Effects of ovariectomy and 17-β estradiol replacement on rat brown adipose tissue mitochondrial function

Steroids 76 (2011) 1051–1056

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

Effects of ovariectomy and 17-␤ estradiol replacement on rat brown adipose tissue mitochondrial function Antònia Nadal-Casellas a,b , Ana M. Proenza a,b , Isabel Lladó a,b , Magdalena Gianotti a,b,∗ a Grup de Metabolisme Energètic i Nutrició, Departament de Biologia Fonamental i Ciències de la Salut, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Cra. Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain b Ciber Fisiopatología Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, Spain

a r t i c l e

i n f o

Article history: Received 16 December 2010 Received in revised form 9 April 2011 Accepted 12 April 2011 Available online 22 April 2011 Keywords: Brown adipose tissue 17-␤ estradiol replacement Mitochondria Ovariectomy Rat Sex

a b s t r a c t Taking into account the sexual dimorphism previously reported regarding mitochondrial function and biogenesis in brown adipose tissue, the aim of the present study was to go further into these differences by investigating the effect of ovariectomy and 17-␤ estradiol (E2) replacement on brown adipose tissue mitochondrial function. In this study, fourteen-week-old control female and ovariectomized female Wistar rats were used. Rats were ovariectomized at 5 weeks of age and were treated every 2 days with placebo (OVX group) or E2 (10 ␮g/kg) (OVX + E2 group) for 4 weeks before sacrifice. We studied the levels of oxidative capacity, antioxidant defence and oxidative damage markers in brown adipose tissue. Moreover, the levels of key elements of mitochondrial biogenesis as well as UCP1 protein levels, as an index of mitochondrial thermogenic capacity, were also determined. In response to ovariectomy, mitochondrial proliferation increased, resulting in less functional mitochondria, since oxidative capacity and antioxidant defences decreased. Although E2 supplementation was able to restore the serum levels of E2 shown by control rats, the treatment reverted the effects of the ovariectomy only in part, and oxidative and antioxidant capacities in OVX + E2 rats did not reach the levels shown by control females. Taking these results into account, we suggest that ovarian hormones are responsible, at least in part, for the sexual dimorphism in BAT mitochondrial function. However, other signals produced by ovary, rather than E2, would play an important role in the control of mitochondrial function in BAT. © 2011 Elsevier Inc. All rights reserved.

1. Introduction At the end of women’s reproductive life, the cessation of ovarian function favours increased body weight, which is associated to adverse metabolic consequences, such as an increase in proinflammatory cytokines, a loss of insulin sensitivity or development of cardiovascular disease, among others [1,2]. However, the mechanism through which menopause per se contributes to weight gain, as well as to the redistribution of fat stores, remains to be fully clarified. Brown adipose tissue (BAT) plays a central role in the control of energy balance in small mammals due to its thermogenic capacity that relies on the presence of the uncoupling protein 1

∗ Corresponding author at: Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Cra. Valldemossa km 7.5, E-07122 Palma de Mallorca, Spain. Tel.: +34 971 173173; fax: +34 971 173184. E-mail addresses: [email protected] (A. Nadal-Casellas), [email protected] (A.M. Proenza), [email protected] (I. Lladó), [email protected] (M. Gianotti). 0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2011.04.009

(UCP1) [3]. BAT thermogenic potential is activated in response to several physiological stimuli, such as cold exposure or an excess of caloric intake [4]. In fact, altered BAT thermogenic activity has been related to the development of obesity in rodents [5]. Recent studies describe the existence of active BAT in adult humans [6–8] and, although it has been suggested that the absence of BAT correlates with obesity also in humans [9], the physiological role of this tissue in human energy homeostasis remains to be elucidated. BAT shows a marked sexual dimorphism in mitochondrial morphology and functionality. BAT of female rats exhibits more differentiated mitochondria than those of males, as their greater size and cristae density, their higher mitochondrial protein, as well as their greater UCP1 levels indicate [10–13]. As a consequence, BAT oxidative and thermogenic capacity in female rats are enhanced compared with males, which could be attributed to a differential regulation of BAT mitochondrial growth cycle between sexes [11]. Recent studies performed in vitro and in vivo point to the mitochondria as a novel important target for the actions of estrogens at several levels [14]. Estrogens regulate the redox status by enhancing antioxidant defences in rodent tissues such as liver or kidney

1052

A. Nadal-Casellas et al. / Steroids 76 (2011) 1051–1056

[15,16] and have been related to a greater mitochondrial oxidative capacity, since they activate the expression of specific proteins of the mitochondrial machinery [17]. In contrast, the effects of steroids on rat BAT function are still controversial. Whereas some studies have indicated that estrogens enhance BAT functionality, as a compensatory mechanism to prevent body weight gain [18,19], others suggest that changes in body weight related to ovarian hormones are not mediated through BAT activity [20,21] and that dietary obesity and estrogens increase energy expenditure through different mechanisms [21]. In addition to sex hormones, it is important to point out that norepinephrine (NE) is one of the main regulators of BAT function [22], whose turnover in BAT is altered by effect of ovariectomy [23]. Mitochondrial biogenesis is a complex event that includes both mitochondrial proliferation and differentiation [24] and requires the coordinated contribution of both mitochondrial and nuclear genome [25]. Peroxisome proliferator-activated receptor gamma coactivator-1␣ (PGC-1␣) is the main regulator of mitochondrial biogenesis process [26]. In short, PGC-1␣ can activate different transcription factors, such as nuclear respiratory factors (NRFs) [27], which regulate the expression of nuclear genes involved in mitochondrial biogenesis, including the mitochondrial transcription factor A (TFAM), a crucial factor for proper mtDNA (mitochondrial DNA) replication and transcription [28–30]. Estrogens have been proposed to modify mitochondrial activity through the regulation of the mitochondrial biogenesis process [17]. Taking into account the sex differences previously reported regarding mitochondrial function and biogenesis in BAT, the aim of this study was to go further into these differences by investigating the effect of the ovariectomy and 17-␤ estradiol (E2) replacement on BAT mitochondrial functionality. To address these issues we determined the levels of markers of oxidative capacity, antioxidant defences and oxidative damage. Moreover, we also studied the levels of key elements of the mitochondrial biogenesis process; as well as UCP1 protein expression as an index of mitochondrial differentiation and thermogenic capacity. 2. Experimental 2.1. Materials Immulite®

2000 Estradiol kit was from Siemens Healthcare Diagnostics. Oligonucleotide primer sequences, LightCycler® 480 SYBR Green I Master for real-time PCR and Tripure® isolation reagent were purchased from Roche Diagnostics (Basel, Switzerland). RT-PCR chemicals were from Applied Biosystems (Lincoln, CA, USA). OxyblotTM Protein Oxidation Detection kit was obtained from Chemicon International (Temecula, CA, USA). Rabbit antisera against TFAM was kindly provided by Dr. H. Inagaki [31]. Anti-COX IV (Cat. num. MS407) was from MitoSciences (Eugene, OR, USA) and anti-UCP1 (Cat. num. UCP12-A) was from Alpha Diagnostic International (San Antonio, TX, USA). The enhanced chemiluminescence Western blotting analysis reagents were supplied by Bio-Rad (Hercules, CA, USA). Routine chemicals were purchased from Sigma–Aldrich (St Louis, MO, USA) and Panreac (Barcelona, Spain). Pelleted diet (A04) was obtained from Panlab (Barcelona, Spain). 2.2. Animals and treatments Animal experiments were performed in accordance with general guidelines approved by EU regulations (86/609/CEE and 2003/65/CE) and our institutional ethics committee. Control female Wistar rats and females ovariectomized (OVX) at 5 weeks of age, to eliminate endogenous ovarian steroid production, were purchased

from Charles River (Barcelona, Spain) and were kept at 22 ◦ C on a 12-h light–dark cycle with free access to water and pelleted standard diet. At ten weeks of age, animals were divided into three experimental groups: control (n = 8), OVX (n = 6) and OVX treated with E2 (OVX + E2) (n = 6). The OVX + E2 rats were given a substitutive dose of E2, which consists of a subcutaneous injection of 10 ␮g/kg/48 h of E2 dissolved in 0.1 mL of corn oil (vehicle) for 4 weeks [32]. In parallel, OVX rats were treated only with the vehicle. After a 12-h period of fasting, rats were sacrificed by decapitation at 14 weeks of age. Blood was collected. Interscapular BAT was dissected and weighed, and a piece was frozen at −80 ◦ C until real-time PCR analysis. 2.3. Sample preparation The serum obtained was frozen at −80 ◦ C until analyzed. Serum levels of E2 were measured by using the Immulite® 2000 Estradiol kit according to the manufacturer’s protocol. Fresh BAT was homogenized with a Teflon/glass homogenizer in STE buffer (250 mM sucrose, 5 mM Tris–HCl and 2 mM EGTA, pH 7.4) in a proportion of 10 mL of buffer per g of tissue and was filtered through a layer of gauze. An aliquot of homogenate was frozen (−20 ◦ C) for semiquantification of mtDNA, determination of enzymatic activities as well as the measurement of the levels of protein, DNA and markers of oxidative damage. Another aliquot was frozen (−20 ◦ C) with protease and phosphatase inhibitors (10 ␮M leupeptin, 10 ␮M pepstatin, 0.2 mM PMSF and 0.2 mM orthovanadate) for Western blot analysis. 2.4. Measurement of protein and DNA content Protein and total DNA levels were determined using the Bradford method [33] and the diaminobenzoic acid method [34], respectively. mtDNA extraction and semi-quantification was carried out in homogenates as previously described [35]. Briefly, real-time PCR was performed to amplify a 162-nt region of the mitochondrial NADH dehydrogenase subunit 4 gene, which is exclusive of mtDNA (primer sequences shown in Table 1). 2.5. Enzymatic activities Cytochrome c oxidase (COX) [36], citrate synthase [37], Mnsuperoxide dismutase (Mn-SOD) [38] and glutathione peroxidase (GPx) [39] activities were assayed by spectrophotometric methods. 2.6. Measurement of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl group levels Levels of TBARS, as an index of lipid peroxides, were measured spectrophotometrically as previously described [40]. Protein carbonyl groups, as an index of protein oxidation, were measured in BAT homogenates by immunoblot detection by using the OxyBlotTM Protein Oxidation Detection kit according to the manufacturer’s protocol with minor modifications [41]. 2.7. Analysis of mRNA levels by real-time PCR Total RNA was obtained from 100 mg of BAT using Tripure® isolation reagent and quantified using a spectrophotometer set at 260 nm. One ␮g of total RNA was reverse transcribed to cDNA for 60 min at 42 ◦ C, followed by 5 min at 60 ◦ C with 25UMuLV reverse transcriptase in a 10 ␮l volume of retrotranscription reaction mixture containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X® -100, 2.5 mM MgCl2 , 2.5 ␮M random hexamers, 10 U RNase inhibitor and 500 ␮M of each dNTP in a Gene Amp 9700 thermal

A. Nadal-Casellas et al. / Steroids 76 (2011) 1051–1056

1053

Table 1 Oligonucleotide primer sequences and conditions used in real-time PCR amplification. Gene

Forward primer (5 –3 ) Reverse primer (5 –3 )

Denaturation temp. (◦ C) Time (s)

Annealing temp. (◦ C) Time (s)

Extension temp. (◦ C) Time (s)

Product length

Tm

PGC-1␣

ATG TGT CGC CTT CTT GCT CT ATC TAC TGC CTG GGG ACC TT ATG GGG AAG GTG AAG GTC GGA G TCG CCC CAC TTG ATT TTG GAG G TAC ACG ATG AGG CAA CCA AA GGT AGG GGG TGT GTT GTG AG

95 (10) 95 (10) 95 (10)

65 (10) 62 (10) 62 (10)

72 (12) 72 (12) 72 (12)

179 pb

83.4

265 pb

89.0

161 pb

80.0

GADPH mtDNA

PGC-1␣, peroxisome proliferator-activated receptor-gamma coactivator-1; GADPH, glyceraldehyde 3-phosphate dehydrogenase; mtDNA, mitochondrial DNA (gene: NADH dehydrogenase).

cycler (Applied Biosystems, Lincoln, CA, USA). Each cDNA solution was diluted 1/10 and aliquots were frozen (−80 ◦ C) until the PCRs were carried out. Real-time PCR was performed for the mRNAs of peroxisomeproliferator-activated receptor-gamma co-activator 1 alpha (PGC1␣) and one housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GADPH). Oligonucleotide primer sequences (Table 1) were obtained from Primer3 and tested with IDT OligoAnalyser 3.0. Finally, a basic local alignment search tool (NCBI Blast) revealed that the primer sequence homology was obtained only for the target genes. Real-time PCR was performed using LightCycler® 480 SYBR Green I Master technology on a LightCycler® 480 System II rapid thermal cycler (Roche Diagnostics, Basel, Switzerland). Each reaction contained 5 ␮l of LightCycler® 480 SYBR Green I Master (containing FastStart Taq DNA polymerase, dNTP mix, reaction buffer, MgCl2 and SYBR Green I dye), 0.5 ␮M of the sense and antisense specific primers, 2.5 ␮l of the cDNA dilution in a final volume of 10 ␮l. The amplification program consisted of a preincubation step for denaturation of the template cDNA (95 ◦ C, 5 min), followed by 40 cycles consisting of a denaturation, annealing, and extension step in conditions shown in Table 1. After each cycle, fluorescence was measured at 72 ◦ C. Product specificity was confirmed in initial experiments by agarose gel electrophoresis and routinely by melting curve analysis. 2.8. Western blot analysis In order to quantify the protein levels of COX IV, TFAM and UCP1, 15–50 ␮g of protein from BAT homogenates was fractionated on SDS-PAGE gels and electrotransferred onto a nitrocellulose filter. A Ponceau S staining was performed in order to check for correct loading and electrophoretic transfer. The membranes were blocked for 1 h and incubated overnight with the corresponding antibodies. Development of immunoblots was performed using an enhanced chemiluminescence kit. Bands were visualized with the ChemiDoc XRS system (Bio-Rad, CA, USA) and analyzed with the image analysis program Quantity One© (Bio-Rad, CA, USA). Bands revealed an apparent molecular mass of 16, 25 and 32 kDa for COX IV, TFAM and UCP1, respectively. 2.9. Statistical analysis All data are expressed as the mean values ± SEM of 6–8 animals per group. Real-time PCR data normalization was performed using the GenEx Standard software (MultiDAnalises) taking into account the efficiency of the reaction. The stability and suitability of GAPDH as a reference gene was validated by NormFinder application of GenEx software. Statistical differences between groups were analyzed by Student’s t-test and a p-value less than 0.05 was considered statistically significant. All statistical analyses were performed using a statistical software package (SPSS 17.0 for Windows, Inc., Chicago, IL, USA).

Table 2 Effects of ovariectomy and E2 supplementation on serum levels of E2, body weight, and BAT weight and composition. Control E2 levels (pg/mL) BW (g) Adiposity indexa BAT weight (g) (g/100g BW) Protein (mg/g tissue) DNA (mg/g tissue)

94.5 199 4.62 0.280 0.141 88.7 1.86

± ± ± ± ± ± ±

OVX 7.4 3 0.13 0.011 0.004 2.9 0.09

76.5 265 6.27 0.295 0.112 71.5 1.36

OVX + E2 ± ± ± ± ± ± ±

7.7 6a 0.29a 0.017 0.007a 1.6a 0.07a

117 244 5.65 0.299 0.122 73.1 1.45

± ± ± ± ± ± ±

16b 6a,b 0.35a 0.027 0.008a 3.5a 0.17a

OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol; BW, body weight. Values are means ± SEM of 6–8 animals per group. Student’s t-test (p < 0.05): a indicates different from control rats and b indicates different from OVX rats. a Adiposity index is the sum of inguinal, gonadal, mesenteric and retroperitoneal adipose tissue weights relative to 100 g of BW.

3. Results 3.1. E2 levels, body weight and BAT weight and composition Serum levels of E2 were slightly lower in OVX rats than in controls (Table 2), although these differences were not statistically significant. In response to E2 supplementation, serum levels of E2 increased, reaching the values shown by control rats. Body weight and adiposity index increased with ovariectomy compared to control rats (33.2% and 35.7%, respectively). Although adiposity index was no altered in response to E2 replacement, body weight decreased in OVX + E2 group with respect to OVX rats, but remained higher than in control females (22.6%). BAT weight was similar in all experimental groups, but decreased with ovariectomy when it is expressed relative to body weight. BAT protein and DNA levels also decreased with ovariectomy and kept these values with E2 supplementation. 3.2. Oxidative enzyme activities and COX IV protein levels Both COX and citrate synthase activities decreased with ovariectomy in comparison to control rats (Table 3). All in all, no effect of the E2 treatment was found either in COX or in citrate synthase activities. COX IV protein levels tended to decrease in both OVX and Table 3 Effects of ovariectomy and E2 supplementation on BAT markers of oxidative capacity.

COX (A.U./g tissue) Citrate synthase (I.U./g tissue) COX IV protein levels (%)

Control

OVX

OVX + E2

565 ± 13 105 ± 4 100 ± 10

513 ± 22a 93.8 ± 4.4a 77.7 ± 8.3

524 ± 15a 93.3 ± 5.8a 73.8 ± 12.8

OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol; COX (cytochrome c oxidase) and citrate synthase activities are expressed as A.U. (arbitrary units) and I.U. (international units), respectively. COX IV (COX subunit IV) protein levels are expressed in A.U per g of BAT and the mean value of control rats was set as 100%. Values are means ± SEM of 6–8 animals per group. Student’s t-test (p < 0.05): a indicates different from control rats.

1054

A. Nadal-Casellas et al. / Steroids 76 (2011) 1051–1056

Table 4 Effects of ovariectomy and E2 supplementation on BAT antioxidant enzyme activities and oxidative damage markers. Control GPx (I.U./g tissue) SOD (I.U./g tissue) GPx/Mn-SOD ratio TBARS (nmol/g tissue) Carbonyl groups (%)

6.89 0.922 1.00 272 100

± ± ± ± ±

OVX 0.42 0.137 0.08 13 10

5.29 0.487 1.23 247 93.1

Table 6 Effects of ovariectomy and E2 supplementation on BAT markers of thermogenic capacity.

OVX + E2 ± ± ± ± ±

0.39a 0.030a 0.08a 10 13.9

6.32 0.459 1.84 275 80.6

± ± ± ± ±

0.37b 0.060a 0.24a,b 8a,b 12.1

OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol. GPx (glutathione peroxidase) and SOD (superoxide dismutase) activities are expressed as I.U. (international units). TBARS, thiobarbituric acid-reactive substances. Levels of control rats were set as 1 in the GPx/Mn-SOD ratio and 100% in the carbonyl groups content. Values are means ± SEM of 6–8 animals per group. Student’s t-test (p < 0.05): a indicates different from control rats and b indicates different from OVX rats.

UCP1 protein levels Per g of tissue (%) Per mtDNA (%) UCP1/COX (%)

Control

OVX

OVX + E2

100 ± 17 100 ± 8 100 ± 17

201 ± 23a 125 ± 5 225 ± 28a

172 ± 30a 100 ± 6 187 ± 31a

OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol. Protein levels of UCP1 (uncoupling protein 1) are expressed in arbitrary units per g of BAT and per mtDNA. UCP1/COX ratio is the relationship between UCP1 protein levels and cytochrome c oxidase activity. Levels of control rats were set as 100%. Values are means ± SEM of 6–8 animals per group. Student’s t-test (p < 0.05): a indicates different from control rats.

4. Discussion OVX + E2 in comparison to control females, although differences did not reach statistical significance.

3.3. Antioxidant enzyme activities and oxidative damage markers GPx and SOD activities decreased in OVX group compared to control rats (Table 4). The treatment with E2 reversed the effects of ovariectomy on GPx activity, with both control and OVX + E2 females exhibiting similar activity levels, in contrast, SOD was not affected by the hormonal supplementation. Although GPx/Mn-SOD ratio, which can be considered an index of cellular H2 O2 metabolism, increased in OVX females, this increase was even more noticeable in OVX + E2 rats. TBARS levels increased in OVX + E2 females compared to OVX, reaching the level of control females. No significant differences between experimental groups were observed in the content of protein carbonyl groups.

3.4. Mitochondrial biogenesis markers No significant differences related to hormonal status were found in mRNA levels of PGC-1␣ (Table 5). In response to ovariectomy, TFAM protein levels decreased compared to control rats and E2 supplementation did not manage to recover the TFAM levels of control females. The levels of mtDNA per cell were greater in OVX rats compared to control females, but they were not altered in response to the hormonal replacement. UCP1 protein levels and UCP1/COX ratio, which represents the tendency to uncouple the mitochondrial proton gradient generated by the respiratory chain, markedly increased in both groups of OVX females (Table 6). However, UCP1 protein levels per mtDNA (i.e. per mitochondrion) were not altered by ovariectomy. Representative immunoblots are shown in Fig. 1.

Detailed knowledge of the factors involved in the sex differences observed in mitochondrial function and biogenesis of BAT is of great interest, because of its thermogenic function and its role in controlling body weight in small mammals. In this context, the ovariectomy of rats represents a useful, interesting tool to investigate the involvement of ovarian hormones on this dimorphism. In the present study we observed that ovarian hormones contributed, in part, to the enhanced differentiation of BAT female mitochondria previously described in rats [10–13]. In fact, BAT mitochondrial functionality is impaired in response to ovariectomy. Although serum levels of E2 were restored by the hormonal treatment, the effects of ovariectomy were only partially prevented. These results would point towards the importance of signals other than E2 produced by ovary in the control of mitochondrial function [42,43]. In this study, ovariectomized female rats show a decrease in both oxidative capacity and antioxidant defences. These results agree with previous studies performed in other tissues, such as aorta or brain, in which low levels of estrogens have been related to both decreased activity and expression of enzymes involved in oxidative metabolism and antioxidant systems [42,44,45]. In this sense, the greater oxidative and antioxidant capacities that BAT of female rats exhibits in comparison to males [10–12] can be attributed to ovarian hormones. However, in spite of the decrease in both GPx ad SOD activities shown by OVX rats, oxidative damage was not increased, which contrasts to what has been described in male rats, those levels of oxidative stress markers are higher than in females [46]. These results would confirm that OVX rats still maintain higher antioxidant capacity than males. Nevertheless, the enhanced GPx activity found in response to E2 supplementation is unable to prevent the increase of oxidative damage, which could be explained by the imbalance between antioxidant enzymes, as pointed out by the increased GPx/SOD ratio, suggesting that low

Table 5 Effects of ovariectomy and E2 supplementation on BAT markers of mitochondrial biogenesis process.

PGC-1␣mRNA levels TFAM protein levels (%) mtDNA (%)

Control

OVX

OVX + E2

3.29 ± 0.66 100 ± 9 100 ± 6

3.83 ± 0.48 63.1 ± 5.4a 216 ± 13a

2.97 ± 0.78 66.6 ± 7.8a 198 ± 30a

OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol. mRNA levels of PGC-1␣ (peroxisome proliferator-activated receptor gamma coactivator-1␣) are expressed in A. U. (arbitrary units). Protein levels of TFAM (mitochondrial transcription factor A) are expressed in arbitrary units per g of BAT. Levels of mtDNA (mitochondrial DNA) are expressed in arbitrary units per mg of DNA (C). TFAM and mtDNA levels of control rats were set as 100%. Values are means ± SEM of 6–8 animals per group. Student’s t-test (p < 0.05): a indicates different from control rats.

Fig. 1. Representative immunoblots. OVX, ovariectomized; OVX + E2, OVX treated with 17-␤ estradiol. COX IV, cytochrome c oxidase subunit IV; UCP1, uncoupling protein 1; TFAM, mitochondrial transcription factor A.

A. Nadal-Casellas et al. / Steroids 76 (2011) 1051–1056

SOD activity constitutes a limiting factor in H2 O2 metabolism. The effect of E2 on BAT redox status seems to differ from what has been reported in rodent tissues such as liver or kidney, in which it is improved in response to lower doses of estrogens administered for shorter periods of time [16,47,48] than those used in this study. The ovariectomy-induced decrease of BAT mitochondrial functionality could be explained, at least in part, by an impairment of the mitochondrial biogenesis process. Thus, the failure of ovarian function induces a decrease of TFAM levels, which is accompanied by an increase in the content of mitochondria (i.e. mtDNA levels), since high TFAM levels induce mitochondrial differentiation whereas mtDNA replication is enhanced even by lower TFAM levels [28–30]. However, the profile shown by TFAM levels in response to ovariectomy does not match the one shown by the expression of PGC-1␣, the main regulator of the mitochondrial biogenesis process [26]. In fact, neither ovariectomy nor E2 supplementation altered PGC-1␣ levels in BAT, in agreement with in vitro studies showing that estrogens do not modify PGC-1␣ mRNA levels in brown adipocytes [49]. In this sense, it is important to point out that the lack of PGC-1␣ mRNA variations does not imply an absence of changes in its protein levels, given that post-transcriptional and post-traductional mechanisms controlling levels, localization and activity of PGC-1␣ protein cannot be ruled out [50,51]. Although ovariectomy is able to attenuate the sexual dimorphism previously described in rat BAT mitochondrial functionality [10,46], the markers of oxidative capacity in OVX female rats did not reach the low levels shown by males [46]. These results can be explained by the fact that, in spite of ovariectomy, serum levels of E2 were not significantly decreased, as happens in other studies [52]. In our study, more than 2 months have passed from ovariectomy to sacrifice, thus, compensatory mechanisms that alleviate the effects of ovariectomy, at least partially, could have been activated in the OVX females. In this sense, it is worth noting that in young female OVX rat estrogen production has been reported to shift from ovaries to extragonadal sites when ovarian function fails [53]. Moreover, previous reports showed that circulating estrogen concentration increases gradually with time after ovariectomy in rats, which is attributed to greater aromatase activity in adipose tissue, among others [54]. This idea is reinforced by the fact that, in contrast to what happens to BAT [55], OVX rats exhibit a marked increase of white adipose tissue mass, which could contribute to estrogen replacement. On the other hand, it is important to note that the markers of mitochondrial functionality were not manifestly affected by the hormonal supplementation. However, given that the hormonal treatment was able to restore the levels of E2 shown by the control rats, it could be suggested that the light effect of the hormonal treatment on mitochondrial oxidative capacity can be attributed of a lack of other signals also produced by the ovary rather than E2, which also play an important role in the control of mitochondrial function [42,43]. In addition to ovary signals, other regulatory factors may be involved in the control of mitochondrial function, especially of thermogenic capacity. Unlike what happens with oxidative and antioxidant capacities, ovariectomy emphasizes the sex differences in UCP1 protein levels, which were noticeably increased in both OVX and OVX + E2 groups. Taking into account that UCP1 mRNA expression decreases in rats devoid of estrogens [56], the existence of a post-transcriptional regulatory mechanism should be considered. In fact, ␤3-AR, which is coupled to the induction of UCP1 synthesis [57], is increased in brown adipocytes in response to ovariectomy (data not shown). The UCP1 protein levels per mitochondrion remain unaltered in response to ovariectomy, but given that the mitochondrial pool found in OVX rats increased, UCP1 protein content in BAT of OVX females increased, which contrasts with the notable body weight gain (33% compared to control females) traditionally attributed to enhanced energy efficiency [18,19]. However, our results are in

1055

agreement with previous studies performed also in rats using models of dietary obesity, in which the body weight increases in spite of the higher UPC1 protein content [46,58]. Moreover, the body weight decreased in OVX + E2 rats, whereas UCP1 protein levels were not altered, thus the role of UCP1 as a body weight regulator does not seem to be crucial in this context. In this sense, the increased oxidative capacity of other tissues such as the muscle [59], or the mobilization of visceral fat [60] could play a more important role than BAT thermogenesis in the loss of weight associated with hormonal treatment in rats, in agreement with previous reports suggesting that changes in body weight associated to ovarian steroids are not mediated through BAT activity [21]. All in all, although the increased UCP1 levels could be understood at first as a compensatory mechanism to prevent body weight gain, the increased UCP1/COX ratio reflects that the greater thermogenic potential is accompanied by a reduced oxidative capacity, thus the greater UCP1 protein levels are not be enough to prevent obesity in OVX females. Moreover, the overweight of OVX rats could be explained by the fact that an important part of their UCP1 is inactive, which would agree with a previous study showing that obesity elevates UCP3 levels in rat skeletal muscle but not its activity [61]. In summary, ovarian hormones are responsible, at least in part, for the sex differences described in BAT mitochondria. In fact, in response to ovariectomy, oxidative capacity and antioxidant defences decreased. However, sexual dimorphism in BAT mitochondrial functionality cannot be exclusively attributed to E2, since despite the fact that serum E2 levels were restored after hormonal treatment, markers of oxidative and antioxidant capacities did not reach the levels of control rats. Thus, other signals produced by ovary, rather than E2, would play an important role in the control of mitochondrial function in BAT. Conflict of interest The authors declare no conflict of interest. Acknowledgements We thank Dr. Hidetoshi Inagaki for providing the antiserum against TFAM. This work was supported by Fondo de Investigaciones Sanitarias of the Spanish Government (PI060293) and by Conselleria d’Innovació i Energia of the Comunitat Autónoma de les Illes Balears (PROGECIB-1C). A. Nadal-Casellas was funded by a grant from the Comunitat Autònoma de les Illes Balears. References [1] Teede HJ, Lombard C, Deeks AA. Obesity, metabolic complications and the menopause: an opportunity for prevention. Climacteric 2010;13:203–9. [2] Gaspard U. Hyperinsulinaemia, a key factor of the metabolic syndrome in postmenopausal women. Maturitas 2009;62:362–5. [3] Heaton GM, Wagenvoord RJ, Kemp Jr A, Nicholls DG. Brown-adipose-tissue mitochondria: photoaffinity labelling of the regulatory site of energy dissipation. Eur J Biochem 1978;82:515–21. [4] Lowell BB, Spiegelman BM. Towards a molecular understanding of adaptive thermogenesis. Nature 2000;404:652–60. [5] Feldmann HM, Golozoubova V, Cannon B, Nedergaard J. UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality. Cell Metab 2009;9:203–9. [6] Saito M, Okamatsu-Ogura Y, Matsushita M, Watanabe K, Yoneshiro T, NioKobayashi J, et al. High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity. Diabetes 2009;58:1526–31. [7] Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, et al. Functional brown adipose tissue in healthy adults. N Engl J Med 2009;360:1518–25. [8] Zingaretti MC, Crosta F, Vitali A, Guerrieri M, Frontini A, Cannon B, et al. The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue. Faseb J 2009;23:3113–20. [9] Nedergaard J, Cannon B. The changed metabolic world with human brown adipose tissue: therapeutic visions. Cell Metab 2010;11:268–72.

1056

A. Nadal-Casellas et al. / Steroids 76 (2011) 1051–1056

[10] Rodriguez-Cuenca S, Pujol E, Justo R, Frontera M, Oliver J, Gianotti M, et al. Sex-dependent thermogenesis, differences in mitochondrial morphology and function, and adrenergic response in brown adipose tissue. J Biol Chem 2002;277:42958–63. [11] Justo R, Frontera M, Pujol E, Rodriguez-Cuenca S, Llado I, Garcia-Palmer FJ, et al. Gender-related differences in morphology and thermogenic capacity of brown adipose tissue mitochondrial subpopulations. Life Sci 2005;76:1147–58. [12] Valle A, Garcia-Palmer FJ, Oliver J, Roca P. Sex differences in brown adipose tissue thermogenic features during caloric restriction. Cell Physiol Biochem 2007;19:195–204. [13] Valle A, Santandreu FM, Garcia-Palmer FJ, Roca P, Oliver J. The serum levels of 17beta-estradiol, progesterone and triiodothyronine correlate with brown adipose tissue thermogenic parameters during aging. Cell Physiol Biochem 2008;22:337–46. [14] Chen JQ, Cammarata PR, Baines CP, Yager JD. Regulation of mitochondrial respiratory chain biogenesis by estrogens/estrogen receptors and physiological, pathological and pharmacological implications. Biochim Biophys Acta 2009;1793:1540–70. [15] Hamden K, Carreau S, Ellouz F, Masmoudi H, El FA. Protective effect of 17betaestradiol on oxidative stress and liver dysfunction in aged male rats. J Physiol Biochem 2007;63:195–201. [16] Enli Y, Oztekin O, Pinarbasili RD. The nitroxide tempol has similar antioxidant effects as physiological levels of 17beta-oestradiol in reversing ovariectomyinduced oxidative stress in mice liver and kidney. Scand J Clin Lab Invest 2009;69:526–34. [17] Mattingly KA, Ivanova MM, Riggs KA, Wickramasinghe NS, Barch MJ, Klinge CM. Estradiol stimulates transcription of nuclear respiratory factor-1 and increases mitochondrial biogenesis. Mol Endocrinol 2008;22:609–22. [18] Kemnitz JW, Glick Z, Bray GA. Ovarian hormones influence brown adipose tissue. Pharmacol Biochem Behav 1983;18:563–6. [19] Yoshioka K, Yoshida T, Wakabayashi Y, Nishioka H, Kondo M. Reduced brown adipose tissue thermogenesis of obese rats after ovariectomy. Endocrinol Jpn 1988;35:537–43. [20] Richard D. Effects of ovarian hormones on energy balance and brown adipose tissue thermogenesis. Am J Physiol 1986;250:R245–9. [21] Guyard B, Fricker J, Brigant L, Betoulle D, Apfelbaum M. Effects of ovarian steroids on energy balance in rats fed a highly palatable diet. Metabolism 1991;40:529–33. [22] Lafontan M, Barbe P, Galitzky J, Tavernier G, Langin D, Carpene C, et al. Adrenergic regulation of adipocyte metabolism. Hum Reprod 1997;12(Suppl. 1):6–20. [23] Yoshida T, Nishioka H, Yoshioka K, Kondo M. Reduced norepinephrine turnover in interscapular brown adipose tissue of obese rats after ovariectomy. Metabolism 1987;36:1–6. [24] Ostronoff LK, Izquierdo JM, Enriquez JA, Montoya J, Cuezva JM. Transient activation of mitochondrial translation regulates the expression of the mitochondrial genome during mammalian mitochondrial differentiation. Biochem J 1996;316(Pt 1):183–91. [25] Fernandez-Silva P, Enriquez JA, Montoya J. Replication and transcription of mammalian mitochondrial DNA. Exp Physiol 2003;88:41–56. [26] Puigserver P, Spiegelman BM. Peroxisome proliferator-activated receptorgamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 2003;24:78–90. [27] Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999;98:115–24. [28] Scarpulla RC. Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg Biomembr 1997;29:109–19. [29] Garstka HL, Schmitt WE, Schultz J, Sogl B, Silakowski B, Perez-Martos A, et al. Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA. Nucleic Acids Res 2003;31:5039–47. [30] Maniura-Weber K, Goffart S, Garstka HL, Montoya J, Wiesner RJ. Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells. Nucleic Acids Res 2004;32:6015–27. [31] Inagaki H, Hayashi T, Matsushima Y, Lin KH, Maeda S, Ichihara S, et al. Isolation of rat mitochondrial transcription factor A (r-Tfam) cDNA. DNA Seq 2000;11:131–5. [32] Busserolles J, Mazur A, Gueux E, Rock E, Rayssiguier Y. Metabolic syndrome in the rat: females are protected against the pro-oxidant effect of a high sucrose diet. Exp Biol Med (Maywood) 2002;227:837–42. [33] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [34] Thomas PS, Farquhar MN. Specific measurement of DNA in nuclei and nucleic acids using diaminobenzoic acid. Anal Biochem 1978;89:35–44. [35] Justo R, Oliver J, Gianotti M. Brown adipose tissue mitochondrial subpopulations show different morphological and thermogenic characteristics. Mitochondrion 2005;5:45–53.

[36] Chrzanowska-Lightowlers ZM, Turnbull DM, Lightowlers RN. A microtiter plate assay for cytochrome c oxidase in permeabilized whole cells. Anal Biochem 1993;214:45–9. [37] Nakano K, Tarashima M, Tachikawa E, Noda N, Nakayama T, Sasaki K, et al. Platelet mitochondrial evaluation during cytochrome c and dichloroacetate treatments of MELAS. Mitochondrion 2005;5:426–33. [38] Quick KL, Hardt JI, Dugan LL. Rapid microplate assay for superoxide scavenging efficiency. J Neurosci Methods 2000;97:139–44. [39] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69. [40] Slater TF, Sawyer BC. The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro Inhibitory effects of free-radical scavengers and other agents. Biochem J 1971;123:823–8. [41] Guevara R, Santandreu FM, Valle A, Gianotti M, Oliver J, Roca P. Sex-dependent differences in aged rat brain mitochondrial function and oxidative stress. Free Radic Biol Med 2009;46:169–75. [42] Irwin RW, Yao J, Hamilton RT, Cadenas E, Brinton RD, Nilsen J. Progesterone and estrogen regulate oxidative metabolism in brain mitochondria. Endocrinology 2008;149:3167–75. [43] Kasapovic J, Pajovic SB, Pejic S, Martinovic JV. Effects of estradiol benzoate and progesterone on superoxide dismutase activity in the thymus of rats. Physiol Res 2001;50:97–103. [44] Stirone C, Duckles SP, Krause DN, Procaccio V. Estrogen increases mitochondrial efficiency and reduces oxidative stress in cerebral blood vessels. Mol Pharmacol 2005;68:959–65. [45] Strehlow K, Rotter S, Wassmann S, Adam O, Grohe C, Laufs K, et al. Modulation of antioxidant enzyme expression and function by estrogen. Circ Res 2003;93:170–7. [46] Nadal-Casellas A, Proenza AM, Gianotti M, Lladó I. Brown adipose tissue redox status in response to dietary-induced obesity-associated oxidative stress in male and female rats. Stress 2010 [Epub ahead of print]. [47] Borras C, Sastre J, Garcia-Sala D, Lloret A, Pallardo FV, Vina J. Mitochondria from females exhibit higher antioxidant gene expression and lower oxidative damage than males. Free Radic Biol Med 2003;34:546–52. [48] Kiray M, Ergur BU, Bagriyanik A, Pekcetin C, Aksu I, Buldan Z. Suppression of apoptosis and oxidative stress by deprenyl and estradiol in aged rat liver. Acta Histochem 2007;109:480–5. [49] Rodriguez-Cuenca S, Monjo M, Gianotti M, Proenza AM, Roca P. Expression of mitochondrial biogenesis-signaling factors in brown adipocytes is influenced specifically by 17beta-estradiol, testosterone, and progesterone. Am J Physiol Endocrinol Metab 2007;292:E340–6. [50] Dominy Jr JE, Lee Y, Gerhart-Hines Z, Puigserver P. Nutrient-dependent regulation of PGC-1alpha’s acetylation state and metabolic function through the enzymatic activities of Sirt1/GCN5. Biochim Biophys Acta 2010;1804: 1676–83. [51] Coste A, Louet JF, Lagouge M, Lerin C, Antal MC, Meziane H, et al. The genetic ablation of SRC-3 protects against obesity and improves insulin sensitivity by reducing the acetylation of PGC-1{alpha}. Proc Natl Acad Sci U S A 2008;105:17187–92. [52] Pantaleao TU, Mousovich F, Rosenthal D, Padron AS, Carvalho DP, Costa VM. Effect of serum estradiol and leptin levels on thyroid function, food intake and body weight gain in female Wistar rats. Steroids 2010. [53] Simpson ER. Sources of estrogen and their importance. J Steroid Biochem Mol Biol 2003;86:225–30. [54] Zhao H, Tian Z, Hao J, Chen B. Extragonadal aromatization increases with time after ovariectomy in rats. Reprod Biol Endocrinol 2005;3:6. [55] Heine PA, Taylor JA, Iwamoto GA, Lubahn DB, Cooke PS. Increased adipose tissue in male and female estrogen receptor-alpha knockout mice. Proc Natl Acad Sci U S A 2000;97:12729–34. [56] Pedersen SB, Bruun JM, Kristensen K, Richelsen B. Regulation of UCP1 UCP2, and UCP3 mRNA expression in brown adipose tissue, white adipose tissue, and skeletal muscle in rats by estrogen. Biochem Biophys Res Commun 2001;288:191–7. [57] Chaudhry A, Granneman JG. Differential regulation of functional responses by beta-adrenergic receptor subtypes in brown adipocytes. Am J Physiol 1999;277:R147–53. [58] Nedergaard J, Raasmaja A, Cannon B. Parallel increases in amount of (3H)GDP binding and thermogenin antigen in brown-adipose-tissue mitochondria of cafeteria-fed rats. Biochem Biophys Res Commun 1984;122: 1328–36. [59] Campbell SE, Febbraio MA. Effect of ovarian hormones on mitochondrial enzyme activity in the fat oxidation pathway of skeletal muscle. Am J Physiol Endocrinol Metab 2001;281:E803–8. [60] Clegg DJ, Brown LM, Woods SC, Benoit SC. Gonadal hormones determine sensitivity to central leptin and insulin. Diabetes 2006;55: 978–87. [61] Chou CJ, Cha MC, Jung DW, Boozer CN, Hashim SA, Pi-Sunyer FX. High-fat diet feeding elevates skeletal muscle uncoupling protein 3 levels but not its activity in rats. Obes Res 2001;9:313–9.