) compared withthe CON diet (16.7 kJ/g), the daily energy intake did not differ between thegroups. The insufficient intake of micro- and macronutrients by the HFD group,caused by the lower consumption of diet, could seriously affect the outcomes ofthe study. Thus, to ensure comparable intakes of micro- and macronutrientsbetween the groups, the ingredients (except for starch and lard) were normalizedaccording to the energy density of each diet, as presented in Table 1. In addition,because the HFD is highly susceptible to oxidation, the content of tert-butyl-hydroquinone was increased [12].

At the end of the 12-wk diet protocol, after 10 to 12 h of fasting, rats receivedintramuscular injections of ketamine hydrochloride (100 mg/kg of body mass)associated with xylazine hydrochloride (50 mg/kg of body mass) and then were

euthanized by decapitation. Blood was collected in dry tubes, to obtain serum, orin tubes containing ethylenediaminetetraacetic acid (10%), to obtain plasma.After centrifugation (3000 rpm at 4�C for 15 min), serum and plasma werecollected and stored at �80�C. Immediately after exsanguination, the peritonealmacrophages were collected under aseptic conditions. Intestines were washedwith saline solution to remove fecal contents before carcass chemical analyses.All rats were sacrificed at 09:00 and 11:00 h.

Fatty acid composition analysis of the diets

Esterification of fatty acids was performed according to the official methodsof the American Oil Chemists’ Society [13]. Analysis of fatty acid methyl esterswas carried out using an Agilent 7890A series gas chromatograph (AgilentTechnologies, Santa Clara, CA, USA) equipped with a split injection port, flameionization detector, and a fused silica capillary column (DB-23, J&W 122-2361;60-m � 0.25-mm inner diameter; film thickness 0.15 mm; Agilent Technologies).The samples (1.0 mL) were injected in split mode (split ratio 1:50). Helium wasused as a carrier gas, and the fatty acids were separated using a 1�C/min gradientfrom 140 to 225�C. The injector temperature was set at 250�C and the detectortemperature was set at 260�C. Peak identification was performed by comparingthe relative retention times with those of a commercial standard mixture of fattyacid methyl esters (Sigma Chemical Company, St. Louis, MO, USA). The resultswere expressed as percentages of the total fatty acids present.

Blood analyses

Serum glucose was determined using the Glucose HK Liquiform commercialkit (Labtest Diagn�ostica S/A, Belo Horizonte, MG, Brazil). Measurements ofplasma insulin, plasminogen activator inhibitor-1, IL-6, and TNF-awere obtainedusing the Luminex-based bead array method using the LINCOplex simultaneousmulti-analyte detection system (Linco Research, Inc., St. Charles, MO, USA),according to themanufacturer’s instructions. The homeostasis model assessmentfor insulin resistance index was calculated using the following formula: fastingserum insulin (mU/mL) � fasting plasma glucose (mmol/L)/22.5.

Carcass chemical analyses

The water, extractable lipids, protein, and ash contents present in thecarcasses of rats were determined as described in another study [14].

Table 2Food intake, weight gain, fat pads, body composition, blood biomarkers, andperitoneal cell counts from rats fed a control diet or high-fat diet for 12 wk

CON HFD

Mean SD Mean SD

Total food intake (g) 1882 197 1364y 99Total food intake (kJ) 31 455 3287 31 938 2092Initial body weight (g) 312.7 17.1 317.4 16.3Final body weight (g) 517.5 36.4 574.5 64.4Weight gain (%) 65.5 7.0 80.7* 15.1Fat pads weight (g):Peri-epididymal 9.3 1.9 15.6y 3.1Retroperitoneal 8.9 2.2 19.8y 3.1

Body composition (g)Extractable lipids 88.5 19.4 128.7* 32.8Lean mass 447.4 72.6 445.1 77.1Moisture 330.8 49.7 330.3 43.7Protein 93.8 18.8 102.0 12.6Ash 15.8 4.5 17.5 5.8

Blood:TNF-a (ng/L) 3.27 1.14 2.52 1.64IL-6 (ng/L) 78.3 40.5 71.2 32.7PAI-1 (ng/L) 178.3 32.4 187.2 77.9Glucose (mmol/L) 5.61 0.82 6.23 0.42Insulin (pmol/L) 155.8 47.5 204.2* 54.4HOMA-IR score 5.70 2.15 8.13* 2.23

Peritoneal cells (� 106)Total cellularity 23.2 6.4 17.3* 1.9Mononuclear cells 15.5 3.6 12.0* 2.2Eosinophils 1.32 0.54 1.50 0.57Mastocytes 1.19 0.59 1.43 0.68Neutrophils 0.64 0.58 0.50 0.41

CON, control diet; HFD, high-fat diet; HOMA-IR, homeostasis model assessmentfor insulin resistance; IL-6, interleukin-6; PAI-1, plasminogen activator inhibitor-1; TNF-a, tumor necrosis factor-aValues are presented as mean and SD (n ¼ 10–12/group).

* P < 0.05.y P < 0.001, mean values significantly different from those of the control

group.

M. C. Borges et al. / Nutrition 29 (2013) 443–449 445

Cell culture

Peritoneal macrophages were obtained by washing the peritoneal cavitywith 60 mL of sterile, pyrogen-free RPMI-1640 medium (pH 7.4) supplementedwith 10% fetal bovine serum, glutamine 2 mmol/L, 100 U/mL penicillin, and 100mg/mL streptomycin (Cultilab, Campinas, SP, Brazil). Cells were spun down (1500rpm for 10 min at 4�C) and resuspended twice in RPMI-1640 culture medium.Cell viability was determined by trypan blue exclusion. Cultures rich in macro-phages were obtained by incubating cells in 6- or 24-well polystyrene cultureplates for 2 h at 37�C in a 5% CO2 humidified air environment. Non-adherent cellswere removed by vigorous washing three times with RPMI-1640 medium.Macrophages were incubatedwith LPS 1 mg/mL (Escherichia coli, serotype 055:B5,Sigma Chemical Company). A 30-min incubation with LPS was used to assessNF-kB binding capacity, IKK-b phosphorylation (Ser 176/181) and expression, andIkB-a phosphorylation (Ser 32) and expression. A 4-h incubation with LPS wasused to evaluate IL-1b, IL-6, and TNF-amRNA expression. A 24-h incubation withLPS was used to determine IL-1b, IL-6, IL-10, TNF-a, and NO concentrations in theculture supernatant. The entire procedurewas executed under aseptic conditionsand all materials used were sterile and free of pyrogen. Total cells were deter-mined using a Neubauer chamber, and the different types of cells present werequantified in cytocentrifuge smears stained using the standard May-Grünwald-Giemsa solutions.

Western blot analysis

Extraction of total protein, electrophoresis, and immunoblotting were per-formed as described previously [6]. Cocktails of protease and phosphataseinhibitors (Sigma Chemical Company) were added to the extraction buffer toavoid sample degradation.

Electrophoretic mobility shift assay

The NF-kB binding capacity was assessed by electrophoretic mobility shiftassay. Nuclear extracts from peritoneal adherent cells (5 � 106 cells/well) wereobtained as previously described [15], and protein concentrationwas determinedaccording to the Bradford method [16]. The NF-kB binding capacity was evalu-ated as previously described [17].

Quantitative polymerase chain reaction

Total RNA from each sample (6 � 106 cells) was isolated using Trizol (Invi-trogen Life Technologies, Rockville, MD, USA) and reverse transcribed to cDNAusing the High Capacity RNA-to-DNA Master Mix kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer’s instructions. Real-time reversetranscription polymerase chain reaction was performed using the ROTOR GENE3000 (Corbett Research,Mortlake, NSW, Australia). The following oligonucleotideprimers (Invitrogen Life Technologies) were used: glyceraldehyde-3-phosphatedehydrogenase gene, 50-GACATCATCCCTGCATCC-30 and 50-CCTGCTTCACCACCTTCTT-30; IL-1b gene, 50-AGGCAGTGTCACTCATTGTGGC-30 and 50-TCACATGGGTCAGACAGCACG-30; IL-6 gene, 50-CCTTCTTGGGACTGATGTTGTTGAC-30 and 50-GGGTGGTATCCTCTGTGAAGTCTCC-30; and TNF-a gene, 50-GCGTGTTCATCCGTTCTCTA-30 and 50-GAGCCACAATTCCCTTTCTAA-30 . Each reaction consisted of 10 ng ofcDNA, 0.1 mmol/L each of forward and reverse primers, and 1� Kapa SYBR FastqPCR Kit (Kapabiosystems, Boston, MA, USA). The relative amount of targetmRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was calcu-lated according to the method described by Pfaffl [18], which takes into accountthe amplification efficiency of quantitative polymerase chain reaction for eachtarget gene.

Cytokine and NO determination in culture supernatants

The IL-1b, IL-6, and TNF-a concentrations in culture supernatants weremeasured by the Luminex-based bead array method using the LINCOplexsimultaneous multi-analyte detection system (Linco Research, Inc.) according tothe manufacturer’s instructions. The concentration of IL-10 in the culturesupernatants was assayed by enzyme-linked immunosorbent assay using the RatIL-10 Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Min-neapolis, MN, USA). NO production was determined according to the Griesscolorimetric method [19].

Statistical analysis

Before analysis, results were checked for distribution normality and homo-scedasticity. Means from the CON and HFD groups were compared by unpairedStudent’s t test or an equivalent non-parametric test (Mann–Whitney). Theresults from the assays using peritoneal macrophages were analyzed with one-way analysis of variance, Tukey post hoc, or an equivalent non-parametric test(Kruskal–Wallis, Dunn post hoc). Statistical analyses were performed using

GraphPad Prism 5.01 (GraphPad Software, Inc., San Diego, CA, USA), and the levelof significance adopted was 0.05.

Results

Food intake, weight gain, and body composition

The consumption of an HFD led to greater weight gain (P <

0.05) and to 70% and 120% increases in peri-epididymal andretroperitoneal fat pad, respectively (P < 0.001; Table 2). Therewas also a trend of an increase final body weight in the HFDgroup (P ¼ 0.05; Fig. 1B, Table 2). In addition, the HFD-fed ratshad greater extractable lipids (P < 0.05) in the carcass comparedwith the CON rats. The increased adiposity of the HFD group wasnot associated with hyperphagia because there were no differ-ences in energy intake between the groups (Table 2). In fact, foodintake (grams) in the HFD group was lower than in the CONgroup during the entire study (Fig. 1A), corresponding toa decrease of approximately 28% at the end of the study (P <

0.001). However, because there was no difference in energyintake, the consumption of protein, fiber, andmicronutrients wassimilar between the groups (Table 2).

Blood biomarkers

There were no effects of the HFD on blood plasminogen acti-vator inhibitor-1, TNF-a, IL-6, or glucose. However, the HFD-fed

0 2 4 6 8 10 12

0

100

200

300

400

500

600

700

CON

HFD

** *

*

Time (weeks)

we

igh

t (

g)

A

B

0 2 4 6 8 10 12

0

5

10

15

20

25

30

CON

HFD

* * * * * * * * * * * *

Time (weeks)

g/d

Fig. 1. Food intake (A) and body weight (B) curves of rats fed the CON or HFD for12 wk. Values are presented as mean and SD (n ¼ 15/group). * P < 0.05, differencefrom CON. CON, control diet; HFD, high-fat diet.

M. C. Borges et al. / Nutrition 29 (2013) 443–449446

rats had increased plasma insulin levels and homeostasis modelassessment for insulin resistance indices comparedwith the CONrats (P < 0.05; Table 2).

Cytokines and NO production

The HFD-fed rats showed a decrease in total cells ob-tained from the peritoneal cavity, which was associated witha decreased mononuclear cell count (P < 0.05; Table 2). Theperitoneal macrophages of the HFD group displayed a lowerproduction of proinflammatory mediators, such as IL-1b (P <

0.05) and IL-6 (P < 0.01), in response to the LPS challengecompared with the CON group (Fig. 2A, B). Also, the incu-bation with LPS led to an increase in NO production only inthe CON group (Fig. 2D). When cells were not incubated withLPS, the concentration of IL-1b, IL-6, and NO did not differbetween the groups (Fig. 2A, B, D). To further elucidate theimpact of the HFD on the peritoneal macrophage inflam-matory response, the IL-10 concentration in the culturesupernatant was determined. In contrast to the CON group (P< 0.05), no increase in IL-10 production was observed inLPS-stimulated macrophages in the HFD group (Fig. 2E).Furthermore, there were no differences between the groupsin the TNF-a supernatant concentration (Fig. 2C) and in theIL-6/IL-10 ratio (Fig. 2F).

Cytokines mRNA expression and NF-kB activation

Incubation with LPS resulted in significant increases in IL-1band in IL-6mRNA expression in the CON group (P< 0.05), but notin the HFD group (Fig. 3A, B). No effect of diet or LPS stimulationwas observed on the relative expression of TNF-amRNA (Fig. 3C).There were no differences between the groups in NF-kB DNA-binding capacity (Supplemental Fig. 1). Nevertheless, when NF-kB activation after LPS stimulationwas calculated as a percentageof values of non-stimulated cells, a decrease in NF-kB activation(�20%) was noticed in the HFD group in contrast with anincrease in the CON group (30%, P < 0.01; Fig. 3D).

NF-kB signaling pathway

In LPS-elicited cells, the phosphorylation of IKK-b was lowerin the HFD group compared with the CON group (P < 0.05;Fig. 4B). Furthermore, LPS stimulation decreased IkBa expression(P < 0.05; Fig. 4G) and increased the ratio between phosphory-lated and total IKK-b (P < 0.001; Fig. 4D) in CON rats but not inthe HFD rats. The ratio between phosphorylated and total IkBawas increased equally in the two groups after LPS incubation (P<0.01; Fig. 4H). There was no influence of HFD or LPS on IkBaphosphorylation or IKK-b expression (Fig. 4C, F).

Discussion

In the present study, consumption of the HFD resulted ina consistent increase in adiposity and a 30% decrease in insulinsensitivity. These effects occurred in the absence of hyperphagia,which underscores the importance of diet quality, independentof energy intake. In accordance with the present results, otherstudies have not observed a hyperphagic behavior driven by anHFD intake [20,21], which implies that overconsumption is notthe sole mechanism by which an HFD intake increases adiposity.

It is important to emphasize that previous studies had alreadyidentified that an HFD, even when nutrient adjusted, producesdetrimental effects on adiposity and on insulin sensitivity [22,23]. In addition, such studies have demonstrated thenutrient-adjusted, semi-purified HFD induce an inflammatorymilieu in metabolic tissues, leading to liver steatosis [22–24],increased infiltration of macrophages in adipose tissue [25], andincreased expression/production of proinflammatory mediators,such as PAI-1, monocyte chemoattractant protein-1, and TNF-a,in white adipose tissue [24,25].

The infiltration of macrophages into white adipose tissue isa well-defined phenomenon in obese subjects and in HFD-fedrodents. In addition, it is currently recognized that adiposetissue-infiltrated macrophages have decreased IL-10 expressionand increased expression of proinflammatory mediators, such asTNF-a, IL-6, inducible NO synthase, and CC receptor 2 [8,26].These observations indicate that macrophages might play anactive role in the chronic inflammation and metabolic abnor-malities observed in HFD-fed animals. In contrast to these find-ings, the results of the present study indicate that 1) there isa significant decrease in peritoneal mononuclear cell counts,possibly indicating a decrease in the macrophage population,and that 2) peritoneal macrophages show a blunted inflamma-tory response when challenged with LPS (1 mg/mL), which isillustrated by the failure to produce sufficient amounts of IL-1b,IL-6, IL-10, and NO after 24 h of incubation. Although dose– andtime–response curves were not evaluated, it seems that macro-phages of HFD-treated rats were less responsive to LPS stimu-lation. Thus, although peritoneal macrophages probably do not

A B C

D E F

0

10000

20000

30000

40000

50000

aa

b

c

-

CON

-

HFDCONHFD

+LPS

Group

+

IL-6

(n

g/l)

0

20

40

60

80

-

CON

-

HFDCONHFD

+LPS

Group

+

TN

F-

(n

g/l)

0

100

200

300

400

a

a

b

c

-

CON

-

HFDCONHFD

+LPS

Group

+

IL-1

(n

g/l)

0

200

400

600

800

a

a,b

b

Group

LPS - - +

CON HFD CON HFD

+

a,b

NO

(m

ol/l)

0

100

200

300

400

a

a,b

b

a,b

-

CON

-

HFDCONHFD

+LPS

Group

+

IL-1

0 (

ng

/l)

0

100

200

300

400

500

IL-6

/IL

-1

0 r

atio

-

CON

- +

HFDCONHFD

+

Group

LPS

Fig. 2. Concentrations of IL-1b (A), IL-6 (B), TNF-a (C), NO (D), and IL-10 (E), and the IL-6/IL-10 ratio (F) in the culture supernatants of peritoneal macrophages from rats fedthe CON or HFD for 12 wk and incubated (þ) or not (�) with LPS for 24 h. Values are presented as mean and SD (n ¼ 8–10/group). Bars without a common letter differ (P <

0.05). CON, control diet; HFD, high-fat diet; IL, interleukin; LPS, lipopolysaccharide; NO, nitric oxide; TNF-a, tumor necrosis factor-a.

M. C. Borges et al. / Nutrition 29 (2013) 443–449 447

contribute to the development of HFD-induced chronic inflam-mation, their inflammatory response was deeply affected byconsumption of an HFD.

Corroborating our results, another study found that perito-neal macrophages from HFD-fed mice showed decreased

A

C

0

3

6

9

12

15

a a

b

a,b

Group

LPS - -

CON HFDCONHFD

+ +

mR

NA

IL

-1

(A

U)

0.0

0.5

1.0

1.5

2.0

2.5

Group

LPS - -

CON HFDCONHFD

+ +

mR

NA

T

NF

- (A

U)

Fig. 3. Relative mRNA expressions of IL-1b (A), IL-6 (B), and TNF-a (C) and NF-kB acand incubated (þ) or not (�) with LPS for 4 h (A–C) or 30 min (D). Values are presented a(D) ** P < 0.01, difference from control. AU, arbitrary units; CON, control diet; HFD, higtumor necrosis factor-a.

production of IL-1b and IL-6 when incubated ex vivo with Por-phyromonas gingivalis, a bacterium that causes periodontitis [10].Similar to the present study, the HFD intake did not increaseblood IL-6 and TNF-a levels. Surprisingly, no impact of the HFDwas observed in peritoneal macrophage TNF-a production in our

B

D

-60

-40

-20

0

20

40

60

**CON HFD

% N

F-

B activatio

n

0

5

10

15

20

a

a,b

b

a,b

Group

LPS - -

CON HFDCONHFD

+ +

mR

NA

IL

-6

(A

U)

tivation (D) of peritoneal macrophages from rats fed the CON or HFD for 12 wks mean and SD (n ¼ 6/group). (A–C) Bars without a common letter differ (P < 0.05).h-fat diet; IL, interleukin; LPS, lipopolysaccharide; NF-kB, nuclear factor-kB; TNF-a,

Fig. 4. Phosphorylated IKK-b (B) and IkB-a (F), IKK-b (C), and IkB-a (G) expression and phosphorylated/total IKK-b (D) and IkB-a (H) ratio of peritoneal macrophages from ratsfed the CON or HFD for 12 weeks and incubated (þ) or not (�) with LPS for 30 min. (A, E) After densitometric analysis, blots were normalized to b-actin values. Values arepresented as mean and SD (n ¼ 8–12/group). Bars without a common letter differ (P < 0.05). CON, control diet; HFD, high-fat diet; IL, interleukin; IKK, complex IkB kinase; IkB,inhibitor of kB; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor-a.

M. C. Borges et al. / Nutrition 29 (2013) 443–449448

study. However, considering that TNF-a is an early response genecompared with IL-1b or IL-6 [27], the peritoneal macrophagesfrom the HFD group could catch up with the CON cells in TNF-a production because of the long period of LPS incubation,masking any existing effect of the HFD.

Our data point to an involvement of alterations at the tran-scriptional level in the HFD-induced blunted inflammatoryresponse of LPS-challenged peritoneal macrophages becausethese cells did not increase IL-1b and IL-6 mRNA expression inresponse to LPS. Therefore, we investigated whether this findingcould be associated with a downregulation of the NF-kBsignaling pathway. The fact that LPS stimulation did not lead tothe expected decrease in IkB-a expression in the HFD groupsuggests that the lower IKK-b activation resulted in lowerphosphorylation and the consequent lower degradation of IkB-a.As a result, after LPS incubation, a 20% decrease in NF-kB bindingcapacity was observed in macrophages from the HFD group incontrast to a 30% increase in the cells from the CON group. This

downregulation of the NF-kB signaling pathway resulting froman HFD intake might be related to decreased serine–threoninekinase (Akt) activation and to epigenetic changes in NF-kBpromoters [28,29].

The mechanisms by which an HFD impairs the inflammatoryresponse in peritoneal macrophages remains elusive; however,we hypothesize that it may be linked to the development oftolerance consequent to the chronic exposure to ligands ofreceptors that signal inflammatory pathways, such as toll-likereceptor (TLR)-4 [30]. There is evidence that not only thechronic intake of a lard-based HFD leads to increased circulatinglevels of LPS and long-chain saturated fatty acids [31–33], but italso decreases the expression of TLR-2 and TLR-4 in in vitro bonemarrow-differentiated macrophages [28].

Our results suggest that long-term consumption of an HFD byrats decreases the capacity of peritoneal macrophages to mountan adequate inflammatory response to a bacterial component.Peritoneal macrophages from leptin-deficient obese (ob/ob) mice

M. C. Borges et al. / Nutrition 29 (2013) 443–449 449

failed to increase phagocytic and candidacidal activity after aninflammatory stimulus (thioglycolate injection) compared withlean mice. These immune defects could not be restored byin vitro leptin treatment, suggesting the obesity might bedetrimental to resident macrophage function independent ofleptin [34]. To our knowledge, there is no study that has evalu-ated the impact of an HFD on phagocytosis and killing of peri-toneal macrophages; however, the decreased response to LPSstimulation observed in our study and the increased suscepti-bility of HFD-fed animals to bacterial infections [10] suggest thatan HFD may interfere with resident macrophage function. Thus,the blunted inflammatory response observed in the presentstudy may be somewhat involved in the impaired innateimmune response observed in HFD-fed animals. The associationbetween obesity/HFD and impaired innate immune response iscritical, considering the worldwide trend to Westernized dietarypatterns and the progressive increases in the proportion ofelderly and obese subjects [35]. In addition, the implicationsmight be particularly worrisome for low-/middle-income coun-tries in which chronic and infectious diseases coexist [36].

In summary, the long-term consumption of an HFD, evenwithout hyperphagia, significantly influenced adiposity andinsulin sensitivity. Peritoneal macrophages from HFD-fed ratsshowed a decreased production of cytokines and NO anda decreased activation of the NF-kB signaling pathway whenchallenged with 1 mg/mL of LPS, indicating an impairment in theinflammatory response.

Acknowledgments

The authors thank I. S. O. Pires and M. C. Ferreira for technicalassistance.

Supplementary data

Supplementary data related to this article can be found onlineat http://dx.doi.org/10.1016/j.nut.2012.06.008.

References

[1] Buettner R, Scholmerich J, Bollheimer LC. High-fat diets: modeling themetabolic disorders of human obesity in rodents. Obesity 2007;15:798–808.

[2] Kang JH, Tsuyoshi G, Han IS, Kawada T, Kim YM, Yu R. Dietary capsaicinreduces obesity-induced insulin resistance and hepatic steatosis in obesemice fed a high-fat diet. Obesity 2010;18:780–7.

[3] Hariri N, Thibault L. High-fat diet-induced obesity in animal models. NutrRes Rev 2010;23:270–99.

[4] Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. AnnuRev Immunol 2011;29:415–45.

[5] Weisberg SP, Hunter D, Huber R, Lemieux J, Slaymaker S, Vaddi K, et al.CCR2 modulates inflammatory and metabolic effects of high-fat feeding. JClin Invest 2006;116:115–24.

[6] Rogero MM, Borelli P, Fock RA, Borges MC, Vinolo MA, Curi R, et al. Effectsof glutamine on the nuclear factor-kappaB signaling pathway of murineperitoneal macrophages. Amino Acids 2010;39:435–41.

[7] Fock RA, Rogero MM, Vinolo MA, Curi R, Borges MC, Borelli P. Effects ofprotein–energy malnutrition on NF-kappaB signalling in murine peritonealmacrophages. Inflammation 2010;33:101–9.

[8] Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch inadipose tissue macrophage polarization. J Clin Invest 2007;117:175–84.

[9] Ghosn EE, Cassado AA, Govoni GR, Fukuhara T, Yang Y, Monack DM, et al.Two physically, functionally, and developmentally distinct peritonealmacrophage subsets. Proc Natl Acad Sci U S A 2010;107:2568–73.

[10] Amar S, Zhou QD, Shaik-Dasthagirisaheb Y, Leeman S. Diet-induced obesityin mice causes changes in immune responses and bone loss manifested bybacterial challenge. Proc Natl Acad Sci U S A 2007;104:20466–71.

[11] Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratoryrodentsdfinal report of the American Institute of Nutrition ad hoc writingcommittee on the reformulation of the AIN-76a rodent diet. J Nutr1993;123:1939–51.

[12] Pang J, Choi Y, Park T. Ilex paraguariensis extract ameliorates obesityinduced by high-fat diet: potential role of AMPK in the visceral adiposetissue. Arch Biochem Biophys 2008;476:178–85.

[13] American Oil Chemists’ Society (AOCS). Preparation of methyl esters offatty acids. Official method Ce 2–66. In: Official methods and recom-mended practices for American Oil Chemists’ Society. 5th ed. Champaign,IL: American Oil Chemists’ Society; 1998.

[14] Donato J, Pedrosa RG, Cruzat VF, Pires IS, Tirapegui J. Effects of leucinesupplementation on the body composition and protein status of ratssubmitted to food restriction. Nutrition 2006;22:520–7.

[15] Rong YQ, Baudry M. Seizure activity results in a rapid induction of nuclearfactor-kappa B in adult but not juvenile rat limbic structures. J Neurochem1996;67:662–8.

[16] Bradford MM. Rapid and sensitive method for quantitation of microgramquantities of protein utilizing principle of protein-dye binding. Anal Bio-chem 1976;72:248–54.

[17] Vinolo MA, Rodrigues HG, Hatanaka E, Sato FT, Sampaio SC, Curi R.Suppressive effect of short-chain fatty acids on production of proin-flammatory mediators by neutrophils. J Nutr Biochem 2011;22:849–55.

[18] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45.

[19] Green LC, Deluzuriaga KR, Wagner DA, Rand W, Istfan N, Young VR. Nitratebiosynthesis in man. Proc Natl Acad Sci U S A 1981;78:7764–8.

[20] Bell RR, Spencer MJ, Sherriff JL. Voluntary exercise and monounsaturatedcanola oil reduce fat gain in mice fed diets high in fat. J Nutr1997;127:2006–10.

[21] Ellis J, Lake A, Hoover-Plow J. Monounsaturated canola oil reduces fatdeposition in growing female rats fed a high or low fat diet. Nutr Res2002;22:609–21.

[22] Bose M, Lambert JD, Ju J, Reuhl KR, Shapses SA, Yang CS. The major greentea polyphenol, (�)-epigallocatechin-3-gallate, inhibits obesity, metabolicsyndrome, and fatty liver disease in high-fat–fed mice. J Nutr2008;138:1677–83.

[23] Safwat GM, Pisan�o S, D’Amore E, Borioni G, Napolitano M, Kamal AA, et al.Induction of non-alcoholic fatty liver disease and insulin resistance byfeeding a high-fat diet in rats: does coenzyme Q monomethyl ether havea modulatory effect? Nutrition 2009;25:1157–68.

[24] Kalupahana NS, Claycombe K, Newman SJ, Stewart T, Siriwardhana N,Matthan N, et al. Eicosapentaenoic acid prevents and reverses insulinresistance in high-fat diet-induced obese mice via modulation of adiposetissue inflammation. J Nutr 2010;140:1915–22.

[25] DeFuria J, Bennett G, Strissel KJ, Perfield JW II, Milbury PE, Greenberg AS,Obin MS. Dietary blueberry attenuates whole-body insulin resistance inhigh fat-fed mice by reducing adipocyte death and its inflammatorysequelae. J Nutr 2009;139:1510–6.

[26] Lumeng CN, DeYoung SM, Bodzin JL, Saltiel AR. Increased inflammatoryproperties of adipose tissue macrophages recruited during diet-inducedobesity. Diabetes 2007;56:16–23.

[27] Wollenberg GK, Deforge LE, Bolgos G, Remick DG. Differential expression oftumor-necrosis-factor and interleukin-6 by peritoneal-macrophages in-vivo and in culture. Am J Pathol 1993;143:1121–30.

[28] Zhou QD, Leeman SE, Amar S. Signaling mechanisms involved in alteredfunction of macrophages from diet-induced obese mice affect immuneresponses. Proc Natl Acad Sci U S A 2009;106:10740–5.

[29] Zhou QD, Leeman SE, Amar S. Signaling mechanisms in the restoration ofimpaired immune function due to diet-induced obesity. Proc Natl Acad SciU S A 2011;108:2867–72.

[30] Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms,molecules and clinical significance. Trends Immunol 2009;30:475–87.

[31] Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM. Changesin gut microbiota control metabolic endotoxemia-induced inflammationin high-fat diet-induced obesity and diabetes in mice. Diabetes 2008;57:1470–81.

[32] Wang Z, Liu D, Wang F, Liu S, Zhao S, Ling EA, et al. Saturated fatty acidsactivate microglia via Toll-like receptor 4/NF-kappaB signalling. Br J Nutr2012;107:229–41.

[33] Cintra DE, Ropelle ER, Moraes JC, Pauli JR, Morari J, de Souza CT, et al.Unsaturated Fatty acids revert diet-induced hypothalamic inflammation inobesity. PLoS One 2012;7:e30571.

[34] Cousin B, Andr�e M, Casteilla L, P�enicaud L. Altered macrophage-like func-tions of preadipocytes in inflammation and genetic obesity. J Cell Physiol2001;186:380–6.

[35] Agarwal S, Busse PJ. Innate and adaptive immunosenescence. Ann AllergyAsthma Immunol 2010;104:183–90.

[36] Narayan KMV, Ali MK, Koplan JP. Global health: global noncommunicablediseasesdwhere worlds meet. N Eng J Med 2010;363:1196–8.