Research Article Macadamia Oil Supplementation Attenuates Inflammation and Adipocyte Hypertrophy in Obese Mice Edson A. Lima,1 Loreana S. Silveira,2 Laureane N. Masi,3 Amanda R. Crisma,3 Mariana R. Davanso,3 Gabriel I. G. Souza,4 Aline B. Santamarina,5 Renata G. Moreira,6 Amanda Roque Martins,3 Luis Gustavo O. de Sousa,3 Sandro M. Hirabara,7 and Jose C. Rosa Neto1 1 Departamento de Biologia Celular e do Desenvolvimento, Universidade de São Paulo, Avenida Lineu Prestes 1524, Cidade Universitária, 05508-000 São Paulo, SP, Brazil 2 Programa de Pós-Graduação em Ciência da Motricidade, Departamento de Educação Fı́sica, Universidade Estadual Paulista (UNESP), 13506-900 Rio Claro, SP, Brazil 3 Departamento de Fisiologia e Biof́ısica, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-000 São Paulo, SP, Brazil 4Departamento de Ciências Biológicas, Laboratório de Movimento Humano da Universidade São Judas Tadeu, 05503-001 São Paulo, SP, Brazil 5 Departamento de Fisiologia, Disciplina de Fisiologia da Nutrição, Universidade Federal de São Paulo, 04023-901 São Paulo, SP, Brazil 6Departamento de Fisiologia Geral, Instituto de Biociências, Universidade de São Paulo, 05508-090 São Paulo, SP, Brazil 7 Programa de Pós-Graduação em Ciência do Movimento Humano, Instituto de Ciências da Atividade Fı́sica e Esporte, Universidade Cruzeiro do Sul, 01506-000 São Paulo, SP, Brazil Correspondence should be addressed to Edson A. Lima; limaea@hotmail.com Received 25 April 2014; Revised 2 July 2014; Accepted 20 July 2014; Published 22 September 2014 Academic Editor: Fábio Santos Lira Copyright © 2014 Edson A. Lima et al.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Excess of saturated fatty acids in the diet has been associatedwith obesity, leading to systemic disruption of insulin signaling, glucose intolerance, and inflammation. Macadamia oil administration has been shown to improve lipid profile in humans. We evaluated the effect of macadamia oil supplementation on insulin sensitivity, inflammation, lipid profile, and adipocyte size in high-fat diet (HF) induced obesity in mice. C57BL/6 male mice (8 weeks) were divided into four groups: (a) control diet (CD), (b) HF, (c) CD supplemented with macadamia oil by gavage at 2 g/Kg of body weight, three times per week, for 12 weeks (CD + MO), and (d) HF diet supplemented with macadamia oil (HF + MO). CD and HF mice were supplemented with water. HF mice showed hypercholesterolemia and decreased insulin sensitivity as also previously shown. HF induced inflammation in adipose tissue and peritoneal macrophages, as well as adipocyte hypertrophy. Macadamia oil supplementation attenuated hypertrophy of adipocytes and inflammation in the adipose tissue and macrophages. 1. Introduction The role of a diet with a higher content of unsaturated fatty acids, in place or concomitant to a diet with high content of lipids, has been appointed as an effective strategy to control metabolic disorders [1]. Monounsaturated fatty acids (MUFA) rich diet has been reported to decrease plasma total cholesterol and LDL-cholesterol and increase HDL- cholesterol levels [2–5]. Moreover, when saturated fatty acids are replaced by MUFA in the diet of obese women, levels of inflammatory markers decrease, including IL-6 and visfatin in serum [6]. Macadamia nut oil is rich in monounsaturated fatty acids, containing approximately 65% of oleic acid (C18:1) and 18% palmitoleic acid (C16:1) of the total content of fatty Hindawi Publishing Corporation Mediators of Inflammation Volume 2014, Article ID 870634, 9 pages http://dx.doi.org/10.1155/2014/870634 http://dx.doi.org/10.1155/2014/870634 2 Mediators of Inflammation acids [7]. Macadamia oil is the main source of palmitoleic acid in the human diet. Some studies have shown that diet rich in macadamia can improve the lipid profile [2, 8–10], but to date there is no studies on the effect of supplementation of macadamia oil on adipocyte hypertrophy and inflammation. In 2008, Cao and colleagues [11] showed that mice defi- cient in lipid chaperones aP2 and mal1 present increased levels of palmitoleic acid in serum. Elevated levels of cir- culating palmitoleic acid restored sensitivity of insulin in liver and skeletalmuscle, hepatosteatosis, and hyperglycemia, generated by high-fat diet. With this, the authors named this fatty acid as a lipokine, since palmitoleic acid has a hormonal- like effect [11]. The administration of high-fat diet in C57BL6 mice induces metabolic perturbations similar to those observed in humans. In fact, consumption of the high levels of saturated fatty acids is associated with overweight, visceral obesity, inflammation, dyslipidemia, and insulin resistance, in skele- tal muscle, liver, and adipose tissue [12–17]. Saturated FFA promotes inflammation by interaction with toll-like receptor 4 (TLR4), activating NF𝜅B, JNK, and AP-1 pathways [18, 19]. A low grade inflammation is established with increase in plasma levels of IL-6, IL-1𝛽, prostaglandins, TNF-𝛼, and leptin and decrease in the production and secretion of adi- ponectin, IL-10, and IL-4 [20, 21]. The increase in local inflammation is potentiated by the recruitment of macro- phages to adipose tissue and polarization ofM2macrophages (macrophages type 2) toM1macrophages (macrophages type 1) [16, 22, 23]. The aim of our study was to evaluate the effect of mac- adamia oil supplementation, rich in MUFA (palmitoleic and oleic acids), on adipose tissue and peritoneal macrophages inflammation in mice fed a balanced diet or high-fat diet rich in saturated fatty acids. We measured glucose uptake (2-6 deoxyglucose uptake) and mRNA content of proteins (GLUT-4; IRS-1) involved in insulin signaling in soleus muscle.The contents of IL-10, IL-6, TNF-𝛼, and IL-1𝛽 in peri- tonealmacrophages and adipose tissue were also determined. The adipocyte size was also evaluated. 2. Materials and Methods 2.1. Animals. All experiments were performed according to protocols approved by the Animal Care and Use Committee of the Institute of Biomedical Sciences, University of São Paulo. C57BL/6 male mice (8 weeks old) were used in this study. Animals were housed with light-dark cycle of 12-12 h and temperature of 23 ± 2∘C. Animals were divided into four groups: (a) control diet (CD), (b) high-fat diet (HFD), (c) control diet supplemented with macadamia nut oil (Vital Âtman, Uchoa, SP, Brazil) (CD + MO), and (d) high-fat diet supplemented with macadamia oil (HF + MO). Control groupswere run concomitantly.The oil composition is shown in Table 1. During the first 4 weeks preceding the induction of obesity by HFD, all groups were ad libitum fed a control diet (76% carbohydrates, 9% fat, and 15% proteins). Similar protocol has been used in our previous studies [24, 25]. CD + MO and HF + MO were supplemented by oral gavage at 2 g per Kg of body weight, three times per week, during Table 1: Fatty acid composition of macadamia oil. Fatty acid % C12:0 lauric acid 0.09 C14:0 myristic acid 0.82 C16:0 palmitic acid 8.45 C16:1n7 palmitoleic acid 19.11 C17:0 heptadecanoic acid 0.28 C16:2n4 9,12-hexadecadienoic acid 0.02 C16:3n4 6,9,12-hexadecatrienoic acid 0.06 C18:0 stearic acid 3.90 C18:1n9 oleic acid 56.35 C18:1n7 vaccenic acid 3.09 C18:2n6 linoleic acid (LA) 1.35 C18:3n3 linolenic acid (ALA) 0.12 C20:0 arachidic acid 2.79 C20:1n9 gondoic acid 2.18 C20:1n11 gadoleic acid 0.12 C22:0 behenic acid 0.75 C22:1n9 erucic acid 0.22 C22:5n3 eicosapentaenoic acid 0.30 SFA 16.08 MUFA 80.01 PUFA 1.83 PUFA n3 0.42 PUFA n6 1.35 n3/n6 0.31 SFA = saturated fatty acids, sum of C12:0, C14:0, C16:0, C17:0, C18:0, C20:0, and C22:0; MUFA = monounsaturated fatty acids, sum of C16:1, C18:1n7, C18:1n9, C20:1n9, C20:1n11, and C22:1n9; PUFA = polyunsaturated fatty acids, sum of C16:3n4, C18:2n6, C18:3n3, and C22:5n3; PUFA n3 = sum of C18:3n3 and C22:5n3; PUFA n6 = C18:2n6. 12 weeks. This dosage of oil was chosen based on previous studies from our group using different oils with no signs of hepatic toxicity [24]. CD and HF diet received water at the same dose. 2.2. Serum Parameters Analysis. Serum triacylglycerol, total cholesterol, LDL-cholesterol, and HDL-cholesterol were determined by colorimetric assays (Labtest Diagnostics, Lagoa Santa, MG, Brazil). Serum glucose and insulin were measured using LABTEST colorimetric assay and radioim- munoassay (Millipore, Billerica, MA, USA), respectively, as described by Masi et al. (2012) [24]. The HOMA index was determined by calculating fasting serum insulin (𝜇U/mL) × fasting plasma glucose (mmol L−1)/22.5. Leptin and adi- ponectin were measured using the protocol of the manufac- turing R&D system. 2.3. GTT and ITT. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were carried out in all groups after 6 h fasting at the end of the 10th and 11th weeks of treatment, respectively. Themethodologies used for GTT and ITTwere similar to that described by Masi et al. (2012) [24]. 2.4. Insulin Responsiveness in Incubated Soleus Muscle. Ani- mals were euthanized on CO 2 chamber and soleus muscles rapidly and carefully isolated and weighed (8–10mg). This protocol was described in [24, 25]. Mediators of Inflammation 3 Table 2: Primer sequences of the genes studies for real-time PCR. Primer name Forward Reverse RPL-19 5-AGC CTG TGA CTG CCA TTC-3 5-ACC CTT CCT CTT CCC TAT GC-3 GLUT-4 5-CAT TCC CTG GTT CAT TGT GG-3 5-GAA GAC GTA AGG ACC CAT AGC-3 IRS-1 5-CTC AGT CCC AAC CAT AAC CAG-3 5-TCC AAA GGG CAC CGT ATT G-3 CPT-1 5-CCT CCG AAA AGC ACC AAA AC-3 5-GCT CCA GGG TTC AGA AAG TAC-3 PGC1-a 5-CAC CAA ACC CAC AGA AAA CAG-3 5-GGG TCA GAG GAA GAG ATA AAG TTG-3 Perilipin 5 5-CAT GAC TGA GGC TGA GCT AG-3 5-GAG TGT TCA TAG GCG AGA TGG-3 2.5. Haematoxylin and Eosin Staining. Adipose samples were fixed in formalin and paraffin embedded. Sections were prepared (5 𝜇M) using Leica EG1150H Machine. Haema- toxylin and Eosin (H&E) staining was conducted using Leica Autostainer XL and Leica CV5030. Sections were mounted using DPX media (Fisher Scientific, Ireland) and analyzed using Nikon 80i transmission light microscope. 2.6. Extraction of Fatty Acids from Gastrocnemius Muscle and Gas Chromatographic Analysis. Gastrocnemius muscle fragments (100mg) were subjected to lipid extraction. For this, 0.5mL chloroform/methanol (2 : 1; v/v) was added to 100mg of gastrocnemius sample, well-vortexed and incu- bated at room temperature for 5min. Additional volumes of 1.25mL chloroform and 1.25mL deionized H 2 O were then added, and finally, following vigorous homogenization for 3min, samples were centrifuged at 1200 g for 5min, at room temperature to obtain two phases: aqueous phase in the top and organic phase in the bottom containing. The organic phase was collected, dried, and suspended in isopropanol. Triglyceride contentwas then determined in the homogenate. After that, for fatty acid composition determination, gastroc- nemius lipid extracts were dried using atmospheric N 2 for evaporation of the solvent without fatty acid oxidation. The fractions of neutral and polar lipids were separated from these extracts by using a column chromatography. The polar (phospholipids) and neutral (triglycerides) fractions were methylated (for formation of methyl esters), using acetyl chloride and methanol. The methyl esters were analyzed in a gas chromatographer coupled to a flame ionizer detector (FID) (Varian GC 3900). Fatty acid composition was then determined by using standard mixtures of fatty acids with known retention times (Supelco, 37 Components). For the analysis of fatty acids, a programmed chromatog- raphy was used with the characteristics described below. The reading was initiated at 170∘C temperature for 1 minute and then a ramp of 2.5∘C/min was employed to reach a final temperature of 220∘C that was maintained for 5min. The injector and detector were maintained at 250∘C. We used the CP wax 52 CB column, with a 0.25mm thickness, internal diameter of 0.25mm, and 30mm long, with hydrogen as the carrier gas. 2.7. Analysis of Inflammatory Parameters 2.7.1. Adipokines Content Measurements. Mice were eutha- nized onCO 2 chamber and retroperitoneal adipose tissuewas rapidly collected. About 100mg of retroperitoneal adipose tissue was used for the determination of TNF-𝛼, IL-6, and IL-10 content. Adipose tissue was homogenized in RIPA buffer (0.625% Nonidet P-40, 0.625% sodium deoxycholate, 6.25mM sodium phosphate, and 1mM ethylenediaminete- traacetic acid at pH 7.4), containing 10 g/mL of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were centrifuged at 12.000 g for 10min at 4∘C, supernatant was collected, and protein concentration was determined using Bradford assay (Bio-Rad, Hercules, CA, USA). Bovine serum albumin was used as protein standard. Ex Vivo Adipose Tissue Culture. Retroperitoneal adipose tissue explants (about 100mg)were cultured inDMEMsterile medium (Gibco), containing 10% FBS, 2mM glutamine, streptomycin, and penicillin for 24 h, at 37∘C and 5% CO 2 , humidified air environment. Thereafter, medium culture was collected and used for the determination of IL-1𝛽 and IL-10, using ELISA assays (DuoSet kits, R&D System). 2.7.2. Peritoneal Macrophage Isolation and Culture. Cytokine and nitric oxide (NO) production were evaluated in macrophages obtained by washing the peritoneal cavity with 6mL RPMI culture medium (Gibco), containing 10% FBS and 4mM glutamine. Macrophage-rich cultures (more than 90% of the cells were F4/80+) were obtained by incubating peritoneal cells in 24-well polystyrene culture plates for 2 h at 37∘C in a 5%CO 2 , humidified air environment. Nonadherent cells were removed by washing with RPMI. Adherent cells were then incubated with 2.5 𝜇g/mL of LPS (E. coli, serotype 0111:B4, Sigma Chemical Company, USA) for 24 h [26]. Mediumwas collected for determination of IL-6, IL-10, IL-1B and TNF-𝛼 by ELISA and nitrite content by Griess method [27]. 2.8. Quantitative RT-PCR. Total RNA from the gastrocne- mius muscle was extracted with Trizol reagent (Invitrogen Life Technologies, Grand Island, NY, USA), following the method described by Chomczynski and Sacchi [28]. Reverse transcription to cDNA was performed using the high-capac- ity cDNA kit (Applied Biosystems, Foster, CA, USA). Gene expression was evaluated by real-time PCR [29], using Rotor Gene (Qiagen) and SYBR Green (Invitrogen Life Technolo- gies) as fluorescent dye. Primer sequences are shown in Table 2. Quantification of gene expression was carried out using the RPL-19 gene as internal control, as previously described [30]. 4 Mediators of Inflammation Table 3: Effect of high fat diet, with or without supplementation of macadamia oil, on obesity characteristics. CD CD +MO HF HF + MO Initial body weight (g) 24.26 ± 5.02 24.77 ± 3.37 24.2 ± 3.42 24.4 ± 4.41 Final body weight (g) 26.2 ± 1.99 26.82 ± 3.32 34.49 ± 6.96∗# 34.75 ± 4.96∗# Liver weight (g) 1.17 ± 0.14 1.10 ± 0.23 1.33 ± 0.52 1.24 ± 0.26 Mesenteric adipose tissue weight (g) 0.32 ± 0.14 0.28 ± 0.12 0.55 ± 0.28∗# 0.52 ± 0.23∗# Epididymal adipose tissue weight (g) 0.63 ± 0.14 0.73 ± 0.34 1.45 ± 0.60∗# 1.59 ± 0.70∗# Retroperitoneal adipose tissue weight (g) 0.21 ± 0.06 0.24 ± 0.12 0.53 ± 0.22∗# 0.55 ± 0.17∗# Adiposity index (g) 1.16 ± 0.24 1.25 ± 0.53 2.53 ± 1.01∗# 2.65 ± 1.01∗# Brown adipose tissue weight (g) 0.104 ± 0.06 0.116 ± 0.03 0155 ± 0.06 0.128 ± 0.02 Values represent the means ± S.D. of the data obtained from analysis of 15 animals per group. ∗𝑃 < 0.001 versus CD; #𝑃 < 0.001 versus CD + MO. CD HF CT MO (a) CT MO Adipocyte area 0 2000 4000 6000 8000 CD HF ∗ (𝜇 m 2 ) (b) Figure 1: Effect of MO supplementation on adipose tissue histology. (a) Histological sections stained with H&E. (b) Area of adipocytes. CD = group of animals maintained on control diet; HF = group of animals fed high-fat diet; CD + MO = group of animals fed control diet supplemented with macadamia oil; HF + MO = group of animals fed high-fat diet supplemented with macadamia oil. The data are given as the means ± S.D. ∗𝑃 < 0.05 (𝑛 = 6). 2.9. Statistical Analysis. Results are presented as mean ± S.D. All groups were compared by using two-way ANOVA followed by Bonferroni posttest. Significance level was set at 𝑃 < 0.05. 3. Result 3.1. Characterization of the Experimental Model 3.1.1. Body Composition. Mice fed high-fat diet showed increased body weight gain, hypercholesterolemia, and insu- lin resistance. These modifications were similar to those observed in our previous studies [24, 25]. Animals fed the high-fat diet (HF and HF + MO) for eight weeks showed increased (by 2-fold) body weight gain and visceral adiposity index as compared with CD and CD + MO (Table 3). The weights of the liver and the brown adipose tissue depot were not altered with diet or supplementation (Table 3). Although the visceral adiposity index of mice fed high-fat diet (HF and HF + MO) was greater than in animals that received control diet (CD and CD + MO), the HF group had an increase (by 1,62-fold) of adipocytes size compared to the control diet (Figures 1(a) and 1(b)), with statistical difference not evidenced in HF + MO group. No difference was evidenced by diet or supplementation in LDL-c, NEFA, and glycerol (data not shown). Moreover, the basal glycemia and𝐾itt were increased in both groups treated with high-fat diet (data not shown). Homa-IR index was increased in the HF group (by 3-fold) as compared to the other groups including the HF + MO (Figure 2(a)). This result suggests a beneficial effect of macadamia oil supplementation on insulin responsiveness in the HF group. The peripheral insulin resistance was confirmed by glu- cose uptake in incubated soleus muscle (data not shown), as also shown previously [24, 25]. In addition, both groups treatedwith high-fat diet showed decrease inGLUT-4mRNA expression (Figure 2(b)). The PGC-1, IRS-1, CPT-1 and Per- ilipin 5 mRNA expression were not modulated in our treat- ment. The HF group showed an increase in triacylglycerol Mediators of Inflammation 5 100 80 60 40 20 0 CT MO CD HF HOMA-IR ∗ (a) 1.5 1.0 0.5 0.0 CT MO CD HF GLUT-4 expression ∗ G LU T- 4 /R PL -1 9 (A .U .) (b) 2.5 2.0 1.5 1.0 0.5 0.0 CT MO CD HF (m g/ m g tis su e) Total TAG∗∗ (c) Figure 2: Insulin sensitivity and triacylglycerol content in skeletal muscle after 12 weeks. (a) HOMA-IR: homeostatic model assessment of insulin resistance; (b) GLUT-4 gene expression; (c) triacylglycerol content in gastrocnemius muscle. The data are given as the means ± S.D. In all experiments the animals were previously fasted for 6 hours. CD = group of animals maintained on control diet; HF = group of animals fed high-fat diet; CD +MO = group of animals fed control diet supplemented with macadamia oil; HF +MO = group of animals fed high-fat diet supplemented with macadamia oil. A.U. = arbitrary unit. ∗𝑃 < 0.05; ∗∗𝑃 < 0.01 (𝑛 = 6). content in gastrocnemius muscle, but this effect was blunted in HF + MO (Figure 2(c)). The fatty acid composition in neutral or polar lipid fractions remains unchanged regardless of the diet given and MO supplementation (see Table 1 in Supplementary Material available online at http://dx.doi.org/ 10.1155/2014/870634). 3.2. Macadamia Oil Supplementation Attenuates High-Fat Diet Induced Inflammation. The contents of the anti-inflam- matory cytokine IL-10 were increased in the HF + MO group (approximately 4,09-fold) (Figure 3(a)), while IL-1b concentration in the medium of adipose tissue explants was increased in the HF group (Figure 3(b)). When stimulated with LPS, macrophages from all groups showed increased IL-6 production (by 2.97-fold) (Figure 4(d)), whereas IL-10 and NO production were ele- vated in cells from the HF and CT + MO groups (2.39- and 4.08-fold compared to base line, resp.) (Figures 4(a) and 4(e)). No effect of LPS stimulation was observed on TNF-𝛼 production by macrophages from all groups (Figure 4(c)). Moreover, macrophages from the HF group showed an increase (by 2,41-fold) of IL-1𝛽 production compared to unstimulated cells whereas the supplementation with MO abolished this elevation (Figure 4(d)). Similar results were found in NO production. MO attenuated nitrate production by LPS stimulation on macrophages from the HF group (Figure 4(a)). The production of IL-10 was decreased in the CT group compared to CT + MO and HF groups (by 1,88- fold) (Figure 4(e)). TNF-𝛼 and IL-6 production remained unchanged by diet or supplementation (Figures 4(c) and 4(d)). 6 Mediators of Inflammation IL-10 in RPAT CT MO 0 50 100 150 CT HF (p g/ m g pr ot ei n ad ip os e t iss ue ) ∗ ∗ ∗ (a) IL-1𝛽 in adipose tissue medium CT MO 0 100 200 300 400 CD HF (p g/ m g pr ot ei n ad ip os e t iss ue ) ∗ (b) Figure 3: Inflammatory parameters in adipose tissue homogenate and adipose tissue explant incubation medium. IL10 content in adipose tissue homogenate (a) and IL1-𝛽 in the adipose tissue explant incubationmedium, after 24 hoursmeasured by ELISA (b).The animals received water or macadamia oil orally, 2 g/kg b.w., with or without association with a high-fat diet. CD = group of animals maintained on control diet; HF = group of animals fed high-fat diet; CD + MO = group of animals fed control diet supplemented with macadamia oil; HF + MO = group of animals fed high-fat diet supplemented with macadamia oil. The data are given as the means ± S.D. ∗𝑃 < 0.05 (𝑛 = 5-6). No significant difference was found in serum levels of adiponectin after 12 weeks of treatment (data not shown). As expected, leptin concentration was increased (by 5.69-fold) in the groups fed the high-fat diet (data not shown). The sig- nificant difference between the CD + MO and the CD + CT groups suggests that MO can enhance circulant leptin. 4. Discussion We showed herein that twelve weeks of macadamia oil sup- plementation attenuate the increase in inflammation and adipocyte hypertrophy in mice fed a high-fat diet that exhibit signs of the metabolic syndrome. High consumption of fat, sucrose, and industrialized foods in association with sedentary lifestyle is the main con- tributor to obesity and its related comorbidities, including dyslipidemias, insulin resistance, and cardiovascular diseases; evidence has been accumulated that low grade inflammation plays a key role in the obesity induced comorbidities [11, 31– 37]. The increase in adipocytes size was attenuated by macad- amia oil treatment. The increase in adipocyte diameter has been associated with disturbances in cellular homeostasis, such as insulin resistance, inflammation, and hypoxia [38]. The prevalence of large adipocytes increases leptin produc- tion and secretion, as observed in our study [39].The increase in leptin is associated with an elevation in low grade inflam- mation. Leptin is known to stimulate proinflammatory cytok- ines production in lymphocytes [40–42], monocytes [43], and macrophages [44]. Mice fed the HFD for 8 weeks exhibited increased IL-10 content in retroperitoneal adipose tissue. This result may be associated with the increase in peroxisome proliferator activated receptor- (PPAR-) gamma activity. This nuclear receptor increased the number of small adipocytes and raised the IL-10 [45, 46]. The increase of IL-10 content in adipose tissue leads to macrophage polarization (type 2) that is important for remodeling and tissue repair [47, 48]. Moreover, the increase in IL-10 content in adipocytes is associated with increased insulin sensitivity in adipose tissue [49, 50]. IL-1𝛽 strongly induces the inflammatory response in innate immune cells [51], via JNK andNF𝜅Bpathway [52]. IL- 1𝛽 is also a potent inductor of insulin resistance.This cytokine decreased insulin-stimulated glucose uptake via ERK activa- tion [53]. Patients with high level of the circulating IL-1𝛽 are associatedwith greater risk on development of type 2 diabetes [54]. Adipose tissue and peritoneal macrophages are two sources of IL-1𝛽, and macadamia oil supplementation was effective in decreasing the production of this cytokine in both. However, unexpectedly, the CDM showed an increased IL-1𝛽 production after LPS stimulation in peritoneal macrophages. NO production is increased in LPS-stimulated macro- phages beingmore pronounced inmice fed high-fat diet [24]. We demonstrated herein that the same pattern and the sup- plementation with macadamia oil prevented the production of NO by peritoneal macrophages from HF mice. Other bioactive compounds, such as epigallocatechin gallate and resveratrol [55], decrease NO production by macrophage inhibition through of MAP kinase, JNK, and NF𝜅B signaling [56]. Mediators of Inflammation 7 CD HF 0 10 20 30 40 CD + MO HF + MO ∗∗ N O (𝜇 M ) (a) CD + MO HF + MO ∗∗∗ ∗∗∗ ∗∗∗ ∗ CD HF 0 200 400 600 IL 1𝛽 (p g/ m L) (b) CD HF 0 50 100 150 200 TN F- 𝛼 (p g/ m L) CD + MO HF + MO − LPS + LPS (c) CD + MO HF + MO − LPS + LPS CD HF 0 500 1000 1500 2000 2500 IL -6 (p g/ m L) (d) CD HF 0 200 400 600 800 1000 IL -1 0 (p g/ m L) CD + MO HF + MO − LPS + LPS ∗ ∗ (e) Figure 4: Nitric oxide and cytokine production by peritoneal macrophages. Peritoneal macrophages were collected and cultured for 24 h in the absence (white bars) or presence (black bars) of 2.5𝜇g/mL LPS. Nitric oxide (a), IL1-𝛽 (b), TNF-𝛼 (c), IL-6 (d), and IL-10 (e) were measured. CD = control diet; HF = high-fat diet; CD + MO = control diet + macadamia oil; HF + MO = high-fat diet + macadamia oil. The data are given as the means ± S.D. ∗𝑃 < 0.05; ∗∗∗𝑃 < 0.001 (𝑛 = 5-6). 8 Mediators of Inflammation In conclusion, macadamia oil supplementation attenu- ated inflammation and adipocyte hypertrophy in obese mice. Conflict of Interests Theauthors declare that they have no conflict of interests with the presented data. Acknowledgments The authors are grateful to Professor Rui Curi for the revision of the paper and for his constant support and encourage- ment. This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cient́ıfico e Tecnológico (CNPQ). References [1] C. Arapostathi, I. P. Tzanetakou, A. D. Kokkinos et al., “A diet rich in monounsaturated fatty acids improves the lipid profile of mice previously on a diet rich in saturated fatty acids,” Angiology, vol. 62, no. 8, pp. 636–640, 2011. [2] J. Hiraoka-Yamamoto, K. Ikeda, H. Negishi et al., “Serum lipid effects of a monounsaturated (palmitoleic) fatty acid-rich diet based on macadamia nuts in healthy, young japanese women,” Clinical and Experimental Pharmacology and Physiology, vol. 31, supplement 2, pp. S37–S38, 2004. [3] R. P. Mensink, P. L. Zock, A. D. M. Kester, and M. B. Katan, “Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials,” The American Journal of Clinical Nutrition, vol. 77, no. 5, pp. 1146– 1155, 2003. [4] T. A. Nicklas, J. S. Hampl, C. A. Taylor, V. J. Thompson, and W. C. Heird, “Monounsaturated fatty acid intake by children and adults: temporal trends and demographic differences,”Nutrition Reviews, vol. 62, no. 4, pp. 132–141, 2004. [5] L. G. Gillingham, S. Harris-Janz, and P. J. H. Jones, “Dietary monounsaturated fatty acids are protective against metabolic syndrome and cardiovascular disease risk factors,” Lipids, vol. 46, no. 3, pp. 209–228, 2011. [6] F. Haghighatdoost, M. J. Hosseinzadeh-Attar, A. Kabiri, M. Eshraghian, and A. Esmaillzadeh, “Effect of substituting satu- rated with monounsaturated fatty acids on serum visfatin levels and insulin resistance in overweight women: a randomized cross-over clinical trial,” International Journal of Food Sciences and Nutrition, vol. 63, no. 7, pp. 772–781, 2012. [7] L. S. Maguire, S. M. O’Sullivan, K. Galvin, T. P. O’Connor, and N. M. O’Brien, “Fatty acid profile, tocopherol, squalene and phytosterol content ofwalnuts, almonds, peanuts, hazelnuts and the macadamia nut,” International Journal of Food Sciences and Nutrition, vol. 55, no. 3, pp. 171–178, 2004. [8] A. E. Griel, Y. Cao, D. D. Bagshaw, A. M. Cifelli, B. Holub, and P. M. Kris-Etherton, “A Macadamia nut-rich diet reduces total and LDL-cholesterol in mildly hypercholesterolemic men and women,” Journal of Nutrition, vol. 138, no. 4, pp. 761–767, 2008. [9] J. D. Curb, G. Wergowske, J. C. Dobbs, R. D. Abbott, and B. Huang, “Serum lipid effects of a high-monounsaturated fat diet based on macadamia nuts,” Archives of Internal Medicine, vol. 160, no. 8, pp. 1154–1158, 2000. [10] N. R. Matthan, A. Dillard, J. L. Lecker, B. Ip, and A. H. Lichtenstein, “Effects of dietary palmitoleic acid on plasma lipo- protein profile and aortic cholesterol accumulation are similar to those of other unsaturated fatty acids in the f1b golden syrian hamster,” The Journal of Nutrition, vol. 139, no. 2, pp. 215–221, 2009. [11] H. Cao, K. Gerhold, J. R. Mayers, M. M. Wiest, S. M. Watkins, and G. S. Hotamisligil, “Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism,” Cell, vol. 134, no. 6, pp. 933–944, 2008. [12] M. D. Jensen, “Role of body fat distribution and the metabolic complications of obesity,” Journal of Clinical Endocrinology and Metabolism, vol. 93, no. 11, pp. S57–S63, 2008. [13] C. L. Kien, “Dietary interventions for metabolic syndrome: role of modifying dietary fats,” Current Diabetes Reports, vol. 9, no. 1, pp. 43–50, 2009. [14] V. T. Samuel, K. F. Petersen, and G. I. Shulman, “Lipid-induced insulin resistance: unravelling the mechanism,”The Lancet, vol. 375, no. 9733, pp. 2267–2277, 2010. [15] D.-H.Han, P. A.Hansen,H.H.Host, and J. O.Holloszy, “Insulin resistance of muscle glucose transport in rats fed a high-fat diet: a reevaluation,” Diabetes, vol. 46, no. 11, pp. 1761–1767, 1997. [16] M. F. Gregor and G. S. Hotamisligil, “Inflammatory mecha- nisms in obesity,” Annual Review of Immunology, vol. 29, pp. 415–445, 2011. [17] H. H. Lim, S. O. Lee, S. Y. Kim, S. J. Yang, and Y. Lim, “Anti- inflammatory and antiobesity effects of mulberry leaf and fruit extract on high fat diet-induced obesity,” Experimental Biology and Medicine, vol. 238, no. 10, pp. 1160–1169, 2013. [18] S. M. Reyna, S. Ghosh, P. Tantiwong et al., “Elevated toll-like receptor 4 expression and signaling in muscle from insulin- resistant subjects,”Diabetes, vol. 57, no. 10, pp. 2595–2602, 2008. [19] J. Jin, X. Zhang, Z. Lu et al., “Acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations inmacro- phages,” American Journal of Physiology: Endocrinology and Metabolism, vol. 305, no. 7, pp. E853–E867, 2013. [20] H. Waki and P. Tontonoz, “Endocrine functions of adipose tissue,” Annual Review of Pathology, vol. 2, pp. 31–56, 2007. [21] N. Ouchi, J. L. Parker, J. J. Lugus, and K. Walsh, “Adipokines in inflammation andmetabolic disease,”Nature Reviews Immunol- ogy, vol. 11, no. 2, pp. 85–97, 2011. [22] S. P. Weisberg, D. McCann, M. Desai, M. Rosenbaum, R. L. Leibel, andA.W. Ferrante Jr., “Obesity is associatedwithmacro- phage accumulation in adipose tissue,” Journal of Clinical Inves- tigation, vol. 112, no. 12, pp. 1796–1808, 2003. [23] D. Patsouris, P.-P. Li, D. Thapar, J. Chapman, J. M. Olefsky, and J. G. Neels, “Ablation of CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals,” Cell Metabolism, vol. 8, no. 4, pp. 301–309, 2008. [24] L. N. Masi, A. R. Martins, J. C. R. Neto et al., “Sunflower oil supplementation has proinflammatory effects and does not reverse insulin resistance in obesity induced by high-fat diet in C57BL/6 mice,” Journal of Biomedicine and Biotechnology, vol. 2012, Article ID 945131, 9 pages, 2012. [25] M. A. R. Vinolo, H. G. Rodrigues, W. T. Festuccia et al., “Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice,” The American Journal of Physiology—Endocrinology and Metabolism, vol. 303, no. 2, pp. E272–E282, 2012. Mediators of Inflammation 9 [26] J. M. Papadimitriou and I. Van Bruggen, “The effects of mal- nutrition on murine peritoneal macrophages,” Experimental and Molecular Pathology, vol. 49, no. 2, pp. 161–170, 1988. [27] N. P. Sen and B. Donaldson, “Improved colorimetric method for determining nitrate and nitrate in foods,” Journal of the Association of Official Analytical Chemists, vol. 61, no. 6, pp. 1389–1394, 1978. [28] P. Chomczynski and N. Sacchi, “Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction,” Analytical Biochemistry, vol. 162, no. 1, pp. 156–159, 1987. [29] R. Higuchi, G. Dollinger, P. S. Walsh, and R. Griffith, “Simulta- neous amplification and detection of specific DNA sequences,” Bio/Technology, vol. 10, no. 4, pp. 413–417, 1992. [30] W. Liu and D. A. Saint, “Validation of a quantitative method for real time PCR kinetics,” Biochemical and Biophysical Research Communications, vol. 294, no. 2, pp. 347–353, 2002. [31] M. Matsuda and I. Shimomura, “Increased oxidative stress in obesity: Implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and cancer,” Obe- sity Research and Clinical Practice, vol. 7, no. 5, pp. e330–e341, 2013. [32] C. Fernandes-Santos, R. E. Carneiro, L. de SouzaMendonca,M. B. Aguila, andC. A.Mandarim-de-Lacerda, “Pan-PPAR agonist beneficial effects in overweight mice fed a high-fat high-sucrose diet,” Nutrition, vol. 25, no. 7-8, pp. 818–827, 2009. [33] J. C. Fraulob, R. Ogg-Diamantino, C. Fernandes-Santos, M. B. Aguila, and C. A. Mandarim-de-Lacerda, “A mouse model of metabolic syndrome: insulin resistance, fatty liver and Non- Alcoholic Fatty Pancreas Disease (NAFPD) in C57BL/6 mice fed a high fat diet,” Journal of Clinical Biochemistry and Nutri- tion, vol. 46, no. 3, pp. 212–223, 2010. [34] G. D. Pimentel, A. P. S. Dornellas, J. C. Rosa et al., “High-fat diets rich in soy or fish oil distinctly alter hypothalamic insulin signaling in rats,” Journal of Nutritional Biochemistry, vol. 23, no. 7, pp. 822–828, 2012. [35] L. Chen, D. J. Magliano, and P. Z. Zimmet, “The worldwide epidemiology of type 2 diabetes mellitus—present and future perspectives,” Nature Reviews Endocrinology, vol. 8, no. 4, pp. 228–236, 2012. [36] C. J. Rebello, F. L. Greenway, and J. W. Finley, “A review of the nutritional value of legumes and their effects on obesity and its related co-morbidities,” Obesity Reviews, vol. 15, no. 5, pp. 392– 407, 2014. [37] K. E. Wellen and G. S. Hotamisligil, “Inflammation, stress, and diabetes,” Journal of Clinical Investigation, vol. 115, no. 5, pp. 1111– 1119, 2005. [38] S. L. Gray and A. J. Vidal-Puig, “Adipose tissue expandability in the maintenance of metabolic homeostasis,” Nutrition Reviews, vol. 65, supplement 1, pp. S7–S12, 2007. [39] T. S. Higa, A. V. Spinola, M. H. Fonseca-Alaniz, and F. S. Anna Evangelista, “Comparison between cafeteria and high-fat diets in the induction of metabolic dysfunction in mice,” Interna- tional Journal of Physiology, Pathophysiology and Pharmacology, vol. 6, no. 1, pp. 47–54, 2014. [40] H. Kwon and J. E. Pessin, “Adipokines mediate inflammation and insulin resistance,” Frontiers in Endocrinology, vol. 4, article 71, 2013. [41] G. M. Lord, G. Matarese, J. K. Howard, R. J. Baker, S. R. Bloom, and R. I. Lechler, “Leptin modulates the T-cell immune response and reverses starvation- induced immunosuppres- sion,” Nature, vol. 394, no. 6696, pp. 897–901, 1998. [42] P. Marzullo, A. Minocci, P. Giarda et al., “Lymphocytes and immunoglobulin patterns across the threshold of severe obe- sity,” Endocrine, vol. 45, no. 3, pp. 392–400, 2014. [43] J. Santos-Alvarez, R. Goberna, and V. Sánchez-Margalet, “Human leptin stimulates proliferation and activation of human circulating monocytes,” Cellular Immunology, vol. 194, no. 1, pp. 6–11, 1999. [44] S. C. Acedo, S. Gambero, F. G. P. Cunha, I. Lorand-Metze, and A. Gambero, “Participation of leptin in the determination of the macrophage phenotype: An additional role in adipocyte andmacrophage crosstalk,” In Vitro Cellular and Developmental Biology - Animal, vol. 49, no. 6, pp. 473–478, 2013. [45] A. Okuno, H. Tamemoto, K. Tobe et al., “Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats,” Journal of Clinical Investigation, vol. 101, no. 6, pp. 1354–1361, 1998. [46] E. Rigamonti, G. Chinetti-Gbaguidi, and B. Staels, “Regulation of macrophage functions by PPAR- 𝛼, PPAR- 𝛾, and LXRs in mice and men,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 28, no. 6, pp. 1050–1059, 2008. [47] S. Fujisaka, I. Usui, A. Bukhari et al., “Regulatory mechanisms for adipose tissue M1 and M2 macrophages in diet-induced obese mice,” Diabetes, vol. 58, no. 11, pp. 2574–2582, 2009. [48] A.Mantovani, A. Sica, S. Sozzani, P. Allavena, A. Vecchi, andM. Locati, “The chemokine system in diverse forms of macrophage activation and polarization,” Trends in Immunology, vol. 25, no. 12, pp. 677–686, 2004. [49] E.-G. Hong, J. K. Hwi, Y.-R. Cho et al., “Interleukin-10 prevents diet-induced insulin resistance by attenuating macrophage and cytokine response in skeletal muscle,” Diabetes, vol. 58, no. 11, pp. 2525–2535, 2009. [50] C. N. Lumeng, J. L. Bodzin, and A. R. Saltiel, “Obesity induces a phenotypic switch in adipose tissue macrophage polarization,” Journal of Clinical Investigation, vol. 117, no. 1, pp. 175–184, 2007. [51] H. Tilg and A. R. Moschen, “Inflammatory mechanisms in the regulation of insulin resistance,”MolecularMedicine, vol. 14, no. 3-4, pp. 222–231, 2008. [52] M. A. McArdle, O. M. Finucane, R. M. Connaughton, A. M. McMorrow, andH.M. Roche, “Mechanisms of obesity-induced inflammation and insulin resistance: insights into the emerging role of nutritional strategies,” Frontiers in Endocrinology, vol. 4, article 52, Article ID Article 52, 2013. [53] J. Jager, T. Grémeaux, M. Cormont, Y. Le Marchand-Brustel, and J.-F. Tanti, “Interleukin-1𝛽-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression,” Endocrinology, vol. 148, no. 1, pp. 241– 251, 2007. [54] J. Spranger, A. Kroke,M.Möhlig et al., “Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based european prospective investigation into can- cer and nutrition (epic)-potsdam study,”Diabetes, vol. 52, no. 3, pp. 812–817, 2003. [55] Y. Zhong, Y.-S. Chiou, M.-H. Pan, and F. Shahidi, “Anti- inflammatory activity of lipophilic epigallocatechin gallate (EGCG) derivatives in LPS-stimulated murine macrophages,” Food Chemistry, vol. 134, no. 2, pp. 742–748, 2012. [56] S. Han, J. H. Lee, C. Kim et al., “Capillarisin inhibits iNOS, COX-2 expression, and proinflammatory cytokines in LPS- induced RAW 264.7 macrophages via the suppression of ERK, JNK, and NF-𝜅B activation,” Immunopharmacology and Immunotoxicology, vol. 35, no. 1, pp. 34–42, 2013. 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