Effects of Ilex paraguariensis (yerba mate) treatment on leptin resistance and inflammatory parameters in obese rats primed by early weaning

Life Sciences 115 (2014) 29–35

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Effects of Ilex paraguariensis (yerba mate) treatment on leptin resistance and inflammatory parameters in obese rats primed by early weaning Natália da Silva Lima, Elaine de Oliveira, Ana Paula Santos da Silva, Lígia de Albuquerque Maia, Egberto Gaspar de Moura, Patricia Cristina Lisboa ⁎ Laboratory of Endocrine Physiology, Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, 20551-030, Brazil

a r t i c l e

i n f o

Article history: Received 26 May 2014 Accepted 8 September 2014 Available online 18 September 2014 Keywords: Ilex paraguariensis Obesity Inflammation Leptin Programming

a b s t r a c t Aims: We evaluated the effects of yerba mate treatment over 30 days on body weight, food intake, hypothalamic leptin action and inflammatory profile in adult rats that were weaned early. Main methods: To induce early weaning, the teats of lactating rats were blocked with a bandage to interrupt milk access for the last 3 days of lactation (EW group). Control offspring had free access to milk throughout lactation. On postnatal day (PN) 150, EW offspring were subdivided into: EW and M groups were treated with water and mate aqueous solution (1 g/kg BW/day, gavage), respectively, for 30 days. Control offspring received water by gavage. On PN180, offspring were killed. Key findings: EW group presented hyperphagia; higher adiposity; higher NPY and TNF-α expression in the ARC nucleus; higher TNF-α and IL-1β levels in the adipose tissue; and lower IL-10 levels in the adipose tissue. These characteristics were normal in M group. As expected, the leptin injection in control offspring caused lower food intake. However, EW group exhibited no change in food intake after the leptin injection, indicating leptin resistance. In contrast, M group had a normal response to the leptin injection. Significance: Thirty days of mate treatment prevented the development of hyperphagia, overweight, visceral obesity and central leptin resistance. This beneficial effect on the satiety of M offspring most likely occurred after the improvement of inflammatory markers in the hypothalamus and adipocytes, which suggests that Ilex paraguariensis plays an important role in the management of obesity by acting on the inflammatory profile. © 2014 Published by Elsevier Inc.

Introduction Nutritional, hormonal or environmental alterations in early life can contribute to the onset of some diseases in adulthood, including obesity, type 2 diabetes, dyslipidemia and cardiovascular diseases. This phenomenon is known as metabolic programming (Barker, 2003; Moura et al., 2008); more recently, it has been termed developmental plasticity (Gluckman and Hanson, 2007). Our group showed that pharmacological and physical early weaning models cause overweight, higher visceral adiposity, hyperphagia, central leptin resistance, hypertriglyceridemia, insulin resistance and other metabolic diseases in offspring at adulthood (Bonomo et al., 2007; Moura et al., 2009; Lima et al., 2011, 2014). An imbalance between energy intake and energy expenditure, such as a hypercaloric diet or sedentary lifestyle, leads to increased body fat storage, resulting in obesity (Riccardi et al., 2004). The regulation of energy balance occurs in specific regions of the central nervous system, including the hypothalamus, which is capable of controlling body ⁎ Corresponding author at: Departamento de Ciências Fisiológicas, 5° andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ, 20551-030, Brazil. Tel.: +55 21 28688334; fax: +55 21 28688029. E-mail address: [email protected] (P.C. Lisboa).

http://dx.doi.org/10.1016/j.lfs.2014.09.003 0024-3205/© 2014 Published by Elsevier Inc.

weight (BW) through the regulation of energy intake and expenditure (Schwartz et al., 2000; Morton et al., 2006). Obesity is associated with a higher proinflammatory status in the hypothalamus with an increase in the IκB kinase-β/nuclear factor-κB (IKKβ/NFκB) pathway (De Souza et al., 2005). Zhang et al. (2008) showed a link between hypothalamic leptin resistance and the IKKβ/NFκB pathway when they observed that this pathway increases the expression suppressor of cytokine signaling 3 (SOCS3), which has an inhibitory action on the signaling leptin pathway, in the hypothalamus. SOCS3 is stimulated by tumor necrosis factor-alpha (TNF-α), an activator of IKKβ/NF-κB. In addition, the signal transducer and activator of transcription 3 (STAT3) of classical leptin signaling, which increases SOCS3 transcription, is controlled by NF-κB. Beverages such as chimarrão (green dried leaves prepared with hot water), tererê (green dried leaves prepared with cold water) and mate tea (roasted leaves prepared with hot water or used to produce soft drinks) that contain yerba mate, a native plant from subtropical regions, are widely consumed in South America (Bastos et al., 2007; Heck and de Mejia, 2007). Studies have shown the potential beneficial effects of Ilex paraguariensis on obesity; these effects are most likely due to the plant's biologic compounds, such as flavonoids (quercetin and rutin), phenolic acids (chlorogenic and caffeic acids), caffeine and saponins. Even when combined with a high-fat diet, Ilex paraguariensis seems to contribute to

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the loss of BW and to improve insulin resistance (Arçari et al., 2009). Recently, we showed that yerba mate treatment in obese rats primed by early weaning was able to normalize hyperphagia, BW, total body fat, and neuropeptide Y (NPY) and SOCS-3 content in the hypothalamus (Lima et al., 2014). Obesity is a chronic and slightly inflammatory state, and this inflammation seems to be directly related to the development of leptin resistance, particularly in the hypothalamus. Arçari et al. (2011) showed that a high-fat diet caused an up-regulation of the TNF-α and interleukin 6 genes in the liver of the obese mouse, and these genes were downregulated via the NF-κB pathway after 8 weeks of yerba mate extract treatment, indicating that yerba mate may have an anti-inflammatory properties. Considering that yerba mate can influence the inflammatory profile but has not been evaluated in the hypothalamus or adipose tissue, we examined whether the leptin resistance observed in adult obese rats that were programmed by early weaning normalized the inflammatory cytokine profile in the plasma, hypothalamus and adipose tissue after 30 days of yerba mate treatment by observing the effects on food intake, fat mass and central leptin action.

contents in the sample were quantified using high-performance liquid chromatography. The analysis of soluble powder yerba mate indicated 41.20 ± 80 mg l− 1 chlorogenic acid, 21 ± 44 ml l−1 caffeine and 8.57 ± 10 mg l−1 theobromine; the concentrations of quercetin and rutin were not determined. On PN 150, four EW offspring from each litter were randomly subdivided into the following two groups: EW + Mate (M, n = 20) — rats received 1 g/kg BW of yerba mate aqueous solution (Lima et al., 2014); and EW + water (EW, n = 20) — rats received pure water. The C offspring (n = 20) were also randomly chosen from each litter and received pure water. The animals received mate tea or water once per day for 30 days by intragastric gavage to guarantee total ingestion. On PN 180, all animals were killed by quick decapitation with no prior anesthesia (because anesthesia affects hormone and lipid metabolism). Blood, brain tissue and retroperitoneal and subcutaneous white adipose tissues (RWAT and SWAT, respectively) were collected. Plasma and tissue samples were frozen at −80 °C until analysis.

Material and methods

A leptin resistance test was performed using a recombinant mouse leptin (PeproTech, Rocky Hill, NJ, USA) dissolved in saline (0.9% wt./ vol.) and injected as a bolus at a dose of 0.5 mg/kg BW intraperitoneally as previously reported (Passos et al., 2004; Bonomo et al., 2007). On PN 180, the rats in the C, EW and M groups were divided into the following groups: leptin (CLEP, EWLEP and MLEP, respectively) or saline (CSAL, EWSAL and MSAL, respectively). We used 10 pups for each group. The animals were placed into individual cages with free access to water and were fasted for 12 h before the test. After injection, each rat was returned individually to its cage with access to 100 g of standard chow and placed in a dark room. Food intake was measured by weighing the food at 1, 3 and 5 h after the leptin or saline injection.

Animals Procedures involving the animals and their care conformed to institutional guidelines (CEUA/017/2009 and CEUA/057/2011) and were in compliance with national and international laws and guidelines for the use of animals in biomedical research. The experiments were performed to minimize the number of rats and their suffering, following the ethical doctrine of the three “R”s — reduction, refinement and replacement. Wistar rats were kept under well-controlled conditions of temperature (25 ± 1 °C) with artificial light–darkness cycles (lights on at 0700 h and lights off at 1900 h). Virgin female rats (3 months old) were caged with male rats at a ratio of 2:1 for 5 days. After mating, each female rat was placed in an individual cage with free access to food and water until delivery. Because pregnant rats produced 10 to 12 pups, we only used dams that produced a litter size of 10 pups to avoid the influence of litter size on the programming effect. At birth, to maximize lactation performance, the litters were adjusted to 6 male pups per dam. Experimental design: programming by early weaning On the 1st postnatal (PN) day, 20 lactating rats were randomly separated into two groups: EW (early weaning, n = 10) — mothers were lightly anesthetized with thiopental (0.06 mg/ml/100 g) and wrapped with a teat bandage (physical barrier) to interrupt lactation for the last 3 days of the period; and C (control, n = 10) — mothers with pups that had standard weaning, i.e., a 21-day lactation period. The EW and C groups had free access to a standard rodent diet, and pups had free access to drinking water. To facilitate food intake by the pups, the food pellets were placed directly inside the cage. After weaning, the EW and control offspring had free access to water and a standard diet. Their BW was recorded until PN 180, and the 24hour food intake was evaluated with animals in individual cages after 12 h of fasting. Oral treatment with the aqueous solution of yerba mate The roasted yerba mate solution was prepared fresh each day by dissolving instant mate tea powder (Leão Jr, Curitiba-PR, Brazil - lot A326/06) in distilled water (330 mg/ml) using a homogenizer. Previously, a sample of this lot was analyzed by our group (Kaezer et al., 2012) and described by Lima et al. (2014). The total phenolic (4.33 ± 0.01 g l−1) content was estimated using the Folin–Ciocalteu method. The chlorogenic acid (610 ± 15 mg l−1), caffeine (508 ± 79 mg l−1), theobromine (99 ± 11 mg l−1), quercetin and rutin (both undetected)

Leptin resistance test — feeding study

Body composition evaluation Total fat mass (g), body mass (g), body fat (%) and trunk fat (%) were measured by dual-energy X-ray absorptiometry (DXA). The rats were anesthetized with an intraperitoneal injection of a 2:1 solution of ketamine hydrochloride (Cetamin®, 50 mg/ml) and xylazine hydrochloride (Xilazin®, 20 mg/ml) at a dose of 0.1 ml/100 g BW and then subjected to DXA (Lukaski et al., 2001) using a Lunar DXA 200368 GE instrument (Lunar, Wisconsin, USA) with specialized software (Encore 2008, Version 12.20 GE Healthcare). The DXA technician was blinded to the experimental protocol. Microdissection of the hypothalamic arcuate (ARC) nucleus The punch technique was used to obtain the ARC nucleus using the bregma as a reference (Palkovits, 1973; Paxinos and Watson, 2007; Helena et al., 2009). Briefly, two subsequent sections of 1000 μM were made: from − 0.6 mm to − 1.6 mm for PVN microdissection (not used) and from −1.6 mm to −2.6 mm for microdissection of the ARC nucleus. The dissection of the ARC nucleus was performed in the second section using a 2-mm ‘square puncher’ needle that was centered on the third ventricle, approximately 1 mm dorsal to the base of the brain (Franco et al., 2012). After removal of the ARC nucleus, the tissue samples were kept at −80 °C until the performance of western blot assays. Western blot analysis To obtain cell extracts, the ARC nucleus was homogenized in 50 μl of ice-cold RIPA buffer pH 7.4 (50 mM TRIS, 150 mM NaCl, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 30 mM sodium pyrophosphatase, 5 mM-EDTA and 1% Triton X-100) with complete/ EDTA-free protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). After homogenization, the ARC was stored at −20 °C. NPY,

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POMC and TNF-α (Sigma Aldrich, St. Louis, MO, USA; Santa Cruz Biotechnology Inc., CA, USA; and AbCam Inc., Cambridge, MA, USA, respectively) contents were analyzed by western blot as described previously (Lima et al., 2014) using actin (EMD Millipore Corporation, Billerica, MA, USA) as the internal control. Protein concentrations were determined using the Bradford method. Immunoassay (ELISA) The levels of TNF-α and IL-10 were determined using enzymelinked immunosorbent assay (ELISA) kits for rat, with intra-assay variations of less than 10% for both assays (USCN Life Science & Technology Co., Ltd., Beijing, China) and with detection limits of 5.4 pg/ml and 2.7 pg/ml, respectively. Real-time PCR The expression levels of the leptin, TNF-α, IL-10, IL-1β, CEBP-β and PPARγ genes were quantified as previously described (Nobre et al., 2011). Total RNA was isolated from RWAT and SWAT using a commercial kit (RNeasy lipid tissue mini kit, Qiagen, Hilden, Germany). For quantitative real-time PCR analysis, reverse transcription was performed on 1 μg of total RNA for all tissues using a Super Script III kit. The products were amplified in an Applied Biosystems 7500 real-time PCR System (Life Technologies Co., Frederick, MD, USA) using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Briefly, after initial denaturation at 50 °C for 2 min and 95 °C for 10 min, the leptin and adiponectin reactions were cycled 40 times using the following parameters: 95 °C for 15 s, 53 °C for 30 s and 70 °C for 45 s. The IL-10, IL-1β, TNF-α, CEBPβ, and PPARγ reactions were cycled 44 times using the following parameters: 95 °C for 15 s, 55 °C for 60 s and 72 °C for 60 s. The product purity was confirmed by agarose gel analysis. The relative mRNA levels (2ΔCt) were determined by comparing the PCR cycle threshold (Ct) between groups, after normalization to the housekeeping gene 36β4. The data were expressed as fold induction over the control group, which was set to 100%. The sequences of the forward and reverse primers used in RT-PCR are described in Table 1. For these measurements, we used five to six offspring per group. Statistical analysis Graph Pad Prism 5 was used for statistical analyses and graphics (Graph Pad Software, Inc., La Jolla, CA, USA). The results are reported as means ± SEM. The experimental data were analyzed by one-way ANOVA followed by Newman–Keuls multiple comparison tests, with the exception of the protein content, which was measured by western Table 1 Sequences of forward and reverse primers used in RT-PCR. Genes

Forward

Leptin 5′-CATCTGCTGGCCTTCTCCAA-3′ Adiponectin 5′-CAAGGGAACTTGTGCAGG-3′ IL-10 5′-GACCCATGAGAGTCTTCACA AC-3′ IL-1 β 5′-GAGTCACAGAAGGAGTGGA-3′ TNF-α CEBPβ PPAR 36β4

Reverse 5′-ATCCAGGCTCTCTGGCTTCTG-3′ 5′-CACCCTTAGGACCAAGAAG-3′ 5′-GATCTTAGCTAACGGGAGCA AC-3′ 5′-ACAGTGAGGAATGTCCACAA AC-3’ 5′-GGTTGTCTTTGAGATCCATGC-3′

5′-TCTCAAAACTCGAGTGACAA GC-3′ 5′-ATGCAATCCGGATCAAACGT-3′ 5′-CCGCAGGAACATCTTTAAGT GA-3′ 5′-ATTCTGGCCCACCAACTT 5′-TGGAAGCCTGATGCTTTATCCC CGG-3′ CA-3′ 5′-TGTTTGACAACGGCAGCA 5′-CCGAGGCAACAGTTGGGTA-3′ TTT-3′

IL-10: interleukin 10; IL-1β: interleukin 1 beta; TNF-α: tumor necrosis factor alpha, CEBPβ: CCAAT/enhancer binding protein beta, PPARγ: peroxisome proliferator-activated receptor gamma, 36β4: internal control.

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blot and analyzed with non-parametric tests (Kruskal–Wallis and Dunn). The significance level was set at p b 0.05. Results As expected, the BW of the early-weaned offspring was significantly higher than that of the controls from PN 150 to PN 180, with an increase in overweight status of approximately 9% (p b 0.05; Fig. 1A). In addition, on PN 180 (Fig. 1B), the EW group showed higher food intake than the controls (+16%, p b 0.05). The M group had lower food intake than the EW group (−10%, p b 0.05). As shown in Table 2, body mass, body fat mass, trunk fat and total fat mass were higher in the EW group (+ 16, + 15%, + 17% and + 26%, p b 0.05, respectively) than in the C group, and the yerba mate treatment prevented these alterations (− 16%, − 22%, − 23% and − 30%, p b 0.05, respectively) in the M group. Regarding the feeding study, the effectiveness of the anorexigenic effect of leptin was observed in the control group, in which leptin treatment (CLEP) significantly suppressed food intake compared with that in the CSAL group (approximately −20%, p b 0.05) for all 3 periods studied (Fig. 1C depicts only the data from 5 h after the injection because the highest observed suppression occurred at that time point). The EWLEP group did not show any alteration in food intake compared with the EWSAL group, thus demonstrating resistance to the anorexigenic action of leptin. However, the MLEP group displayed lower food intake than the MSAL group (− 28%, p b 0.05), suggesting that the leptin action was normalized by the yerba mate treatment. The NPY content in the ARC nucleus was higher in the EW group (2fold increase, p b 0.05; Fig. 2A), and there was no change in the M group. However, the EW group showed unchanged POMC content in the ARC nucleus compared with the C group (Fig. 2B), and the M group showed higher hypothalamic POMC content than the two other groups (+92% vs. C and +64% vs. EW, p b 0.05). Despite the tendency toward an increase in plasma TNF-α levels in the EW group (+ 57%) and a reduction in plasma TNF-α levels in the M group (− 48%), the levels did not differ significantly among the groups (p = 0.2354; Fig. 3A). The EW offspring showed unchanged plasma IL-10 levels compared with the C group, but the M group showed higher plasma IL-10 levels than the other groups (+ 96% vs. the C group and +70% vs. the EW group, p b 0.05; Fig. 3B). In the ARC nucleus (Fig. 4), the EW group showed higher TNF-α content compared with the C group (+75%, p b 0.05), and the 30-day yerba mate treatment was able to normalize the TNF-α content relative to that observed in the EW group (−44%, p b 0.05). As shown in Table 3, the mRNA expression of leptin, CEBP-β and PPARγ showed no change in RWAT among the 3 groups. However, the M offspring presented lower leptin mRNA levels in SWAT (−50% vs. C and −53% vs. EW, p b 0.05). The EW group had higher TNF-α and IL1β levels in RWAT (1.1-fold and 3.7-fold increases vs. C, respectively; p b 0.05) but lower IL-10 levels (−81%, p b 0.0); these changes were reversed by the yerba mate treatment in the M group. Discussion Previously, we showed that early weaning 3 days before the end of the standard lactation period leads to certain endocrine and metabolic changes in adult rats (Lima et al., 2011, 2013). Recently, we also demonstrated that 30 days of treatment with yerba mate extract has a protective effect for some dysfunctions in these animals, including visceral obesity, total body fat mass, hyperphagia, and hypertriglyceridemia (Lima et al., 2014). Our results corroborate the previous findings of Kang et al. (2012) in C57BL/6J mice that were fed a high-fat diet and treated with yerba mate extract at doses of 0.5 g/kg, 1 g/kg BW and 2 g/kg BW for 4 weeks. In addition, we showed that changes in food intake could be associated with changes in the hypothalamic leptin signaling pathway, as yerba mate restored the higher SOCS-3 and NPY

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(A)

(B) 40

Body weight (g)

*

Food intake of 24h (g)

500 #

450

400 200

100

*

#

EW

M

20 10 0

0

Relative food intake 5h after leptin injection (g/body weight)

C

(C)

30

EW

C

M

2.5 2.0 #

*

1.5 1.0 0.5 0.0 C SAL

C LEP

EW SAL EW LEP

M SAL

M LEP

Fig. 1. Body weight (A) and 24 h food intake (B) at PN180 in rats that were normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Five hours relative food intake after leptin or saline injection (C) of adult offspring breastfed for 21 days (CSAL, CLEP), early weaned (EWSAL, EWLEP) or EW treated for 30 days with yerba mate (MSAL, MLEP). Values represent mean ± SEM of 10 rats per group. *p b 0.05 versus C and CSal, respectively; #p b 0.05 versus EW and MSal, respectively.

contents in the total hypothalamus to normal levels during adulthood (Lima et al., 2014). Here, we evaluated the effects of an Ilex paraguariensis aqueous solution (yerba mate) on adiposity; central leptin resistance; NPY, POMC and TNF-α levels in the ARC; plasma cytokine levels; adipose tissue mRNA expression of the adipokines TNF-α, IL-10, IL-1β and leptin; and mRNA expression of CEBPβ and PPARγ, which are transcription factors involved in adipogenesis. All these parameters are directly related to obesity and energy imbalance, and most were altered in the EW group, although these alterations were reversed by the mate treatment. Corroborating our previous findings, the EW group showed higher BW and 24-h food intake; the yerba mate treatment normalized these changes. In part, this normalization in BW can be explained by the observed lower food intake in the EW + M group. However, we cannot exclude the possibility that mate increased the metabolic rate or locomotion. Previous reports (Andrade et al., 2012) do not support this last hypothesis, as treatment with 2 g/kg BW of yerba mate extract in rats and rabbits over 12 weeks did not produce signs of changes in behavior, respiration or locomotion. Here, the evaluation of body composition with DXA analysis was similar to the previously published carcass method (Lima et al., 2014). The EW group showed higher body mass, body fat mass, trunk fat and total fat mass, and the M group showed normalization of these parameters, suggesting that treatment

Table 2 Body composition of offspring at PN180. Groups

Body mass (g) Body fat (%) Trunk fat (%) Total fat mass (g)

C

EW

M

Mean

SEM

Mean

SEM

Mean

SEM

395.8 23.5 22.5 91.0

8.6 0.8 0.8 4.3

461.1* 27.07* 26.3* 114.9*

11.1 1.5 1.6 9.4

386.8# 21.2# 20.2# 79.7#

6.8 0.9 0.9 3.8

Body composition by DXA at PN180 of rats that were normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Values represent mean ± SEM of 10 rats per group. *p b 0.05 versus C; #p b 0.05 versus EW.

with Ilex paraguariensis has a beneficial effect on weight loss and body composition. In fact, other studies showed similar effects of yerba mate treatment on obese mice fed a high-fat diet (Arçari et al., 2009, 2011), and Ilex paraguariensis attenuated the progression of atherosclerosis in rabbits fed a cholesterol-rich diet (Mosimann et al., 2006). In our study, the EW offspring showed no change in leptin mRNA expression in RWAT despite their hyperleptinemia (Lima et al., 2014), which was not prevented by the mate treatment. However, the mate treatment decreased leptin mRNA expression in SWAT, which could explain the intermediary leptin serum levels that were observed in this group, which did not differ from those in the control and EW groups (Lima et al., 2014). The action of yerba mate on leptin serum levels is controversial: some authors showed no effect (Pimentel et al., 2013), others showed a clear decrease in serum leptin (Kang et al., 2012; Pang et al., 2008), and Hussein et al. (2011) showed an increase in serum leptin. These contradictory results may be related to differences in the initial obesity models, species, treatment period and yerba mate concentration. Previously, through western blot analysis, we showed that EW offspring had lower hypothalamic JAK2 and pSTAT3 contents and higher SOCS3 content, which indicate leptin resistance (Lima et al., 2014); mate treatment only normalized the SOCS3 content. To confirm the leptin resistance in the EW group, we tested the anorexigenic effect of leptin on PN 180 by injecting leptin or saline in all animals (divided into 6 randomized groups) and weighing the leftover food at 1, 3 and 5 h following the injection. The CLEP group had lower food intake after the leptin injection than the CSAL group, demonstrating a normal response to the anorexigenic effect of leptin in the C group. However, the EWLEP group showed no difference in food intake compared with the EWSAL group, indicating that these offspring had resistance to the anorexigenic action of leptin. These results corroborate our previous published paper describing adult offspring primed by a pharmacological early weaning model in which prolactin was inhibited by bromocriptine (Bonomo et al., 2007). Furthermore, the MLEP group presented the same profile as the CLEP group (lower food intake than the MSAL group). This finding shows that yerba mate treatment was able to restore leptin action in these animals.

N.S. Lima et al. / Life Sciences 115 (2014) 29–35

(A)

(B)

1.0

*

POMC content in ARC POMC/Actin

NPY content in ARC (NPY/Actin)

33

0.8 0.6 0.4 0.2 0.0

2.0

* #

1.5 1.0 0.5 0.0

C

EW

M

C

EW

M

(C)

Fig. 2. NPY (A) and POMC (B) contents in arcuate nucleus (ARC) and representative blots (C) at PN180 in rats normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Values represent mean ± SEM of 8 rats per group. *p b 0.05 versus C; #p b 0.05 versus EW.

Leptin is able to decrease NPY, which is an orexigenic neurotransmitter that is abundant in the brain (Billington et al., 1994). Previously, we demonstrated that obese adult rats primed by physical early weaning presented higher NPY content in the total hypothalamus (Younes-Rapozo et al., 2012). This increase possibly contributes to hyperphagia, and we showed that yerba mate treatment was able to inhibit the increase in NPY (Lima et al., 2014). In the ARC, the EW group showed high NPY levels, in agreement with previous data, whereas the M group showed no change compared with the EW and control groups, indicating a less marked effect in the ARC than in the total hypothalamus. This difference in NPY expression in the M group (total hypothalamus vs. ARC nucleus) can be explained as follows: the alteration in NPY may be nucleus-specific (such as a higher level of NPY in the paraventricular nucleus). Furthermore, no significant change was observed in the POMC content in the EW group, corroborating the results of Younes-Rapozo et al. (2012). However, treatment with Ilex paraguariensis increased the POMC content in the ARC nucleus; the anorexigenic effect of POMC may explain the normophagia that was observed in the M group. Both NPY and POMC results strongly suggest that yerba mate restored leptin action on the hypothalamic neuropeptides involved in the control of food intake. Obesity is a chronic inflammatory state (Hotamisligil, 2006) characterized by increased levels of inflammatory cytokines (Yudkin et al.,

(B)

40

80

30

60

IL-10 (pg/ml)

TNF-α (pg/ml)

(A)

1999) such as TNF-α. NF-κB signaling activation in the cytosol leads to free NF-κB translocation to the nucleus, resulting in the activation of several genes, including TNF-α and IL-1β (Andreakos et al., 2005). Zhang et al. (2008) showed that central leptin resistance has a link with the inflammatory IKKβ/NFκB pathway because TNF-α, an activator of IKKβ/NF-κB, stimulates the mRNA expression and protein content of SOCS3 in the hypothalamus, which is an inhibitory protein of the leptin pathway (negative feedback of JAK2). Therefore, NF-κB increases SOCS3 expression and activation, and this integration between the inflammatory and leptin pathways can result in the deregulation of food intake control. In the present study, we observed higher TNF-α content in the ARC nucleus, which can contribute to higher SOCS3 levels in the total hypothalamus (Lima et al., 2014) and, consequently, to the central leptin resistance, hyperphagia and obesity that were observed in the EW group. These findings are interesting because serum TNF-α tended only to increase, suggesting that the changes in the hypothalamus and adipose tissue preceded the plasma alterations. In fact, Milanski et al. (2012) showed that hypothalamic inflammation in obese rats led to leptin and insulin resistance. The suppression of TNF-α with a specific antibody was able to restore the hypothalamic sensitivity to leptin and improve insulin signaling in the liver. This result was accompanied by lower liver steatosis and lower hepatic expression of markers of steatosis in these animals (Milanski et al., 2012).

20 10

* #

40 20 0

0 C

EW

M

C

EW

M

Fig. 3. Serum TNF-α (A) and IL-10 (B) at PN180 in rats normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Values represent mean ± SEM of 8 rats per group. *p b 0.05 versus C; #p b 0.05 versus EW.

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TNF-α content in ARC (TNFα/Actin)

(A)

(B) 1.0

*

0.8 0.6

#

0.4 0.2 0.0 C

EW

M

Fig. 4. TNF-α (A) content in arcuate nucleus (ARC) and representative blot (B) at PN180 of rats normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Values represent mean ± SEM of 8 rats per group. *p b 0.05 versus C; #p b 0.05 versus EW.

Arçari et al. (2011) demonstrated that an aqueous extract of Ilex paraguariensis decreased the mRNA expression of TNF-α, IL-6 and inducible nitric oxide synthase (iNOS) in the liver in vivo after 8 weeks of treatment but produced no change in the TNF-α and IL-16 serum levels. Moreover, Gosmann et al. (2012) showed that phenolic compounds from Ilex paraguariensis decreased the mRNA expression of TNF-α in 3T3-L1 culture. Furthermore, Anhê et al. (2012) showed that quercetin (flavonoid present in yerba mate) decreased TNF-α and iNOS mRNA expression in the skeletal muscle of ob/ob mice. In the present study, the yerba mate treatment improved the inflammatory profile, including the restoration of pro-inflammatory TNF-α in the ARC and RWAT, whereas the higher anti-inflammatory IL-10 levels in plasma and RWAT could explain the improvement in leptin action in these animals. In the adipogenesis process, the following three steps are necessary: cell cycle arrest, clonal expansion and the differentiation and activation of C/EBP-α, C/EBP-β and C/EBP-δ (CCAAT/enhancer binding protein alpha, beta and gamma), followed by the activation of PPARγ2 (peroxisome proliferator-activated receptor, gamma 2), all of which have actions on different genes associated with adipogenesis (Mandrup and Lane, 1997; Morrison and Farmer, 2000; Rosen et al., 2000). PPARγ is the most important regulator of adipogenesis (Farmer Stephen, 2006). Barak et al. (1999) showed that PPARγ deficiency inhibits the adipogenesis in mice. Yerba mate has shown anti-adipogenic effects in vivo and in vitro. Gosmann et al. (2012) showed that phenolic compounds of Ilex paraguariensis (such as chlorogenic acid, rutin, metasaponin 4, and fresh saponin) inhibited adipogenesis in 3T3-L1 preadipocytes by decreasing C/EBPα and PPARγ2 expression. In vitro, 50 μg/ml yerba mate and chlorogenic acid as well as 500 μg/ml rutin were able to inhibit C/EBPα and PPARγ2 expression in 3T3-L1 cells (Arçari et al., 2013). A high fat diet causes higher C/EBPα mRNA expression in mice, and treatment with yerba mate for 8 weeks decreased the expression of this transcriptional factor (Arçari et al., 2013). Despite these results,

we found no changes in CEBPβ or PPARγ mRNA expression in the RWAT of the EW group. Again, the relatively short treatment period in our study compared with other studies could explain the different results. The EW animals may have had only hypertrophy but not hyperplasia of fat cells, which could explain the unaltered mRNA levels of the transcriptional factors involved in adipogenesis. Conclusion Obesity is an inflammatory condition, and yerba mate treatment exhibits anti-inflammatory properties. Ilex paraguariensis treatment contributed to decreased TNF-α content in the ARC, which improved the leptin signaling pathway and increased the POMC content in the ARC. In this way, mate improved food intake in EW rats, restoring BW and preventing fat accumulation. Additionally, the peripheral inflammatory profile was improved by mate treatment, as shown by an increase in IL-10, an anti-inflammatory cytokine, and a decrease in proinflammatory cytokines (TNF-α and IL-1β) in adipose tissue. In summary, our data reinforce the beneficial effect of Ilex paraguariensis on body composition, leptin action and the inflammatory profile in obese rats primed by early weaning, highlighting the possible therapeutic use of yerba mate for obesity management. Conflict of interest statement On behalf of all authors, the corresponding author states that there is no conflict of interest.

Acknowledgments All authors are grateful to Miss Monica Moura and Mr. Ulisses Risso Siqueira for technical assistance. Financial support

Table 3 Expression of cytokines and adipogenesis markers in white adipose tissue of offspring at PN180. mRNAs

Leptin/36β4 in RWAT (AU) Leptin/36β4 in SWAT (AU) TNF-α/36β4 in RWAT (AU) IL-1β/36β4 in RWAT (AU) IL-10/36β4 in RWAT (AU) CEBPβ/36β4 in RWAT (AU) PPARγ/36β4 in RWAT (AU)

C

EW

M

Mean

SEM

Mean

SEM

Mean

SEM

1.18 1.00 1.01 1.39 1.46 1.11 1.22

0.4 0.2 0.2 0.6 0.7 0.3 0.5

0.84 1.06 2.10* 6.52* 0.28* 1.80 1.46

0.2 0.1 0.6 2.1 0.2 0.2 1.0

0.69 0.49*# 1.09# 1.32# 3.53# 1.34 0.64

0.1 0.1 0.2 0.5 1.4 0.4 0.5

mRNA expressions in retroperitoneal white adipose tissue (RWAT) and subcutaneous white adipose tissue (SWAT) at PN180 of rats that were normally breastfed for 21 days (C), early weaned (EW) or EW that received yerba mate for 30 days (M). Values represent mean ± SEM of 5 to 6 rats per group. *p b 0.05 versus C; #p b 0.05 versus EW.

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