Abstract The effects of acute and chronic acclimationto cold on uncoupling protein 1 (UCP1) levels, as well ason GDP-binding to mitochondria, cytochrome c oxidaseactivity and mitochondrial protein concentration inbrown adipose tissue (BAT) of intact male and femalerats have been analyzed. Results reveal that females ratsare more sensitive to cold because their threshold tem-perature for the thermogenic response is set at a highervalue (around 22°C) than that of males (around 18°C),hence leading to differences in BAT UCP1 levels be-tween the sexes at different environmental temperatures.In vitro experiments showed that steroid hormones, β-es-tradiol, estrone and progesterone, can reduce norepi-nephrine-induced UCP1 synthesis in brown adipocytesdifferentiated in primary culture. Thus the different sex-associated response of cold-induced thermogenesis inrats does not appear to be explained by a direct action ofsex steroids upon the adipocyte, implying that other fac-tors in the thermogenic regulatory system must be in-volved.

Key words Brown adipocytes · β-Estradiol ·Thermogenesis · Uncoupling protein 1 (UCP1)

Introduction

Brown adipose tissue (BAT) is the main site of non-shiv-ering thermogenesis, which specifically functions to pro-duce heat in neonates and in cold-acclimated rodents.BAT thermogenic activity depends on the presence of theuncoupling protein 1 or UCP1, an inner-mitochondrialmembrane protein usually expressed only in BAT inmammals, which functions by dissipating the proton gra-dient generated by the respiratory chain and thereby un-coupling oxidative phosphorylation [4, 18, 19, 27].

BAT thermogenesis is physiologically regulated. Thistissue is richly sympathetically innervated, and release ofnorepinephrine (the main physiological regulator), whichoccurs for example during cold exposure or after chronicoverfeeding [28], stimulates BAT recruitment and thusincreases its thermogenic potential, implying stimulationof brown adipocyte cell proliferation, mitochondriogene-sis and an increase in UCP1 levels – the latter through anincrease in UCP1 mRNA expression [9, 26, 27] and sta-bility [20]. The effect of norepinephrine on thermogenicactivity is mainly mediated by β-adrenergic receptors (β-AR), and in culture systems β3-AR in particular hasbeen shown to be the main AR mediating the stimulatoryaction of norepinephrine on UCP1 synthesis and activityin mature cells [3, 25]. Thus, in response to an augment-ed demand placed on BAT to produce heat, its thermo-genic activity increases. Conversely, thermoneutralityand fasting lower BAT activity and depress the metabolicrate [5, 31]. A diminished response of BAT to food in-take and to exposure to the cold has been observed inpractically all genetic forms of obesity (see [9]). Hence,it is generally accepted that BAT is of particular rele-vance in small mammals to the regulation both of tem-perature and whole energy balance.

In general, sex differences in energy metabolism arediscussed on the basis of differences in the respectivelevels and actions of the sex steroid hormones. In partic-ular, it has been shown that physiological situations suchas pregnancy and lactation, where there is a need for en-hancing metabolic efficiency in order to face the addi-tional nutritional requirements of the developing pups,the increased food intake is not followed by stimulationof diet-induced thermogenesis [32]. This depressed ther-mogenesis has been related to the presence of female sexsteroids, progesterone and estradiol, acting directly uponBAT and resulting in a reduction in oxygen consumption[21]. These effects were shown not to be dependent onchanges in the turnover of norepinephrine in BAT, but itwas suggested that in some way estradiol causes dissoci-ation between BAT’s thermogenic activity and its sym-pathetic activation [15]. However, whether the altera-

S. Quevedo · P. Roca · C. Picó · A. Palou (✉)Departament de Biologia Fonamental i Ciències de la Salut.Bioquímica i Biologia Molecular. Universitat de les Illes Balears.Ctra. Valldemossa km 7.5, Palma de Mallorca E-07071, Spaine-mail: dbfapo0@ps.uib.esTel.: +34-71-173170, Fax: +34-71-173184

Pflügers Arch – Eur J Physiol (1998) 436:689–695 © Springer-Verlag 1998

O R I G I N A L A RT I C L E

Santiago Quevedo · Pilar Roca · Catalina PicóAndreu Palou

Sex-associated differences in cold-induced UCP1 synthesisin rodent brown adipose tissue

Received: 23 March 1998 / Received after revision: 20 May 1998 / Accepted: 21 May 1998

tions are associated with changes in the levels of UCP1or affected by any other molecular step in the thermo-genic system has not been previously investigated.

We chose therefore to follow the variation of BATUCP1 levels as a key parameter in the pathway that reg-ulates thermogenesis in rodents at the cellular level. Invitro it has been shown that estradiol can reduce norepi-nephrine-induced UCP1 synthesis in brown adipocytesdifferentiated in primary culture, but that this does notaccount for the different thermogenic responses betweenthe sexes. The effects of submitting rats to different tem-perature conditions revealed that females are more sensi-tive to the cold because their threshold temperature is setat a higher value compared with the males. These sexdifferences in the responsiveness to cold are reflected invivo in differences in BAT UCP1 levels.

Materials and methods

Materials

[8-3H]GDP and [U-14C]sucrose were obtained from Amersham.GDP, ferrocytochrome c, (±)-arterenol bitartrate salt (norepineph-rine), 17β-estradiol, estrone and progesterone were obtained fromSigma. Newborn calf serum and DMEM were obtained from ICNand human insulin (Actrapid) from Novo Industries. Other reagentswere supplied by Sigma, and routine chemicals used were fromMerck and Panreac.

In vivo experiments

Animals

Male and female Wistar rats 16 weeks old were obtained fromCRIFFA, Spain. They were acclimated to 22°C, with a 12-hlight/12-h dark cycle and with free access to a standard chow diet(Panlab). Two sets of experiments were performed:

Experiment 1: acute exposure to low temperature. Four groups offive to six animals each were studied: male and female rats weremaintained at 22°C and male and female rats were exposed to thecold (4°C) for 24 h.Experiment 2: chronic exposure to different temperatures. Sixgroups of five to six animals each were studied: male and femalerats were maintained at 22°C and male and female rats were accli-mated to thermoneutrality (28°C) or the cold (18°C) for 7 days.

Sample obtention

In both experiments, animals were killed by decapitation at thestart of the light cycle, after the indicated temperature acclimation,and the interscapular BAT (IBAT) was excised, weighed and pro-cessed. A small piece of IBAT (approx. 100 mg) was frozen in liq-uid N2 and stored at –70°C for the determination of UCP1 mRNA.The rest of the IBAT depot was homogenized in HEPES/sucrosebuffer (250 mM sucrose/1 mM HEPES/0.2 mM EDTA buffer, pH7.0) in a Teflon/glass homogenizer (ten strokes) for isolation ofmitochondria. The homogenate was filtered through a layer ofgauze. An aliquot was used for determination of total protein con-tent [2]. The rest was used for mitochondrial isolation, followingbasically Nedergaard and Cannon [17]. Briefly, nuclei and cell de-bris were first removed by centrifuging at 800 g for 10 min at 4°C.The pellet was washed and the resulting supernatant was centri-fuged at 8500 g for 10 min at 4°C. The pellet containing the puri-fied mitochondria was resuspended in 200 µl of the homogenizing

buffer and used for determining total mitochondrial protein, cyto-chrome c oxidase (COX) activity (measured by a spectophotomet-ric method [33] to monitor changes in absorbance during the oxi-dation of reduced ferrocytochrome at 37°C), specific GDP-bind-ing [13] and UCP1 levels by immunoblotting.

Immunoblotting for UCP1

One to two micrograms of IBAT mitochondrial protein was frac-tionated by sodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE, 10% polyacrylamide) according to Laemmli[12] and electrotansferred onto a nitrocellulose filter as described[23]. Rabbit polyclonal antiserum raised against purified rat UCP1obtained in our laboratory [23] was used as primary antibody.Blocking and development of the immunoblots were performedusing an ECL western blotting analysis system (Amersham). Thebands were analyzed by scanner photodensitometry and quantifiedusing the BioImage program (Millipore).

RNA extraction and slot-blot analysis of UCP1 mRNAand β-actin mRNA

Total RNA was isolated from IBAT using guanidinium hydrochlo-ride [10]. The purified total RNA was dissolved in diethylpyrocar-bonate-treated water and RNA concentration determined using aspectrophotometer set at 260 nm. Specific mRNA levels for UCP1and β-actin were determined as previously described [13], usingprobes labelled with UCP1 or β-actin cDNA digoxigenin 11-dUTP,respectively. In brief, aliquots of 5 µg total RNA were immobilizedon a positively charged nylon membrane (Boehringer Mannheim)using slot-blot apparatus (Bio-Rad). Prehybridization (1 h) and hy-bridization (16 h) were carried out at 65°C. Immunological chemi-luminescence detection was performed using a Boehringer kit.Bands were scanned and quantified as described above.

In vitro experiments

Cell culture

BAT precursor cells from cervical, axillary and interscapularbrown fat tissue of 4-week-old male NMRI mice (obtained fromCRIFFA, Spain) were prepared, inoculated and cultured as earlierdescribed [26] in 6-well multidish plates. The culture mediumconsisted of DMEM supplemented with 10% newborn calf serum,4 nM insulin, 10 mM HEPES, 50 IU/ml of penicillin, 50 µg/mlstreptomycin, 2 mM glutamine and 25 µg/ml sodium ascorbate.The cells were grown at 37°C in an atmosphere of 8% CO2 in air.The culture medium was changed on the 1st, 3rd and 6th day afterinoculation. Cells were treated from day 3 with 17β-estradiol, es-trone or progesterone (added on days 3 and 6 after inoculation) atthe doses specified in figure legends, and from day 6 with differ-ent concentrations of norepinephrine (10–9 to 10–5 M). Cells wereharvested on day 7 (after 24 h of norepinephrine treatment) as pre-viously described [24], which involved removing the culture me-dium before the cells were rinsed with ice-cold phosphate-buf-fered saline (PBS: 137 mM NaCl, 2.7 mM KCl and 10 mM phos-phate buffer, pH 7.4). The cells were scraped into 1.5 ml of PBSwith a rubber policeman and transferred into Eppendorf tubes. Thecell suspensions were pelleted, resuspended with PBS, and thensonicated. The cell lysates were stored at –20°C prior to proteinand UCP1 measurements, as described above. For UCP1 measure-ment, 20 µg of total cell protein was run per lane.

Statistics

All data are presented as mean values ± SEM. In in vivo experi-ments, differences between groups were assessed by one-way ortwo-way analysis of variance (ANOVA) and least significant dif-ference (LSD) post hoc comparisons and student’s t-test.

690

Results and discussion

The sex steroids estradiol and progesterone have beenshown to have an inhibitory effect on BAT thermogenicactivity in rats, reflected in the decreased responsivenessof brown adipocytes to norepinephrine [21]. In view ofthe evidence of the involvement of steroids in BAT ther-mogenic activity, gender-associated differences in thethermogenic response to stimulation with norepinephrineare to be expected, affecting the levels of the molecularbasis of non-shivering thermogenesis, i.e., the UCP1.Thus, in the following experiment we measured IBATUCP1 levels in male and female rats acclimated to thetemperature of the animal house (22°C) and after acuteexposure (24 h) to the cold (4°C).

In vivo induction of UCP1 in maleand female rats preacclimated at 22°C after acuteexposure to a low temperature (4°C)

Table 1 shows body mass and total tissue mass, total andmitochondrial protein concentration and specific COXactivity of IBAT in male and female rats at 22°C and af-ter 24 h at 4°C. Body mass and IBAT mass of male andfemale rats were not affected significantly by acute coldexposure, as reported [14]. No significant differenceswere found in IBAT mass between male and female rats,although total and mitochondrial protein concentrationsin IBAT were higher in female than in male rats, at bothtemperatures, and cold exposure led to a significant in-crease in IBAT mitochondrial protein concentration infemale rats. No changes between male and female ratswere found in terms of specific COX activity, which re-mained unchanged after the acute treatment.

As expected [14], 24 h of cold exposure (4°C) led to asignificant increase in specific UCP1 levels in IBATfrom female rats and a more marked increase in thatfrom male rats (see Fig. 1), with the UCP1 concentra-

tions of both sexes reaching similar levels at this temper-ature. Surprisingly, male and female animals acclimatedto 22°C had different specific IBAT UCP1 levels, whichwere for female rats double that for male rats.

A similar profile was observed for the specific GDPbinding to mitochondria (Fig. 2). Female rats acclimatedto 22°C had higher specific GDP binding than males,which remained unchanged after cold exposure. Con-versely, the GDP binding of male rats exposed to 4°C in-creased, reaching similar levels to those of female rats atthis temperature.

UCP1 mRNA levels followed a similar tendency asUCP1 levels and GDP binding, increasing in both groups

691

Fig. 1 Effect of acute exposure to cold on specific uncouplingprotein 1 (UCP1) content of IBAT in male (open bars) and female(shaded bars) rats. Rats were treated as indicated in Table 1 and1–2 µg of IBAT mitochondrial protein was used for immunoblotanalysis of UCP1, as described in Materials and methods, and theUCP1 content was quantified by densitometric scanning of immu-noblots. Mean value of male rats at 22°C was set to 100 and theother values expressed relative to this. Data represent means ±SEM (4°C, n=5–6; 22°C, n=11–12). Significant differences(P<0.05), Student’s t-test: *, males versus females; ∆, 22°C versus4°C. ANOVA significances (P<0.05): T, effect of temperature;SxT, interaction of sex and temperature

Parameter Temperature (°C) ANOVA

22 4

Males Females Males Females

Body mass (g) 465±13 269±3* 502±28 274±6* SIBAT mass (mg) 296±19 300±15 324±42 301±23Total protein (mg/g of tissue) 32.1±2.8 44.2±3.0* 29.3±4.2 49.0±2.1* SMitochondrial protein 15.0±1.2 25.7±0.8* 17.7±1.0 33.4±2.5*∆ S, T(mg/g of tissue)Specific COX activity 3.17±0.16 2.99±0.06 2.90±0.23 2.90±0.24(units/mg of mitochondrial protein)

Table 1 Effect of acute exposure to cold on body mass and inter-scapular brown adipose tissue (IBAT) mass, total and mitochondri-al protein concentration and specific cytochrome oxidase c (COX)activity in male and female rats. Rats were preacclimated to 22°Cand remained at 22°C or were transferred to 4°C for 24 h. Units ofCOX activity are given as µmol of ferrocytochrome oxidized/min.Data represent means ± SEM (n=5–6 for the groups at 4°C and

n=11–12 for the groups at 22°C). Data of animals at 22°C werecombined with those obtained at the same temperature in the sec-ond in vivo experiment (Table 2), as the treatment was the sameand no differences between both groups were found. Significantdifferences (P<0.05), Student’s t-test: *, males versus females; ∆,22°C versus 4°C. ANOVA significance differences (P<0.05): S,effect of sex; T, effect of temperature

of rats after exposure to 4°C (Fig. 3). At 22°C, femalerats had higher UCP1 mRNA levels than male rats at thesame temperature, although the difference was not aspronounced as the protein content. At 4°C, no changes inUCP1 mRNA levels were found between the sexes. Thefact that female rats acclimated to 22°C had a UCP1 pro-tein content twofold higher than that of male rats, whileshowing UCP1 mRNA levels increased only by 1.3-foldwould mean that the increased protein levels could notbe the result only of increased gene transcription but alsoof a different specific mRNA and/or protein turnover[20, 24]. In fact, differences in the half-life of UCP1 incultured primary brown adipocytes, depending on theduration of the adrenergic stimulation, and in intactmice, depending on the degree of exposure to the cold,have been previously described [24].

All results obtained so far concerning ucp1 gene ex-pression and UCP1 levels and activity (GDP binding)show a coincident profile, in that at 22°C female ratshave a higher relative thermogenic capacity than malerats, while when acclimated to 4°C the differences be-tween the sexes practically disappears.

Two possibilities may account for the results. Thefirst is that female rats might have a low threshold tem-perature for cold-induced thermogenic response, i.e.,their BAT is already activated at 22°C, whereas male ratsare not (or are less) sensitive to the cold at this tempera-ture. The second consideration raises the possible effectof sex steroids on UCP1 synthesis. Both aspects wereconsidered in consecutive experiments.

In vitro study of the effects of sex steroidson UCP1 synthesis in brown adipocytes

The fact that female rats have a higher thermogenic capac-ity (at 22°C) than males apparently contradicts the results

of Puerta et al. [21] who showed that brown adipocytesisolated from β-estradiol-treated rats have an impaired res-piration rate in response to norepinephrine treatment. Inorder to confirm whether there is a direct effect of estradi-ol upon the adipocyte and whether it could affect UCP1synthesis, we performed the following in vitro experi-ment, in which UCP1 appearance in response to norepi-nephrine treatment was measured in steroid-treated brownadipocytes differentiated in primary culture.

In rats it has not yet been possible to differentiatebrown adipocytes in primary culture that have the capac-ity to express UCP1. However, Néchad et al. [16] de-scribed reproducible conditions for mice. Thus we isolat-ed preadipocytes from male mice allowing them to pro-liferate and differentiate in primary culture (see Materi-als and methods) in the presence of different concentra-tions of sex steroids. Either β-estradiol, estrone or pro-gesterone (10–5 M) was added from day 3 after inocula-tion and cells were harvested at confluence (day 7) after24 h of UCP1 synthesis stimulation by different doses ofnorepinephrine (Fig. 4). In all cases, the maximum UCP1response to norepinephrine was obtained at 10–7 M, as re-ported for ucp1 gene expression [26], with all steroidhormones tested exerting an inhibitory effect. The maxi-mum inhibition was found for β-estradiol, whose inhibi-tory effect was seen at different concentrations (Fig. 5).These results provide evidence that the described depres-sive effect of β-estradiol upon thermogenesis [21] can beexplained through a direct effect on UCP1 synthesis.

However, the above considered sex-associated differ-ences in UCP1 levels in vivo cannot be directly explainedin the light of differences in sex steroids. Other factorsmust be involved in determining the threshold tempera-ture and the degree of sensitivity to cold in both genders.

692

Fig. 2 Effect of acute exposure to cold on specific GDP-bindingto mitochondria of interscapular brown adipose tissue (IBAT) inmale (open bars) and female (shaded bars) rats. Rats were treatedas indicated in Table 1. Data represent means ± SEM (4°C, n=5–6;22°C, n=11–12). Significant differences (P<0.05), Student’s t-test:*, males versus females, ∆, 22°C versus 4°C. ANOVA significanc-es (P<0.05): S, effect of sex; T, effect of temperature; SxT, interac-tion of sex and temperature

Fig. 3 Effect of acute exposure to cold on specific UCP1 mRNAlevels of IBAT in male (open bars) and female (shaded bars) rats.Rats were treated as indicated in Table 1 and 5 µg of total RNAisolated from IBAT was hybridized with DNA probes to UCP1and β-actin, as described in Materials and methods, and the spe-cific mRNA content was quantified by densitometric scanning ofthe slot-blots. Ratios of UCP1 mRNA to β-actin mRNA were cal-culated. Mean value of male rats at 22°C was set to 100 and theother values expressed relative to this. Data represent means ± SEM(4°C, n=5–6; 22°C, n=11–12). Significant differences (P<0.05),Student’s t-test: *, males versus females. ANOVA significances(P<0.05): T, effect of temperature

UCP1 levels in BAT in male and female ratsafter chronic exposure from 22°C to thermoneutrality(28°C) and a lower temperature (18°C)

In view of the different UCP1 response of male and fe-male rats to changes of environmental temperature we de-cided to study the effect of chronic acclimation (7 days)on both groups of rats from 22°C to the temperature ofthermoneutrality (28°C) and to a lower temperature(18°C).

As shown in Table 2, chronic exposure to 18°C led toan increase of IBAT mass in both groups of rats, albeitonly significant for male rats, without affecting theirbody mass. A general tendency of increasing total and

mitochondrial protein concentration in IBAT was associ-ated with the decrease in environmental temperature inboth groups of rats, although, with reference to thermo-neutrality, male rats needed a decrease in the temperatureto 18°C in order to increase both parameters, but in fe-male rats the increase was already produced at 22°C.Thus, at 28°C, no differences were found in total and mi-tochondrial protein concentration in IBAT between maleand female rats, while at 22°C and 18°C both parameterswere significantly higher in female than in male rats.

Figure 6 depicts specific UCP1 levels in the differentexperimental groups. Both male and female rats had, af-ter 7 days at 18°C, higher specific UCP1 levels thanthose acclimated to 28°C or 22°C. Moreover, although at

693

Fig. 4 In vitro effect of sex steroids on norepinephrine-stimulatedappearance of UCP1 in brown adipocytes. Cultured cells weretreated from day 3 with 17β-estradiol (●●), estrone (■)or proges-terone (■■) (10–5 M) and from day 6 with different concentrationsof norepinephrine (10–9–10–5 M). Control cells received the nor-epinephrine treatment only (●). After 24 h of norepinephrinestimulation, cells were harvested and 20 µg of protein were usedfor immunoblot analysis of specific UCP1, as described in Materi-als and methods. Values are expressed as a percentage of the max-imum specific UCP1 value, which was set to 100%. Data repres-ent means of 2–3 experiments performed in duplicate

Fig. 5 In vitro effect of 17β-estradiol on norepinephrine-stimulat-ed UCP1 appearance in brown adipocytes. Cultured cells weretreated from day 3 with different concentrations of 17β-estradiol(βE): 10–7 M (■), 10–6 M (■■) or 10–5 M (●●) and from day 6 withdifferent concentrations of norepinephrine (10–9 to 10–5 M). Con-trol cells received the norepinephrine treatment only (●). After 24h of norepinephrine stimulation, cells were harvested and specificUCP1 analysed as in Fig. 4. Values are expressed as percentage ofthe maximum specific UCP1 value, which was set to 100%. Datarepresent means ± SEM of 3 experiments performed in duplicate

Table 2 Effect of chronic exposure to different temperatures onbody mass and IBAT mass and total and mitochondrial proteinconcentration in male and female rats. Rats were preacclimated to22°C and remained at 22°C or were transferred to 28°C or to 18°Cfor 7 days. Data represent means ± SEM (n=5–6 for groups at28°C and 18°C and n=11–12 at 22°C, as indicated in Table 1) and

analyzed by one- and two-way ANOVA. Significant differences(P<0.05), Student’s t-test: *, males versus females; ∆, superindex,differences between temperatures. ANOVA significances(P<0.05): S, effect of sex; T, effect of temperature; SxT, interac-tion of sex and temperature

Parameter Temperature (°C) ANOVA

28 22 18

Males Females Males Females Males Females

Body mass (g) 433±13 271±10* 465±13 269±3* 512±13 281±2* S, T, SxTIBAT mass (mg) 309±33 286±30 296±19 300±15 430±35 325±12* T

∆22,28

Total protein 36.6±1.5 36.9±3.3 32.1±2.8 49.2±5.9* 42.7±2.3 68.3±10.0* S, T, SxT(mg/g of tissue) ∆22 ∆22,28

Mitochondrial protein 12.9±0.7 14.0±1.4 15.0±1.2 25.7±0.8* 20.5±0.96 38.0±2.1* S, T, SxT(mg/g of tissue) ∆28 ∆22,28 ∆22,28

28°C there was no difference in UCP1 levels betweenmales and females, at both 22°C and 18°C UCP1 levelswere significantly higher in female rats than in male rats.This implies that at 22°C UCP1 synthesis is alreadystimulated in female rats but not in male rats. The sameconclusion can be deduced from results of specific GDPbinding to mitochondria (Fig. 7).

Changes in UCP1 mRNA levels do not serve solely toexplain the above mentioned sex-associated differencesin specific UCP1 levels and specific GDP-binding to mi-tochondria in response to different environmental tem-peratures, in that there were no differences between sex-es at 28°C and 18°C. However, it is noticeable that at22°C female rats had higher UCP1 mRNA levels thanmales (30% increase) (results not shown).

Thus, data obtained in the first experiment of acclima-tion, i.e., acute exposure to a low temperature, showedthat female rats exposed to 4°C have a lower thermogenicresponse compared with male rats (with 126% increase inUCP1 levels for female rats and 333% for male rats), de-spite the fact that at 4°C there are no differences in spe-cific UCP1 levels between male and female rats. Theseresults, together with those of the second experiment ofacclimation, i.e., chronic exposure to different tempera-tures, proved that in female rats, but not in male rats, thethermogenic system is already responsive to the cold at22°C. Thus, the apparent temperature lag in the male rat’sresponse to the cold suggests that they might have a low-er threshold temperature for cold-induced thermogenesisthan females.

Besides norepinephrine, there are other factors in-volved in the BAT response to cold which should be con-sidered for sex-dependent differences in UCP1 synthesis.

It is known that modified plasma levels of thyroid hor-mones, insulin and glucocorticoids are followed bychanges in sympathetic nervous system activity andUCP1 activity measured by GDP-specific binding to iso-lated mitochondria (see [15] and references therein).What is more, although sex hormones acting directly onbrown adipocytes produced lower UCP1 levels (see Fig.4), an indirect positive effect via the central nervoussystem has been suggested [22].

Of particular physiological relevance is triiodothyro-nine (T3) which acts upon BAT in connection with norepi-nephrine, since norepinephrine stimulation markedly in-creases type II 5′-deiodinase (5′D-II) activity of brown ad-ipocytes thus allowing high intracellular generation of T3from thyroxine (T4) [30]. T3 amplifies the transcriptionalactivation of the UCP1 gene by norepinephrine [1] andstabilizes the mature UCP1 mRNA [8], in addition to a di-rect norepinephrine-independent stimulation [8]. Differen-tial regulation of these hormones has been describedamong various tissues [29] with higher pituitary T3 andthyrotropin-releasing hormone (TRH) receptors and activ-ity of 5′D-II in the anterior pituitary gland of female ratsthan in male rats [6]. This has been mainly attributed tothe modulatory effect of estrogens which may be responsi-ble for the sex-dependent regulation of thyrotropin (TSH)secretion [6]. More generally, it has been recently shownthat estrogen-induced increases in pituitary total RNA lev-els, which are dependent on new protein synthesis, aregender specific, are inhibited by T3, and may be mediatedvia specific estrogen-induced changes in protein-DNA in-teractions [34]. In addition, it can also be argued that thehigher plasma T3 and T4 levels in the female rats com-pared to male rats [7, 11] could also contribute to the high-er UCP1 levels in the female rats acclimated at 22°C.

In conclusion, our results demonstrate a different sex-associated response in the cold-induced thermogenesis in

694

Fig. 7 Effect of chronic exposure to different temperatures onspecific GDP-binding to mitochondria of IBAT in male (●●) andfemale (●) rats. Rats were treated as indicated in Table 2. Data re-present means ± SEM (18°C, 28°C n=5–6; 22°C, n=11–12). Sig-nificant differences (P<0.05), Student’s t-test: *, males versus fe-males; ∆, superindex, differences between temperatures. ANOVAsignificance differences (P<0.05): S, effect of sex; T, effect oftemperature

Fig. 6 Effect of chronic exposure to different temperatures onspecific UCP1 content of IBAT in male (●●) and female (●) rats.Rats were treated as indicated in Table 2 and 1–2 µg of IBAT mi-tochondrial protein was used for immunoblot analysis of UCP1, asdescribed in Materials and methods, and the UCP1 content wasquantified by densitometric scanning of immunoblots. Mean valueof male rats at 22°C was set to 100 and the other values expressedrelative to this. Data represent means ± SEM (18°C, 28°C n=5–6;22°C, n=11–12). Significant differences (P<0.05), Student’s t-test:*, males versus females; ∆, superindex, differences between tem-peratures. ANOVA significances (P<0.05): S, effect of sex; T, ef-fect of temperature

rats that does not appear to be explained by a direct ac-tion of sex steroids upon the adipocyte. In determiningthe difference in the response of both sexes to the cold,other factors (e.g., thyroid hormones) must be involved,apparently affecting the threshold temperature at whichBAT is stimulated, which seems to be for male rats ataround 18°C and for females at around 22°C, and henceleading to differences in BAT UCP1 appearance betweenboth sexes at different environmental temperatures.

Acknowledgements This work was funded by DGICYT, Minis-terio de Educación y Ciencia, Spain (grants PB94–1178 andPM97-0074 to A.P.) and by the European Commission (ContractNo. ERBCHRXCT940490).

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