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Thermogenesis is involved in the body-fat lowering effects of resveratrol in rats
Food Chemistry 141 (2013) 1530–1535
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Thermogenesis is involved in the body-fat lowering effects of resveratrol in rats Goiuri Alberdi, Víctor M. Rodríguez, Jonatan Miranda, M. Teresa Macarulla, Itziar Churruca, María P. Portillo ⇑ Nutrition and Obesity Group, Department of Nutrition and Food Science, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain
a r t i c l e Article history: Received 3 October Received in revised Accepted 11 March Available online 13
i n f o 2012 form 29 January 2013 2013 April 2013
Keywords: Skeletal muscle Brown adipose tissue Mitochondria Thermogenesis Uncoupling proteins
a b s t r a c t The effect of resveratrol on thermogenesis in skeletal muscle and interscapular brown adipose tissue (IBAT) was investigated. Rats were fed an obesogenic diet supplemented with resveratrol (30 mg/kg/ day) or not supplemented for 6 weeks. Resveratrol intake led to increased gene expression of mitochondrial-transcription-factor-A (TFAM), mitochondrial-protein-cytochrome-C-oxidase subunit-2 (COX2), sirtuin-1 (SIRT1), peroxisome-proliferator-activated-receptor-b/d (PPARb/d) and proliferator-activatedreceptor-gamma-coactivator1-a (PGC-1a) in IBAT and increased UCP1protein expression; however, peroxisome-proliferator-activated-receptor-a (PPARa) expression remained unchanged. In gastrocnemius muscle, resveratrol increased the gene expression of TFAM and COX2; however, no changes were observed in levels of SIRT1, PGC-1a and PPARb/d. Acetylated-PGC-1a was decreased in the resveratroltreated group, indicating a higher level of activation, and a significant increase of UCP3 protein expression was observed in this group. The increases in UCP protein expression in two important thermogenic tissues after resveratrol treatment may contribute to increased whole-body energy dissipation, which may help to better understand the body-fat lowering effect of this polyphenol. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, there has been a growing interest in using plant compounds with beneficial biological effects on health as functional ingredients. Among these compounds, polyphenols are being extensively studied. One molecule belonging to this chemical group that has garnered attention is resveratrol. Resveratrol is a phytoalexin that exists as two structural isomers: cis and trans. The trans-isomer (Fig. 1) is the main, and more stable, form found in plants. This form is present in grapes, berries and peanuts, is expressed in response to stress, and used as a defence mechanism against fungal, viral and bacterial infections, and damage from exposure to ultraviolet radiation (Langcake & Pryce, 1976; Signorelli & Ghidoni, 2005). Resveratrol is efficiently absorbed upon oral administration. However, owing to its low water solubility, it must be bound to proteins and/or conjugated to remain at high concentration in serum (Delmas et al., 2011). Most resveratrol undergoes rapid ⇑ Corresponding author. Address: Department of Nutrition and Food Science, Faculty of Pharmacy, Paseo de la Universidad, 7, 01006 Vitoria, Spain. Tel.: +34 945 013067; fax: +34 945 013014. E-mail addresses: [email protected] (G. Alberdi), [email protected] (V.M. Rodríguez), [email protected] (J. Miranda), mariateresa.macarulla@ ehu.es (M.T. Macarulla), [email protected] (I. Churruca), mariapuy.portillo@ ehu.es (M.P. Portillo). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.085
and extensive metabolism in enterocytes before entering into the blood. Furthermore, it undergoes rapid first-pass metabolism in the liver (Walle, Hsieh, DeLegge, Oatis, & Walle, 2004; Wenzel & Somoza, 2005). Consequently, resveratrol bioavailability is very low, and the amounts of the parent compound that reach the plasma and tissues are lower than the levels of glucuronide and sulphate metabolites (Andrés-Lacueva, Urpí-Sardá, Zamora-Ros, & Lamuela-Raventós, 2009; Asensi et al., 2002; Walle et al., 2004). Resveratrol is well known for its beneficial effects on cardiovascular function (Bradamante, Barenghi, & Villa, 2004) and is an interesting molecule in obesity research because, in recent years, it has been shown to have body-fat lowering properties in animals (Ahn, Cho, Kim, Kwon, & Ha, 2008; Baile et al., 2011; Baur et al., 2006; Dal-Pan, Blanc, & Aujard, 2010; Macarulla et al., 2009; Rivera, Morón, Zarzuelo, & Galisteo, 2009; Szkudelska, Nogowski, & Szkudelski, 2009) and humans (Timmers et al., 2011). A combination of various mechanisms may explain this effect on body fat accumulation. It has been reported that resveratrol can inhibit adipogenesis in pre-adipocytes (Bai, Pang, Yang, & Yang, 2008; Chen, Li, Li, Shan, & Zhu, 2011; Rayalam, Yang, Ambati, DellaFera, & Baile, 2008) and increase apoptosis in mature adipocytes (Rayalam et al., 2008). Moreover, this compound reduces lipogenesis (Alberdi et al., 2011; Rivera et al., 2009) and increases lipolysis in isolated adipocytes and white adipose tissue (Alberdi et al., 2011; Lasa et al., 2012; Picard et al., 2004; Szkudelska et al.,
G. Alberdi et al. / Food Chemistry 141 (2013) 1530–1535
2009). Increased fatty acid oxidation has also been described in other organs and tissues, such as liver and skeletal muscle, from animals treated with resveratrol (Ahn et al., 2008; Baur et al., 2006; Gómez-Zorita et al., 2012). However, the influence of resveratrol on adaptive thermogenesis, a metabolic process that contributes to increased energy expenditure, has been scarcely studied to date (Barger et al., 2008; Lagouge et al., 2006). Adaptive thermogenesis, also called facultative thermogenesis, is defined as heat production in response to environmental temperature or diet (Lowell & Spiegelman, 2000) and is mediated by uncoupling proteins (UCPs), which are located in the inner mitochondrial membrane. These proteins act as uncouplers of oxidative phosphorylation by dissipating the proton gradient across the membrane and producing heat, rather than being used to drive the synthesis of ATP (Ricquier, Casteilla, & Bouillaud, 1991). UCPs can, in this manner, dissipate surplus caloric energy and can consequently be important regulators of body weight. It has been demonstrated that thermogenesis can be modified by macronutrient diet composition, diet carbohydrate and fat type (Rodríguez, Portillo, Picó, Macarulla, & Palou, 2002; Samec, Seydoux, & Dulloo, 1999), and by several biomolecules present in food stuffs (Dulloo, 2011; Hursel & Westerterp-Plantenga, 2010; Ribot, Portillo, Picó, Macarulla, & Palou, 2007). In this context, thermogenic ingredients may be considered functional agents that could help to prevent a positive energy balance and obesity. The present study aimed to analyse the effects of resveratrol on thermogenesis in interscapular brown adipose tissue (IBAT) and skeletal muscle, two tissues that greatly contribute to thermogenesis. The involvement of this effect in the body-fat lowering effect of this polyphenol was evaluated. 2. Materials and methods 2.1. Animals, diets and experimental design The experiment was conducted with sixteen male Sprague– Dawley rats that were 6-weeks old, had an average body weight of 180 ± 2 g, and were purchased from Harlan Ibérica (Barcelona, Spain). The experiments were performed with the approval of the Ethical Committee of the University of the Basque Country (document reference CUEID CEBA/30/2010), which follows European regulations (European Convention – Strasburg 1986, Directive 2003/65/EC and Recommendation 2007/526/EC). The rats were individually housed in polycarbonate metabolic cages (Techniplast Gazzada, Guguggiate, Italy) and placed in an air-conditioned room (22 ± 2 °C) with a 12 h light–dark cycle. After a 6-d adaptation period, the rats were randomly divided into two dietary groups of eight animals each, a control group and a resveratrol-treated group, and these were fed a commercial obesogenic diet (Harlan Iberica, TD.06415), which contained 22.5% of fat and 20% of sucrose. Resveratrol was added to the diet, as previously reported (Macarulla et al., 2009), to ensure a dose of 30 mg resveratrol/kg body weight/day. All animals had free access to food and water. Food intake and body weight were measured daily. At the end of the experimental period (6 weeks), the animals were sacrificed under anaesthesia (chloral hydrate) by cardiac exsanguination. Blood was collected, and serum was obtained after centrifugation (1000g for 10 min at 4 °C). Gastrocnemius muscles and interscapular brown adipose tissue (IBAT) were dissected and weighed. All samples were immediately frozen.
the manufacturer’s instructions. RNA samples were then treated with a DNA-free kit (Ambion, Applied Biosystems, Austin, TX, USA) to remove any contamination with genomic DNA. The yield and quality of the RNA were assessed by measuring absorbance at 260, 270, 280 and 310 nm and by electrophoresis on 1.3% agarose gels. From each sample, 1.5 lg total RNA was reverse-transcribed to first-strand complementary DNA (cDNA) with iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Sirtuin-1 (SIRT1), proliferator-activated receptor-gamma coactivator 1-a (PGC-1a), transcription the mitochondrial transcription factor A (TFAM), mitochondrial protein cytochrome C oxidase subunit 2 (COX2), peroxisome proliferator-activated receptor a (PPARa) and peroxisome proliferator-activated receptor (PPARb/ d) mRNA levels were quantified, and mitochondrial ribosomal protein (18S) was used as the reference gene. A 9.5 lL aliquot of each diluted cDNA sample was used for polymerase chain reaction amplification in a 25-lL reaction volume. The cDNA samples were amplified on an iCycler-MyiQ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in the presence of SYBRGreen master mix (Applied Biosystems, Austin, TX, USA) and a 300 nM concentration of each of the sense and antisense primers. The PCR parameters were as follows: initial 2 min at 50 °C, denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 60 °C for 30 s. In the case of PGC-1a and SIRT1 in IBAT, the annealing temperature was 63.5 and 66.4 °C, respectively. Specific primers were synthesised commercially (Integrated DNA Technologies, Leuven Belgium), and the sequences are listed in Table 1. The gene expression analysis was performed by the Comparative threshold cycle (Ct) method. The amplification of the 18S sequence was performed in parallel and was used to normalise the values obtained for the target genes. The results were expressed as fold changes of the threshold cycle (Ct) value relative to the controls by the 2DDCt method (Livak & Schmittgen, 2001).
2.3. Western blot analysis For the UCP Western blot, gastrocnemius muscle and IBAT samples obtained from each rat were homogenised in a PBS buffer with
2.2. Extraction and analysis of RNA and semiquantification by reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from 100 mg IBAT and gastrocnemius muscle with Trizol (Invitrogen, Carlsbad, CA, USA) according to
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Fig. 1. Chemical structure of resveratrol (Pubchem web page).
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Table 1 Primers for PCR amplification of each gene studied. SYBRÒ green RT-PCR. Primers COX2 PPARa PPARb/d PGC-1a SIRT1 TFAM 18S
Sense primer 0
Antisense primer 0
5 -AAC AAT TCT CCC AGC TGT CAT TC-3 50 -GAG AAA GCA AAA CTG AAA GCA GAG A-30 50 -GAG GGG TGC AAG GGC TTC TT-30 50 -CCA AAG CTGA AGC CCT CTT GC-30 50 -GAC GAC GAG GGC GAG GAG-30 50 -CAC GAG CCC TGG AGT ACC C-30 50 -CAT CGA GCA GGT CTG TTC CC-30
50 -AGT CAA AGC ATA GGT CTT CAT AGT C-30 50 -GAA GGG CGG GTT ATT GCT G-30 50 -CAC TTG TTG CGG TTC TTC TTC TG-30 50 -GTT TAG TCT TCC TTT CCT CGT GTC C-30 50 -ACA GGA GGT TGT CTC GGT AGC-30 50 -CCA CAT TCC CCG GAA CAG C-30 50 -TAG ATT GGC TTG ACG GAC TTG G-30
Peroxisome proliferator-activated receptor, PPARa and PPARb/d; peroxisome proliferator-activated receptor gamma coactivator, PGC-1a; sirtuin-1, SIRT1; transcription the mitochondrial transcription factor A, TFAM; mitochondrial protein cytochrome C oxidase subunit 2, COX2.
protease inhibitors. The homogenates were spin-dried at 4000 rpm for 3 min. The pellet was resuspended in 300 lL RIPA buffer and vortexed three times for 15 s each time. The homogenates were centrifuged at 16,000 rpm for 10 min at 4 °C. The protein concentration was measured by the Bradford method (Bradford, 1976). Immunoblot analyses were performed with 20 lg tissue extracts separated by electrophoresis in a 10% SDS–polyacrylamide gel and transferred to PVDF membranes. UCP levels were detected via specific antibodies for UCP1 (1:10,000) and UCP3 (1:4000) (Santa Cruz Biotech, CA, USA). Polyclonal rabbit anti-b-actin antibody (1:5000) (Sigma, St. Louis, MO, USA) was used to normalise the signal obtained for total protein extracts. For the PGC-1a Western blot, immunoprecipitation was performed. A total 500 lg of muscle extracts were diluted with three volumes of PBS (with added protease inhibitors). PGC-1a was immunoprecipitated with 1:20 of H-300 antibody (Santa Cruz Biotech, CA, USA) in constant rotation, at 4 °C, overnight. Afterwards, 20 lL Protein G Agarose (Santa Cruz Biotech, CA, USA) was added to each sample, and they were rotated for 4 h at 4 °C. The immunoprecipitated samples were then washed three times with 500 lL PBS buffer. After the final wash, 100 lL 2 SDS sample buffer was added to the samples, which were then boiled. A total of 30 lg of muscle extracts were separated by electrophoresis in a 7.5% SDS–polyacrylamide gel and then transferred to a PVDF membrane. PGC-1a levels and acetylation levels were detected via specific antibodies for PGC-1a (1:30,000) (Santa Cruz Biotech, CA, USA) and acetylated lysine (1:30,000) (Cell Signaling). 2.4. Serum parameters
and COX2, a mitochondrion-encoded protein, which is embedded in the lipid bilayer of the inner mitochondrial membrane, a critical component of the oxidative phosphorylation pathway (P < 0.001), and in SIRT1 (P < 0.05) and PGC-1a (P < 0.05) in IBAT (Fig. 2). By contrast, PPARa and PPARb/d expression levels remained unchanged (Fig. 2). The protein expression level of UCP1 was also increased in animals treated with resveratrol (P < 0.05) (Fig. 5).
Fig. 2. SIRT1, PGC-1a, TFAM, COX2, PPARb/d and PPARa gene expression in interscapular brown adipose tissue from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
Serum non-esterified fatty acid (NEFA) concentration was determined spectrophotometrically with a commercial kit (Roche, Mannhein, Germany). 2.5. Statistical analysis The results are presented as the means ± standard error of the means. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The data were analysed by Student’s t-test. The statistical significance was set at the P < 0.05 level. 3. Results We previously reported, in this cohort of animals, that rats treated with 30 mg/kg/day resveratrol showed a significant reduction in the size of white adipose tissue deposits with no differences in body weight between the groups (Alberdi et al., 2011). However, no changes were found in IBAT and gastrocnemius muscle weights between the experimental groups (Macarulla et al., 2009). In the present study, resveratrol intake led to increased gene expression of TFAM, a mitochondriogenesis marker (P < 0.001),
Fig. 3. SIRT1, PGC-1a, TFAM, COX2 and PPARb/d gene expression in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄⁄⁄ P < 0.001.
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Fig. 4. Acetylation status of PGC-1a in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05.
In gastrocnemius muscle, resveratrol increased the gene expression level of TFAM (P < 0.001) and COX2 (P < 0.001). In contrast, no changes were observed in the gene expression levels of SIRT1, PGC1a or PPARb/d (Fig. 3). The ratio of acetylated-PGC-1a/total PGC-1a protein was significantly reduced in resveratrol-treated rats (P < 0.05) (Fig. 4), suggesting that resveratrol increased deacetylated-PGC-1a, which is the active form. When the UCP3 protein expression level was analysed, a significant increase was found in rats treated with resveratrol (P < 0.05) (Fig. 5). With regard to serum levels of NEFA, no significant differences were found between the experimental groups (0.42 ± 0.08 mmol/L for the control group and 0.31 ± 0.05 mmol/L for the resveratroltreated group). 4. Discussion In a previous study, we analysed the effects of three doses of resveratrol on body fat accumulation in rats fed an obesogenic diet. We observed that a dose of 6 mg/kg/day did not have an effect, a dose of 30 mg/kg/day was effective in reducing body fat and a dose of 60 mg/kg/day did not increase the effect produced by 30 mg/kg/ day (Macarulla et al., 2009). In light of these results, we decided to evaluate which mechanisms in white adipose tissue could explain the significant reduction in body fat induced by 30 mg/kg/day resveratrol. In that study, we analysed the involvement of de novo lipogenesis, fatty acid uptake from circulating triacylglycerols and lipolysis in the body fat-lowering effect of resveratrol (Alberdi et al., 2011). Apart from changes in white adipose tissue triacylglycerol metabolism, modifications in energy balance could also account for the body-fat lowering effect of resveratrol. As we previously reported, and in accordance with the vast majority of published
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Fig. 5. Protein expression of UCP1 in interscapular brown adipose tissue and UCP3 expression in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05.
studies, resveratrol did not affect food intake (Macarulla et al., 2009). A potential increase in thermogenesis could also be considered but, as explained in the introduction, the analysis of the effects of resveratrol on UCPs is scarce in the current literature (Barger et al., 2008; Lagouge et al., 2006). In this context, the present study aimed to determine the potential contribution of thermogenesis changes induced in IBAT and skeletal muscle to the reduction in body fat induced by resveratrol. For this purpose, by using the previously described cohort of animals treated with 30 mg/kg/day resveratrol (Alberdi et al., 2011), we analysed the effects of this polyphenol on the expression of genes related to thermogenesis. IBAT was chosen for this study because it is the main thermogenic tissue in rodents (Cannon & Nedergaard, 1985). Skeletal muscle is also of particular interest because it constitutes the largest mass in the body and is an important site of thermogenesis, not only in rats (Thurlby & Ellis, 1986) but also in humans (Astrup, Bülow, Christensen, Madsen, & Quaade, 1986; Simonsen, Stallknecht, & Bülow, 1993). Both of these tissues have high levels of mitochondrial activity. In the present study, resveratrol induced a significant increase in SIRT1 expression in IBAT and in PGC-1a, the founding member of a family of transcriptional co-activators. PGC-1a is a potent inducer of mitochondrial biogenesis, an important part of the thermogenic program. It has been reported that PGC-1a leads to an activation of nuclear respiratory factor-1 (NRF-1) and a subsequent increase in the synthesis of TFAM, which in turn results in the increased duplication of mitochondrial DNA (Lowell & Spiegelman, 2000; Scarpulla, 2008). A figure is provided to better explain this process (Fig. 6). In the present study, increased PGC-1a expression was indeed accompanied by a very strong increase in TFAM expression, suggesting that resveratrol, under the present experimental conditions, enhanced
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Fig. 6. Activation of thermogenesis: PGC-1a co-activates transcriptional factors assembled on the UCP1 enhancer, thus increasing UCP1 expression. Adapted from Lowell & Spiegelman, 2000. PGC-1 a: proliferator-activated receptor-gamma coactivator 1-a, NFR-1: nuclear respiratory factor-1, PPAR: peroxisome proliferator-activated receptor RXR: retinoid X receptor, RAR: retinoic acid receptor, TR: thyroid receptor, UCP1: uncoupling protein 1.
mitochondriogenesis. This fact was confirmed by the increase in the expression of COX2, a mitochondrion-encoded protein that is a critical component of the oxidative phosphorylation pathway. These results suggest that the increase in the expression of SIRT1 and PGC-1a was involved in mitochondriogenesis. In addition, we analysed the level of UCP1protein expression, the first uncoupling protein discovered, which is exclusively expressed in BAT (Ricquier et al., 1991). It was observed that resveratrol treatment led to an increased amount of this thermogenin. It has been proposed that PGC-1a activates transcriptional factors, such as peroxisome proliferator-activated receptor c (PPARc), retinoic acid receptor (RAR) and thyroid receptor (TR), is assembled on the UCP1 enhancer, and thus increases UCP1 (Fig. 6) (Lowell & Spiegelman, 2000). Consequently, it can be suggested that the increased amount of UCP1 found in rats treated with resveratrol, and the increased mitochondriogenesis observed, were likely to be related to the increase in PGC-1a. These results are in line with those reported by Lagouge et al. (2006) in mice BAT, but they used a very high dose of resveratrol (400 mg/kg/day) and a longer experimental period than that used in the present study (30 mg/kg/day and 6 weeks). Resveratrol induced a strong increase in TFAM and COX2 expressions in skeletal muscle, as it did in BAT, suggesting that in this tissue, the polyphenol also led to increased mitochondriogenesis, but in this case, SIRT1 and PGC-1a expressions were not modified. Nevertheless, taking into account that the regulation of PGC-1a is not only determined by its expression level but also by a number of post-transcriptional modifications, such as deacetylation (Cantó & Auwerx, 2009), and that SIRT1 is activated by the deacetylation of PGC-1a (Howitz et al., 2003), the potential contribution of SIRT1 activation and in turn, of PGC-1a activation, to the increase of mitochondriogenesis in resveratrol-treated rats cannot
be ruled out. To evaluate this possibility, we analysed the ratio of acetylated-PGC-1a/total PGC-1a protein. We found that the ratio was significantly reduced in resveratrol-treated rats, and consequently, that it was indeed activated by SIRT1. This suggests that PGC-1a was involved in the mitochondriogenesis that occurred in skeletal muscle. The expression of UCP3, a protein abundantly, although not exclusively, expressed in skeletal muscle, was significantly increased by resveratrol treatment. The increase in mitochondria and UCP3 is in accord with results observed by Lagouge et al. (2006) in mouse skeletal muscle. However, while they found increased expression of SIRT1 and PGC-1a in this tissue, in the present study, no changes were observed in these parameters. To understand this difference, it should be noted that important differences exist in terms of experimental design. Lagouge et al. (2006) used a very high dose of resveratrol (400 mg/kg/day) for 15 weeks, and we used a lower dose (30 mg/ kg/day) for a shorter time (6 weeks). Moreover, Chen et al. (2011) also observed an increased expression of PGC-1a associated with TFAM up-regulation in rats treated with a higher dose of resveratrol (100 mg/kg/day). Although this is a controversial issue, several papers in the literature suggest a positive correlation between NEFA plasma concentration and UCP expression in tissues (González-Barroso et al., 1996; Samec, Seydoux, & Dulloo, 1998; Weigle et al., 1998). Taking this into account, we analysed serum NEFA concentrations and observed that they did not change after resveratrol treatment, suggesting that the differences in UCP expression levels observed between the experimental groups were not related to plasma NEFA levels. These results are in agreement with an earlier study from our group in which we analysed the effect of dietary lipid source on UCP expression (Rodríguez, Portillo, Picó, Macarulla, & Palou, 2002). The results obtained in the present study show that resveratrol increases the level of UCP protein expression in two important thermogenic tissues, brown adipose tissue and skeletal muscle. This can contribute to increased whole-body energy dissipation and consequently to increased energy expenditure, thus reducing energetic efficiency. This may help to better understand the body-fat lowering effect of resveratrol, at least in rats. New studies are needed to determine whether these changes also take place in humans. With regard to the relevance of the BAT results in humans, it should be noted that although it has been often believed that BAT is lost postnatally within the first few years of human life (Cannon & Nedergaard, 2004), recent studies have demonstrated that healthy adult humans do possess significant depots of metabolically active BAT (Cypess et al., 2009). Acknowledgements This study was supported by Grants from the Ministerio de Ciencia e Innovación (AGL2008-01005-ALI) and Ministerio de Economía y Competitividad (AGL2011-27406-ALI), Instituto de Salud Carlos III (RETIC PREDIMED and CiberObn), Government of the Basque Country (IT386-10 and IT572-13) and University of the Basque Country (UPV/EHU) (ELDUNANOTEK UFI11/32). References Ahn, J., Cho, I., Kim, S., Kwon, D., & Ha, T. (2008). Dietary resveratrol alters lipid metabolism-related gene expression of mice on an atherogenic diet. Journal of Hepatology, 49(6), 1019–1028. Alberdi, G., Rodríguez, V. M., Miranda, J., Macarulla, M. T., Arias, N., Andrés-Lacueva, C., et al. (2011). Changes in white adipose tissue metabolism induced by resveratrol in rats. Nutrition Metabolism (London), 8(1), 29. Andrés-Lacueva, C., Urpí-Sardá, M., Zamora-Ros, R., & Lamuela-Raventós, R. M. (2009). Plant Phenolics and Human Health: Biochemistry, Nutrition and Pharmacology. Hoboken, NJ: John Wiley & Sons, Inc..
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Thermogenesis is involved in the body-fat lowering effects of resveratrol in rats Goiuri Alberdi, Víctor M. Rodríguez, Jonatan Miranda, M. Teresa Macarulla, Itziar Churruca, María P. Portillo ⇑ Nutrition and Obesity Group, Department of Nutrition and Food Science, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria, Spain CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain
a r t i c l e Article history: Received 3 October Received in revised Accepted 11 March Available online 13
i n f o 2012 form 29 January 2013 2013 April 2013
Keywords: Skeletal muscle Brown adipose tissue Mitochondria Thermogenesis Uncoupling proteins
a b s t r a c t The effect of resveratrol on thermogenesis in skeletal muscle and interscapular brown adipose tissue (IBAT) was investigated. Rats were fed an obesogenic diet supplemented with resveratrol (30 mg/kg/ day) or not supplemented for 6 weeks. Resveratrol intake led to increased gene expression of mitochondrial-transcription-factor-A (TFAM), mitochondrial-protein-cytochrome-C-oxidase subunit-2 (COX2), sirtuin-1 (SIRT1), peroxisome-proliferator-activated-receptor-b/d (PPARb/d) and proliferator-activatedreceptor-gamma-coactivator1-a (PGC-1a) in IBAT and increased UCP1protein expression; however, peroxisome-proliferator-activated-receptor-a (PPARa) expression remained unchanged. In gastrocnemius muscle, resveratrol increased the gene expression of TFAM and COX2; however, no changes were observed in levels of SIRT1, PGC-1a and PPARb/d. Acetylated-PGC-1a was decreased in the resveratroltreated group, indicating a higher level of activation, and a significant increase of UCP3 protein expression was observed in this group. The increases in UCP protein expression in two important thermogenic tissues after resveratrol treatment may contribute to increased whole-body energy dissipation, which may help to better understand the body-fat lowering effect of this polyphenol. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In recent years, there has been a growing interest in using plant compounds with beneficial biological effects on health as functional ingredients. Among these compounds, polyphenols are being extensively studied. One molecule belonging to this chemical group that has garnered attention is resveratrol. Resveratrol is a phytoalexin that exists as two structural isomers: cis and trans. The trans-isomer (Fig. 1) is the main, and more stable, form found in plants. This form is present in grapes, berries and peanuts, is expressed in response to stress, and used as a defence mechanism against fungal, viral and bacterial infections, and damage from exposure to ultraviolet radiation (Langcake & Pryce, 1976; Signorelli & Ghidoni, 2005). Resveratrol is efficiently absorbed upon oral administration. However, owing to its low water solubility, it must be bound to proteins and/or conjugated to remain at high concentration in serum (Delmas et al., 2011). Most resveratrol undergoes rapid ⇑ Corresponding author. Address: Department of Nutrition and Food Science, Faculty of Pharmacy, Paseo de la Universidad, 7, 01006 Vitoria, Spain. Tel.: +34 945 013067; fax: +34 945 013014. E-mail addresses: [email protected] (G. Alberdi), [email protected] (V.M. Rodríguez), [email protected] (J. Miranda), mariateresa.macarulla@ ehu.es (M.T. Macarulla), [email protected] (I. Churruca), mariapuy.portillo@ ehu.es (M.P. Portillo). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.085
and extensive metabolism in enterocytes before entering into the blood. Furthermore, it undergoes rapid first-pass metabolism in the liver (Walle, Hsieh, DeLegge, Oatis, & Walle, 2004; Wenzel & Somoza, 2005). Consequently, resveratrol bioavailability is very low, and the amounts of the parent compound that reach the plasma and tissues are lower than the levels of glucuronide and sulphate metabolites (Andrés-Lacueva, Urpí-Sardá, Zamora-Ros, & Lamuela-Raventós, 2009; Asensi et al., 2002; Walle et al., 2004). Resveratrol is well known for its beneficial effects on cardiovascular function (Bradamante, Barenghi, & Villa, 2004) and is an interesting molecule in obesity research because, in recent years, it has been shown to have body-fat lowering properties in animals (Ahn, Cho, Kim, Kwon, & Ha, 2008; Baile et al., 2011; Baur et al., 2006; Dal-Pan, Blanc, & Aujard, 2010; Macarulla et al., 2009; Rivera, Morón, Zarzuelo, & Galisteo, 2009; Szkudelska, Nogowski, & Szkudelski, 2009) and humans (Timmers et al., 2011). A combination of various mechanisms may explain this effect on body fat accumulation. It has been reported that resveratrol can inhibit adipogenesis in pre-adipocytes (Bai, Pang, Yang, & Yang, 2008; Chen, Li, Li, Shan, & Zhu, 2011; Rayalam, Yang, Ambati, DellaFera, & Baile, 2008) and increase apoptosis in mature adipocytes (Rayalam et al., 2008). Moreover, this compound reduces lipogenesis (Alberdi et al., 2011; Rivera et al., 2009) and increases lipolysis in isolated adipocytes and white adipose tissue (Alberdi et al., 2011; Lasa et al., 2012; Picard et al., 2004; Szkudelska et al.,
G. Alberdi et al. / Food Chemistry 141 (2013) 1530–1535
2009). Increased fatty acid oxidation has also been described in other organs and tissues, such as liver and skeletal muscle, from animals treated with resveratrol (Ahn et al., 2008; Baur et al., 2006; Gómez-Zorita et al., 2012). However, the influence of resveratrol on adaptive thermogenesis, a metabolic process that contributes to increased energy expenditure, has been scarcely studied to date (Barger et al., 2008; Lagouge et al., 2006). Adaptive thermogenesis, also called facultative thermogenesis, is defined as heat production in response to environmental temperature or diet (Lowell & Spiegelman, 2000) and is mediated by uncoupling proteins (UCPs), which are located in the inner mitochondrial membrane. These proteins act as uncouplers of oxidative phosphorylation by dissipating the proton gradient across the membrane and producing heat, rather than being used to drive the synthesis of ATP (Ricquier, Casteilla, & Bouillaud, 1991). UCPs can, in this manner, dissipate surplus caloric energy and can consequently be important regulators of body weight. It has been demonstrated that thermogenesis can be modified by macronutrient diet composition, diet carbohydrate and fat type (Rodríguez, Portillo, Picó, Macarulla, & Palou, 2002; Samec, Seydoux, & Dulloo, 1999), and by several biomolecules present in food stuffs (Dulloo, 2011; Hursel & Westerterp-Plantenga, 2010; Ribot, Portillo, Picó, Macarulla, & Palou, 2007). In this context, thermogenic ingredients may be considered functional agents that could help to prevent a positive energy balance and obesity. The present study aimed to analyse the effects of resveratrol on thermogenesis in interscapular brown adipose tissue (IBAT) and skeletal muscle, two tissues that greatly contribute to thermogenesis. The involvement of this effect in the body-fat lowering effect of this polyphenol was evaluated. 2. Materials and methods 2.1. Animals, diets and experimental design The experiment was conducted with sixteen male Sprague– Dawley rats that were 6-weeks old, had an average body weight of 180 ± 2 g, and were purchased from Harlan Ibérica (Barcelona, Spain). The experiments were performed with the approval of the Ethical Committee of the University of the Basque Country (document reference CUEID CEBA/30/2010), which follows European regulations (European Convention – Strasburg 1986, Directive 2003/65/EC and Recommendation 2007/526/EC). The rats were individually housed in polycarbonate metabolic cages (Techniplast Gazzada, Guguggiate, Italy) and placed in an air-conditioned room (22 ± 2 °C) with a 12 h light–dark cycle. After a 6-d adaptation period, the rats were randomly divided into two dietary groups of eight animals each, a control group and a resveratrol-treated group, and these were fed a commercial obesogenic diet (Harlan Iberica, TD.06415), which contained 22.5% of fat and 20% of sucrose. Resveratrol was added to the diet, as previously reported (Macarulla et al., 2009), to ensure a dose of 30 mg resveratrol/kg body weight/day. All animals had free access to food and water. Food intake and body weight were measured daily. At the end of the experimental period (6 weeks), the animals were sacrificed under anaesthesia (chloral hydrate) by cardiac exsanguination. Blood was collected, and serum was obtained after centrifugation (1000g for 10 min at 4 °C). Gastrocnemius muscles and interscapular brown adipose tissue (IBAT) were dissected and weighed. All samples were immediately frozen.
the manufacturer’s instructions. RNA samples were then treated with a DNA-free kit (Ambion, Applied Biosystems, Austin, TX, USA) to remove any contamination with genomic DNA. The yield and quality of the RNA were assessed by measuring absorbance at 260, 270, 280 and 310 nm and by electrophoresis on 1.3% agarose gels. From each sample, 1.5 lg total RNA was reverse-transcribed to first-strand complementary DNA (cDNA) with iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Sirtuin-1 (SIRT1), proliferator-activated receptor-gamma coactivator 1-a (PGC-1a), transcription the mitochondrial transcription factor A (TFAM), mitochondrial protein cytochrome C oxidase subunit 2 (COX2), peroxisome proliferator-activated receptor a (PPARa) and peroxisome proliferator-activated receptor (PPARb/ d) mRNA levels were quantified, and mitochondrial ribosomal protein (18S) was used as the reference gene. A 9.5 lL aliquot of each diluted cDNA sample was used for polymerase chain reaction amplification in a 25-lL reaction volume. The cDNA samples were amplified on an iCycler-MyiQ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in the presence of SYBRGreen master mix (Applied Biosystems, Austin, TX, USA) and a 300 nM concentration of each of the sense and antisense primers. The PCR parameters were as follows: initial 2 min at 50 °C, denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 60 °C for 30 s. In the case of PGC-1a and SIRT1 in IBAT, the annealing temperature was 63.5 and 66.4 °C, respectively. Specific primers were synthesised commercially (Integrated DNA Technologies, Leuven Belgium), and the sequences are listed in Table 1. The gene expression analysis was performed by the Comparative threshold cycle (Ct) method. The amplification of the 18S sequence was performed in parallel and was used to normalise the values obtained for the target genes. The results were expressed as fold changes of the threshold cycle (Ct) value relative to the controls by the 2DDCt method (Livak & Schmittgen, 2001).
2.3. Western blot analysis For the UCP Western blot, gastrocnemius muscle and IBAT samples obtained from each rat were homogenised in a PBS buffer with
2.2. Extraction and analysis of RNA and semiquantification by reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from 100 mg IBAT and gastrocnemius muscle with Trizol (Invitrogen, Carlsbad, CA, USA) according to
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Fig. 1. Chemical structure of resveratrol (Pubchem web page).
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Table 1 Primers for PCR amplification of each gene studied. SYBRÒ green RT-PCR. Primers COX2 PPARa PPARb/d PGC-1a SIRT1 TFAM 18S
Sense primer 0
Antisense primer 0
5 -AAC AAT TCT CCC AGC TGT CAT TC-3 50 -GAG AAA GCA AAA CTG AAA GCA GAG A-30 50 -GAG GGG TGC AAG GGC TTC TT-30 50 -CCA AAG CTGA AGC CCT CTT GC-30 50 -GAC GAC GAG GGC GAG GAG-30 50 -CAC GAG CCC TGG AGT ACC C-30 50 -CAT CGA GCA GGT CTG TTC CC-30
50 -AGT CAA AGC ATA GGT CTT CAT AGT C-30 50 -GAA GGG CGG GTT ATT GCT G-30 50 -CAC TTG TTG CGG TTC TTC TTC TG-30 50 -GTT TAG TCT TCC TTT CCT CGT GTC C-30 50 -ACA GGA GGT TGT CTC GGT AGC-30 50 -CCA CAT TCC CCG GAA CAG C-30 50 -TAG ATT GGC TTG ACG GAC TTG G-30
Peroxisome proliferator-activated receptor, PPARa and PPARb/d; peroxisome proliferator-activated receptor gamma coactivator, PGC-1a; sirtuin-1, SIRT1; transcription the mitochondrial transcription factor A, TFAM; mitochondrial protein cytochrome C oxidase subunit 2, COX2.
protease inhibitors. The homogenates were spin-dried at 4000 rpm for 3 min. The pellet was resuspended in 300 lL RIPA buffer and vortexed three times for 15 s each time. The homogenates were centrifuged at 16,000 rpm for 10 min at 4 °C. The protein concentration was measured by the Bradford method (Bradford, 1976). Immunoblot analyses were performed with 20 lg tissue extracts separated by electrophoresis in a 10% SDS–polyacrylamide gel and transferred to PVDF membranes. UCP levels were detected via specific antibodies for UCP1 (1:10,000) and UCP3 (1:4000) (Santa Cruz Biotech, CA, USA). Polyclonal rabbit anti-b-actin antibody (1:5000) (Sigma, St. Louis, MO, USA) was used to normalise the signal obtained for total protein extracts. For the PGC-1a Western blot, immunoprecipitation was performed. A total 500 lg of muscle extracts were diluted with three volumes of PBS (with added protease inhibitors). PGC-1a was immunoprecipitated with 1:20 of H-300 antibody (Santa Cruz Biotech, CA, USA) in constant rotation, at 4 °C, overnight. Afterwards, 20 lL Protein G Agarose (Santa Cruz Biotech, CA, USA) was added to each sample, and they were rotated for 4 h at 4 °C. The immunoprecipitated samples were then washed three times with 500 lL PBS buffer. After the final wash, 100 lL 2 SDS sample buffer was added to the samples, which were then boiled. A total of 30 lg of muscle extracts were separated by electrophoresis in a 7.5% SDS–polyacrylamide gel and then transferred to a PVDF membrane. PGC-1a levels and acetylation levels were detected via specific antibodies for PGC-1a (1:30,000) (Santa Cruz Biotech, CA, USA) and acetylated lysine (1:30,000) (Cell Signaling). 2.4. Serum parameters
and COX2, a mitochondrion-encoded protein, which is embedded in the lipid bilayer of the inner mitochondrial membrane, a critical component of the oxidative phosphorylation pathway (P < 0.001), and in SIRT1 (P < 0.05) and PGC-1a (P < 0.05) in IBAT (Fig. 2). By contrast, PPARa and PPARb/d expression levels remained unchanged (Fig. 2). The protein expression level of UCP1 was also increased in animals treated with resveratrol (P < 0.05) (Fig. 5).
Fig. 2. SIRT1, PGC-1a, TFAM, COX2, PPARb/d and PPARa gene expression in interscapular brown adipose tissue from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
Serum non-esterified fatty acid (NEFA) concentration was determined spectrophotometrically with a commercial kit (Roche, Mannhein, Germany). 2.5. Statistical analysis The results are presented as the means ± standard error of the means. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The data were analysed by Student’s t-test. The statistical significance was set at the P < 0.05 level. 3. Results We previously reported, in this cohort of animals, that rats treated with 30 mg/kg/day resveratrol showed a significant reduction in the size of white adipose tissue deposits with no differences in body weight between the groups (Alberdi et al., 2011). However, no changes were found in IBAT and gastrocnemius muscle weights between the experimental groups (Macarulla et al., 2009). In the present study, resveratrol intake led to increased gene expression of TFAM, a mitochondriogenesis marker (P < 0.001),
Fig. 3. SIRT1, PGC-1a, TFAM, COX2 and PPARb/d gene expression in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄⁄⁄ P < 0.001.
G. Alberdi et al. / Food Chemistry 141 (2013) 1530–1535
Fig. 4. Acetylation status of PGC-1a in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05.
In gastrocnemius muscle, resveratrol increased the gene expression level of TFAM (P < 0.001) and COX2 (P < 0.001). In contrast, no changes were observed in the gene expression levels of SIRT1, PGC1a or PPARb/d (Fig. 3). The ratio of acetylated-PGC-1a/total PGC-1a protein was significantly reduced in resveratrol-treated rats (P < 0.05) (Fig. 4), suggesting that resveratrol increased deacetylated-PGC-1a, which is the active form. When the UCP3 protein expression level was analysed, a significant increase was found in rats treated with resveratrol (P < 0.05) (Fig. 5). With regard to serum levels of NEFA, no significant differences were found between the experimental groups (0.42 ± 0.08 mmol/L for the control group and 0.31 ± 0.05 mmol/L for the resveratroltreated group). 4. Discussion In a previous study, we analysed the effects of three doses of resveratrol on body fat accumulation in rats fed an obesogenic diet. We observed that a dose of 6 mg/kg/day did not have an effect, a dose of 30 mg/kg/day was effective in reducing body fat and a dose of 60 mg/kg/day did not increase the effect produced by 30 mg/kg/ day (Macarulla et al., 2009). In light of these results, we decided to evaluate which mechanisms in white adipose tissue could explain the significant reduction in body fat induced by 30 mg/kg/day resveratrol. In that study, we analysed the involvement of de novo lipogenesis, fatty acid uptake from circulating triacylglycerols and lipolysis in the body fat-lowering effect of resveratrol (Alberdi et al., 2011). Apart from changes in white adipose tissue triacylglycerol metabolism, modifications in energy balance could also account for the body-fat lowering effect of resveratrol. As we previously reported, and in accordance with the vast majority of published
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Fig. 5. Protein expression of UCP1 in interscapular brown adipose tissue and UCP3 expression in gastrocnemius muscle from control and resveratrol-treated rats. The values are the means for eight animals per group with the standard errors of the means, shown by vertical bars. ⁄P < 0.05.
studies, resveratrol did not affect food intake (Macarulla et al., 2009). A potential increase in thermogenesis could also be considered but, as explained in the introduction, the analysis of the effects of resveratrol on UCPs is scarce in the current literature (Barger et al., 2008; Lagouge et al., 2006). In this context, the present study aimed to determine the potential contribution of thermogenesis changes induced in IBAT and skeletal muscle to the reduction in body fat induced by resveratrol. For this purpose, by using the previously described cohort of animals treated with 30 mg/kg/day resveratrol (Alberdi et al., 2011), we analysed the effects of this polyphenol on the expression of genes related to thermogenesis. IBAT was chosen for this study because it is the main thermogenic tissue in rodents (Cannon & Nedergaard, 1985). Skeletal muscle is also of particular interest because it constitutes the largest mass in the body and is an important site of thermogenesis, not only in rats (Thurlby & Ellis, 1986) but also in humans (Astrup, Bülow, Christensen, Madsen, & Quaade, 1986; Simonsen, Stallknecht, & Bülow, 1993). Both of these tissues have high levels of mitochondrial activity. In the present study, resveratrol induced a significant increase in SIRT1 expression in IBAT and in PGC-1a, the founding member of a family of transcriptional co-activators. PGC-1a is a potent inducer of mitochondrial biogenesis, an important part of the thermogenic program. It has been reported that PGC-1a leads to an activation of nuclear respiratory factor-1 (NRF-1) and a subsequent increase in the synthesis of TFAM, which in turn results in the increased duplication of mitochondrial DNA (Lowell & Spiegelman, 2000; Scarpulla, 2008). A figure is provided to better explain this process (Fig. 6). In the present study, increased PGC-1a expression was indeed accompanied by a very strong increase in TFAM expression, suggesting that resveratrol, under the present experimental conditions, enhanced
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Fig. 6. Activation of thermogenesis: PGC-1a co-activates transcriptional factors assembled on the UCP1 enhancer, thus increasing UCP1 expression. Adapted from Lowell & Spiegelman, 2000. PGC-1 a: proliferator-activated receptor-gamma coactivator 1-a, NFR-1: nuclear respiratory factor-1, PPAR: peroxisome proliferator-activated receptor RXR: retinoid X receptor, RAR: retinoic acid receptor, TR: thyroid receptor, UCP1: uncoupling protein 1.
mitochondriogenesis. This fact was confirmed by the increase in the expression of COX2, a mitochondrion-encoded protein that is a critical component of the oxidative phosphorylation pathway. These results suggest that the increase in the expression of SIRT1 and PGC-1a was involved in mitochondriogenesis. In addition, we analysed the level of UCP1protein expression, the first uncoupling protein discovered, which is exclusively expressed in BAT (Ricquier et al., 1991). It was observed that resveratrol treatment led to an increased amount of this thermogenin. It has been proposed that PGC-1a activates transcriptional factors, such as peroxisome proliferator-activated receptor c (PPARc), retinoic acid receptor (RAR) and thyroid receptor (TR), is assembled on the UCP1 enhancer, and thus increases UCP1 (Fig. 6) (Lowell & Spiegelman, 2000). Consequently, it can be suggested that the increased amount of UCP1 found in rats treated with resveratrol, and the increased mitochondriogenesis observed, were likely to be related to the increase in PGC-1a. These results are in line with those reported by Lagouge et al. (2006) in mice BAT, but they used a very high dose of resveratrol (400 mg/kg/day) and a longer experimental period than that used in the present study (30 mg/kg/day and 6 weeks). Resveratrol induced a strong increase in TFAM and COX2 expressions in skeletal muscle, as it did in BAT, suggesting that in this tissue, the polyphenol also led to increased mitochondriogenesis, but in this case, SIRT1 and PGC-1a expressions were not modified. Nevertheless, taking into account that the regulation of PGC-1a is not only determined by its expression level but also by a number of post-transcriptional modifications, such as deacetylation (Cantó & Auwerx, 2009), and that SIRT1 is activated by the deacetylation of PGC-1a (Howitz et al., 2003), the potential contribution of SIRT1 activation and in turn, of PGC-1a activation, to the increase of mitochondriogenesis in resveratrol-treated rats cannot
be ruled out. To evaluate this possibility, we analysed the ratio of acetylated-PGC-1a/total PGC-1a protein. We found that the ratio was significantly reduced in resveratrol-treated rats, and consequently, that it was indeed activated by SIRT1. This suggests that PGC-1a was involved in the mitochondriogenesis that occurred in skeletal muscle. The expression of UCP3, a protein abundantly, although not exclusively, expressed in skeletal muscle, was significantly increased by resveratrol treatment. The increase in mitochondria and UCP3 is in accord with results observed by Lagouge et al. (2006) in mouse skeletal muscle. However, while they found increased expression of SIRT1 and PGC-1a in this tissue, in the present study, no changes were observed in these parameters. To understand this difference, it should be noted that important differences exist in terms of experimental design. Lagouge et al. (2006) used a very high dose of resveratrol (400 mg/kg/day) for 15 weeks, and we used a lower dose (30 mg/ kg/day) for a shorter time (6 weeks). Moreover, Chen et al. (2011) also observed an increased expression of PGC-1a associated with TFAM up-regulation in rats treated with a higher dose of resveratrol (100 mg/kg/day). Although this is a controversial issue, several papers in the literature suggest a positive correlation between NEFA plasma concentration and UCP expression in tissues (González-Barroso et al., 1996; Samec, Seydoux, & Dulloo, 1998; Weigle et al., 1998). Taking this into account, we analysed serum NEFA concentrations and observed that they did not change after resveratrol treatment, suggesting that the differences in UCP expression levels observed between the experimental groups were not related to plasma NEFA levels. These results are in agreement with an earlier study from our group in which we analysed the effect of dietary lipid source on UCP expression (Rodríguez, Portillo, Picó, Macarulla, & Palou, 2002). The results obtained in the present study show that resveratrol increases the level of UCP protein expression in two important thermogenic tissues, brown adipose tissue and skeletal muscle. This can contribute to increased whole-body energy dissipation and consequently to increased energy expenditure, thus reducing energetic efficiency. This may help to better understand the body-fat lowering effect of resveratrol, at least in rats. New studies are needed to determine whether these changes also take place in humans. With regard to the relevance of the BAT results in humans, it should be noted that although it has been often believed that BAT is lost postnatally within the first few years of human life (Cannon & Nedergaard, 2004), recent studies have demonstrated that healthy adult humans do possess significant depots of metabolically active BAT (Cypess et al., 2009). Acknowledgements This study was supported by Grants from the Ministerio de Ciencia e Innovación (AGL2008-01005-ALI) and Ministerio de Economía y Competitividad (AGL2011-27406-ALI), Instituto de Salud Carlos III (RETIC PREDIMED and CiberObn), Government of the Basque Country (IT386-10 and IT572-13) and University of the Basque Country (UPV/EHU) (ELDUNANOTEK UFI11/32). References Ahn, J., Cho, I., Kim, S., Kwon, D., & Ha, T. (2008). Dietary resveratrol alters lipid metabolism-related gene expression of mice on an atherogenic diet. Journal of Hepatology, 49(6), 1019–1028. Alberdi, G., Rodríguez, V. M., Miranda, J., Macarulla, M. T., Arias, N., Andrés-Lacueva, C., et al. (2011). Changes in white adipose tissue metabolism induced by resveratrol in rats. Nutrition Metabolism (London), 8(1), 29. Andrés-Lacueva, C., Urpí-Sardá, M., Zamora-Ros, R., & Lamuela-Raventós, R. M. (2009). Plant Phenolics and Human Health: Biochemistry, Nutrition and Pharmacology. Hoboken, NJ: John Wiley & Sons, Inc..
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