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Am J Physiol Endocrinol Metab 292: E340-E346, 2007. First published September 5, 2006; doi:10.1152/ajpendo.00175.2006
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Expression of mitochondrial biogenesis-signaling factors in brown adipocytes is influenced specifically by 17beta-estradiol, testosterone, and progesterone

S. Rodríguez-Cuenca,1 M. Monjo,2 M. Gianotti,1 A. M. Proenza,1 and P. Roca1

1Grup de Metabolisme Energètic i Nutrició, Departament de Biologia Fonamental i Ciències de la Salut, Institut Universitari d'Investigació en Ciències de la Salut, Universitat de les Illes Balears, Palma de Mallorca, Spain; and 2Department of Biomaterials, Institute for Clinical Dentistry, University of Oslo, Oslo, Norway

Submitted 12 April 2006 ; accepted in final form 28 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Control of mitochondrial biogenesis in brown adipose tissue (BAT), as part of the thermogenesis program, is a complex process that requires the integration of multiple transcription factors to orchestrate mitochondrial and nuclear gene expression. Despite the knowledge of the role of sex hormones on BAT physiology, little is known about the effect of these hormones on the mitochondrial biogenic program. The aim of this study was to determine the effect of testosterone, 17beta-estradiol, and progesterone on the expression of nuclear factors involved in the control of mitochondrial biogenesis and thermogenic function such as ppar{gamma}, pgc1{alpha}, nrf1, gabpa, and tfam, and also an inhibitor of PI3K-Akt pathway, recently found to be involved in the control of mitochondrial recruitment (pten). For this purpose, an in vitro assay using cell-cultured brown adipocytes was used to address the role of steroid hormones, progesterone, testosterone, and 17beta-estradiol on the mRNA expression of these factors by real-time PCR. Thus 17beta-estradiol seemed to exert a dual effect, activating the PI3K-Akt pathway by inhibiting pten mRNA expression and also inhibiting nrf1 and tfam mRNA expression. Progesterone seemed to positively stimulate mitochondriogenesis and BAT differentiation by increasing the mRNA expression of the gabpa-tfam axis and ppar{gamma}, respectively, but also exerted a negative output by increasing pten mRNA levels. Finally, testosterone inhibited the transcription of pgc1{alpha}, the master factor involved in UCP1 expression and mitochondrial biogenesis. In conclusion, our results support the idea that sex hormones have direct effects on different mediators of the mitochondriogenesis program.

brown adipose tissue; sex steroids; mitochondriogenesis program signaling; nuclear and mitochondrial transcription factors

BROWN ADIPOSE TISSUE (BAT) is a specialized tissue that, in small mammals and newborns, is responsible for nonshivering thermogenesis (15, 39), the main mechanism for thermoregulatory heat production, with uncoupling protein-1 (UCP1) as the principal mediator (4, 5, 23). UCP1 is an inner-membrane mitochondrial protein whose function is to uncouple the respiratory chain from ATP synthesis by dissipating the proton gradient generated by the respiratory chain as heat (32).

The expression of uncoupling proteins and the regulation of mitochondrial biogenesis are critical points for the understanding of the transcriptional basis of adaptive thermogenesis to meet environmental and physiological stimuli such as cold exposure, diet, infection, exercise, and oxidative stress (20).

Control of mitochondrial biogenesis is a complex biological process that requires the integration of multiple transcription factors to orchestrate the programs of mitochondrial and nuclear gene expression involved in energy production (12, 37).

The nuclear respiratory transcriptional factors NRF1 and GABPA (homologous to the human NRF2) are involved in the expression of many genes related to mitochondrial function and biogenesis, including those encoding subunits of the five respiratory complexes and the mitochondrial transcription factor A (TFAM), a nuclear encoded transcription factor that has been considered indicative of mitochondrial differentiation (9, 11, 19, 37).

Peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) is a transcriptional coactivator that has the dual action of stimulating the expression and the transcriptional function of NRF1 and NRF2 (25, 47). Moreover, PGC-1{alpha} participates in the induction of UCP1 expression by interacting with members of the nuclear receptor superfamily such as PPAR{alpha} (1, 46) and PPAR{gamma} (30). Thus PGC-1{alpha} coordinates mitochondrial biogenesis and adaptive thermogenesis response in BAT (3) through the coactivation of NRFs (47) and PPARs (38) and plays an essential role in differentiation-induced mitochondrial biogenesis (43).

The PI3K-Akt (phosphoinositide 3-kinase-Akt murine thyoma viral oncogene homolog 1) pathway, which is linked to radical oxygen species and insulin signaling, converges with several other pathways in the regulation of NRF expression and function (41), thus suggesting the involvement of this pathway and others in the control of mitochondrial biogenesis in response to environmental and physiological stimuli (20) and also in UCP1 expression (44). Thus phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which inhibits the PI3K-Akt pathway, arises as an important modulator involved in mitochondrial biogenesis and the thermogenesis program.

Sex differences in BAT thermogenic capacity between males and females have been described (31, 33, 35, 36). In particular, one of the most important differences was related to mitochondrial recruitment, where female BAT showed larger mitochondria and higher cristae density compared with males (36). To this extent, 17beta-estradiol, testosterone, and progesterone have been proposed as key factors that could account, in part, for these differences. Thus, for example, previous data demonstrated that these hormones modify beta-adrenergic signaling and ucp1 expression in brown adipocytes (24, 34), suggesting the importance of these hormones in the thermogenic program.

To date, some studies have demonstrated effects of androgens and estrogens on gene expression of some subunits of mitochondrial enzymes (10, 45). In the case of estrogens, it has also been demonstrated to increase some mitochodrial encoded genes directly acting on mitochondrial (mt)DNA (6–8). Nevertheless, the information currently available as to the direct effect of 17beta-estradiol, testosterone, and progesterone on BAT mitochondrial recruitment is scarce.

The aim of this study was to determine the effect of 17beta-estradiol, testosterone, and progesterone on the expression of some nuclear factors that are part of the circuits controlling mitochondrial biogenesis and thermogenic function, (16) such as PPAR{gamma}, PGC-1{alpha}, NRF1, GABPA, and TFAM, and also the inositol phosphatase PTEN. For this purpose, an in vitro assay using cell-cultured brown adipocytes was used to address the role of the steroid hormones progesterone, testosterone, and 17beta-estradiol on the mRNA expression of these factors per se but also in cultured adipocytes challenged with norepinephrine (NE) that modulates the expression of several mitochondrial transcription factors (47, 21) and is a key hormone involved in the recruitment of BAT. We hypothesize that sex hormones could exert a coordinate effect with NE in the mitochondrial recruitment.

In addition, we also investigated the expression of these factors in vivo in male and female BAT to analyze its expression as a possible mechanism underlying the sex differences in the thermogenic and mitochondrial recruitment process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Testosterone, 17beta-estradiol, and progesterone were from Sigma (St. Louis, MO). Other cell culture reagents were supplied by Sigma, Cultek (Madrid, Spain), and GIBCO-BRL (Gaithersburg, MD); RNA isolation and PCR chemicals were from Roche Diagnostics (Basel, Switzerland), and routine chemicals were from Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain). The microplate spectrophotometer was supplied by BioTek Instruments (Winooski, VT); the Gene Amp 9700 thermal cycler was supplied by Applied Biosystems (Foster City, CA), and the Lightcycler thermal cycler was supplied by Roche (Basel, Switzerland).

Animals and isolation of BAT. Three-month-old male and female Wistar rats (n = 14) were used for the in vivo assay (obtained from Charles River Laboratories, Wilmington, MA). Animals were housed at 22°C, with a 12:12-h light-dark cycle, with free access to drinking water and standard chow pellets (Panlab, Barcelona, Spain). Animals were killed by decapitation at the start of the light cycle, and interscapular BAT was rapidly removed, frozen in liquid N2, and stored at –70°C until RNA isolation. Animal experiments were performed in accordance with general guidelines approved by our institutional ethics committee and European Union regulations (86/609/EEC).

Primary cultures of brown adipocytes. Brown fat precursor cells were isolated from 4-wk-old male NMRI mice (supplied by Charles River Laboratories) as previously described (26). The cervical, interscapular, and axillar BAT depots were dissected out from each mouse under sterile conditions. The tissue was pooled and incubated in HEPES buffer (pH 7.4, 2 ml/mouse), containing 0.2% (wt/vol) crude collagenase type II. The tissue was digested for 30 min at 37°C and vortexed every 5 min. The digest was poured through a 250-µm silk filter into 10-ml sterile tubes. The solution was then cooled at 4°C for 15–30 min to allow the mature brown fat cells and lipid droplets to float. The infranatant was filtered through a 30-µm silk filter into 10-ml sterile tubes, and precursor cells were collected by centrifugation for 10 min at 700 g, washed in DMEM, pelleted again, and resuspended in 0.5 ml of culture medium per mouse. Two tenths of a milliliter of pooled final precursor cell suspension were inoculated in six-well plates, each well containing 1.8 ml of culture medium. This was day 0. From that moment on, cells were incubated at 37°C in 8% CO2.

For the first 6 days, the preadipocytes were grown in 2 ml of a medium consisting of DMEM supplemented with 10% newborn calf serum, 4 nM insulin, 4 mM glutamine, antibiotics (50 IU/ml penicillin and 50 µg/ml streptomycin), 10 mM HEPES, and 25 µg/ml sodium ascorbate. This medium-containing serum was changed on day 1 (cells were previously washed with DMEM) and day 3. On day 6, the medium was discarded, and a serum-free medium was added consisting of DMEM-F-12 (1:1), 0.5% free fatty acid bovine serum albumin, 4 nM insulin, 4 mM glutamine, antibiotics (50 IU/ml penicillin and 50 µg streptomycin), 10 mM HEPES, and 25 µg/ml sodium ascorbate.

The different treatments were carried out on day 6, when cells presented a differentiated morphology and lipid accumulation. Serum-free medium was used to avoid hormonal interference. Testosterone, 17beta-estradiol, or progesterone (from 10–9 to 10–7 M) were dissolved in ethanol and added to the corresponding wells, with the final ethanol concentration never exceeding 0.01%. An equivalent volume of ethanol was added to untreated controls. On day 7, after 24-h treatment, the cells were exposed to 10–7 M NE for 6 h. Then, the cells were harvested with TriPure for RNA isolation.

RNA isolation. Total RNA was isolated from BAT and cells by use of TriPure reagent, following the instructions of the manufacturer. RNA was determined in triplicate using a microplate spectrophotometer set at 260 nm.

Reverse transcription. Total RNA (1 µg) was reverse transcribed to cDNA at 42°C for 15 min with 25 U MuLV reverse transcriptase in a 10-µl volume of RT reaction mixture containing 10 mM Tris·HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 2.5 µM random hexamers, 10 U RNAase Inhibitor, and 500 µM of each dNTP in a DNA Gene Amp 9700 thermal cycler.

Real-time RT-PCR. The primers used were designed using specific primer analysis software Primer3 (Whitehead Institute for Biomedical Research) and Oligo Analyzer 3.0 (Integrated DNA Technologies), and the specificity of the sequences was analyzed by Fasta in the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/) (Table 1). Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics). Reactions were performed per duplicate in a 10-µl volume containing 3 µl of cDNA sample (diluted 1:10) using 0.5 µM primers, 3 mM of MgCl2, and dNTPs, Taq DNA polymerase, and reaction buffer provided in the LightCycler FastStart DNA Master SYBR Green I mix. All real-time conditions are summarized in Table 1. Product specificity was confirmed in initial experiments by agarose gel electrophoresis and routinely by melting curve analysis. For mathematical analysis, the crossing points (CP) values were used for each transcript. CP is defined as the point at which fluorescence of the transcript rises appreciably above the background fluorescence. The "fit point method" was performed in the LightCycler software 3.3, at which CP was measured at a constant fluorescence level. PCR efficiencies of each amplicon (between 1.7 and 1.9 in our study) were calculated using the following formula: E = (F/F0)Formula , where n and n0 were the crossing point values of F and F0, fluorescence signals of the linear phase of the logarithmic amplification curve.


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  		Table 1. Oligonucleotide primer sequences and amplification conditions

 
Statistics. Statistical PCR data analysis was performed using the Relative Expression Software Tool (REST). The statistical model used was the Pair Wise Fixed Reallocation Randomisation Test (28). Differences in expression between groups, expressed as fold change, were assessed using the means for statistical significance by randomization tests, a proper model to avoid the normal distribution assumption of the data. The level of probability was set at P < 0.05 as statistically significant. This method included the correction of data using 18S that was validated as housekeeping gene.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sex differences in gene expression of mitochondrial biogenic factors in BAT. Female BAT showed a lower expression of nrf1 (0.5-fold change, P = 0.03) compared with males (Fig. 1). Similarly, gabpa was also lower in females compared with males, although it did not reach statistical significance (0.5-fold change, P = 0.09). tfam, a transcription factor regulated by NRFs, followed the same pattern (0.5-fold change in females) but failed to be significant (P = 0.37). There were no differences in the expression of ppar{gamma} (P = 0.24) or pgc1{alpha} (P = 0.24). pten expression was markedly lower in female BAT compared with males (0.4-fold change, P < 0.02).


Figure 1
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  		Fig. 1. Sex differences in the mRNA expression of ppar{gamma}, pgc1{alpha}, nrf1, gabpa, tfam, and phosphatase pten. See text for definitions. mRNA expression of mitochondrial biogenesis-related factors in male and female brown adipose tissue (n = 7). Data represent fold changes of 7 samples/group and were established using each control as 1. Differences in expression between groups were assessed by Pair Wise Fixed Reallocation Randomisation Test. Level of probability was set at P < 0.05 as statistically significant. *Significant differences between males and females.

 
17beta-Estradiol, testosterone, and progesterone have a direct and differential effect on mitochondrial biogenic factor gene expression in cultured brown adipocytes.

Progesterone treatment. Progesterone induced an upregulation of tfam mRNA levels at maximum dosage in both the absence and presence of NE (1.6-fold change, P = 0.02 and P = 0.04, respectively; Fig. 2). This increase was accompanied by an increase in gabpa, although this only reached statistical significance in combination with NE (1.9-fold change, P = 0.05). No changes were observed for nrf1. This progesterone effect in the gabpa-tfam axis was not reflected either in its upstream link pgc1{alpha}.


Figure 2
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  		Fig. 2. Effect of progesterone treatment on mRNA expression in primary culture of brown adipocytes. Differentiated brown adipocytes were either nontreated (open bars) or stimulated (filled bars) for 6 h with norepinephrine (NE; 10–7 M) after 24-h treatment with progesterone (10–9 to 10–7 M). Data represent fold changes of 5–6 samples/group and were established using each control as 1. Differences in expression between groups were assessed by Pair Wise Fixed Reallocation Randomisation Test. Level of probability was set at P < 0.05 as statistically significant. *Significant differences between hormone-treated vs. control (or sex hormone or NE treatment).

 
Progesterone treatment increased the mRNA expression of ppar{gamma} at maximum concentration (2.4-fold change in the absence of NE, P = 0.04). Along the same lines, progesterone exerted an upregulation of pten mRNA in combination with NE treatment (1.4-fold change, P = 0.04).

17beta-Estradiol treatment. Estradiol treatment brought about a dowregulation of nrf1 (Fig. 3), although this was significant only at the lower dosage tested in combination with NE (0.6-fold change, P = 0.04). gabpa was also downregulated, although differences were not statistically significant (P = 0.19). Likewise, tfam mRNA expression was also downregulated at the lower dosage tested in combination with NE (0.5-fold change, P = 0.03). pgc1{alpha} and ppar{gamma} mRNA levels were not modified under estradiol treatment, although ppar{gamma} showed a tendency to be downregulated.


Figure 3
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  		Fig. 3. Effect of 17beta-estradiol treatment on mRNA expression in primary culture of brown adipocytes. Differentiated brown adipocytes were either nontreated (open bars) or stimulated (filled bars) for 6 h with NE (10–7 M) after 24-h treatment with 17beta-estradiol (10–9 to 10–7 M). Data represent fold changes of 5–6 samples/group and were established using each control as 1. Differences in expression between groups were assessed by Pair Wise Fixed Reallocation Randomisation Test. Level of probability was set at P < 0.05 as statistically significant. *Significant differences between hormone-treated vs. control (or sex hormone or NE treatment).

 
pten mRNA expression was downregulated under estradiol treatment (0.8-fold change, P < 0.02) in both the absence and presence of NE (10–7 M).

Testosterone treatment. Testosterone exerted a downregulation of pgc1{alpha} in the absence of NE (~0.4-fold change, P < 0.04) at all the concentrations tested except for 10–8 M, which did not reach statistical signficance; Fig. 4). NE treatment in the presence of testosterone seemed to reverse the androgen effects. Nevertheless, this effect was significant only at maximum testosterone dosage (~2-fold with respect to non-NE-treated cells). Moreover, testosterone did not exert any effect on pgc1{alpha} downstream targets nrf1, gabpa, or tfam mRNA expression.


Figure 4
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  		Fig. 4. Effect of testosterone treatment on mRNA expression in primary culture of brown adipocytes. Differentiated brown adipocytes were either nontreated (open bars) or stimulated (filled bars) for 6 h with NE (10–7 M) after 24-h treatment testosterone (10–9 to 10–7 M). Data represent fold changes of 5–6 samples/group and were established using each control as 1. Differences in expression between groups were assessed by Pair Wise Fixed Reallocation Randomisation Test. Level of probability was set at P < 0.05 as statistically significant. *Significant differences between hormone-treated vs. control (or sex hormone or NE treatment); #significant differences between hormone-treated vs. hormone-treated plus NE.

 
No effect was observed after testosterone treatment on ppar{gamma} levels, although a tendency to be downregulated was observed. Testosterone treatment also induced a downregulation of pten levels at maximum concentration, but this was reversed in the presence of NE.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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Recent studies have provided insights into the pathways regulating mitochondrial biogenesis. The evidence supports a model where regulated coactivators communicate physiological signals to specific transcription factors. These events result in the activation of the genes needed for mitochondrial biogenesis and respiration function (16). In this paper, we provide evidence that 17beta-estradiol, progesterone, and progesterone signaling might play an important role in the modulation of factors involved in the mitochondriogenesis program.

In vivo, female BAT showing lower nfr1 and tfam mRNA expression could apparently be in discrepancy with the higher mitochondrial recruitment and function and higher beta-adrenergic signaling depicted in female BAT (36). Nevertheless, the higher female mitochondrial recruitment could, at least in part, be explained by the lower pten phosphatase mRNA expression in female BAT compared with male despite the low levels of nfr1-tfam mRNA, since the activation of PI3K-Akt pathway, which is inhibited by PTEN phosphatase (18), brings about the phosphorylation and nuclear translocation of NRF1 binding to TFAM and finally increase in mtDNA (29, 41) and UCP1 expression (44). It is relevant to consider the overall effect of these different signal transduction pathways to address the net signal input in the mitochondrial program.

Progesterone treatment exerted an upregulation of tfam mRNA in brown adipocytes in vitro. These results are supported in vivo by a strong correlation between serum progesterone and TFAM protein in BAT of female rats (Frontera M, unpublished data). The upregulation of tfam mRNA by progesterone could be due to the increased gabpa mRNA levels and strongly suggest that progesterone, by means of a coordinated rise in gabpa and tfam, would lead to an enhanced replication and transcription of mtDNA, at least in vitro, conducting to the stimulation of the mitochondriogenesis program. Nevertheless, it must be considered that mRNA expression does not necessary reflect activation of these factors in the frame of the mitochondrial biogenesis.

Moreover, the increase in ppar{gamma} expression found, which could be mainly ppar{gamma}2 (27), after progesterone treatment, could be responsible for the induction of ucp1 mRNA expression (34) and for promoting the differentiation of brown adipocytes (42). Nevertheless, progesterone treatment in adipocytes challenged with NE treatment also brought about an upregulation of pten mRNA levels, suggesting that progesterone may also exert a negative input in the stimulation of mitochondrial program by inhibiting the PI3K-Akt pathway.

17beta-Estradiol seemed to exert a dual effect on the mitochondrial biogenic program. First is inhibiting it, through the downregulation of nrf1, gabpa, and tfam mRNA expression showed in brown adipocytes stimulated with NE. Thus 17beta-estradiol effect may explain, at least partially, the lower nrf1 and tfam mRNA levels shown by female BAT, which is more beta-adrenergic activated than male (36). Second, 17beta-estradiol also exerted a downregulation of pten mRNA levels in cell-cultured brown adipocytes, suggesting an activation of the PI3K-Akt pathway and therefore a mitochondrial recruitment-stimulatory signal. Recently, estradiol has been demonstrated to inactivate PTEN activity by phosphorylation and, hence, influence PTEN activity, suggesting both a chronic and an acute control mechanism of PTEN by estrogens (14). To this extent, it is tempting to speculate that the estradiol signal could be responsible for the sex differences in pten mRNA expression and, hence, contribute to the differences between the sexes in mitochondrial biogenesis and thermogenic program. Nevertheless, it is worth noticing that 17beta-estradiol and testosterone may not necessarily reflect a male or female profile in the mitochondrial biogenesis program framework (22, 40).

Testosterone treatment triggered a marked downregulation of pgc1{alpha} mRNA levels. As PGC-1{alpha} is also directly involved in the transcriptional control of UCP1 (1), this androgen-induced downregulation in pgc1{alpha} mRNA levels could be related to the drop of ucp1 mRNA expression observed in a similar experimental model (34). No coordinate downregulation was observed in either nrf1 or gabpa mRNA, two PGC-1 downstream targets, indicating a possible effect of sex hormones on its expression independently of PGC-1 action. Thus it could be hypothesized that testosterone could exert its negative effect on thermogenesis mainly by affecting a short-term response such as UCP1 recruitment rather than a long-term response such as the mitochondrial biogenesis process, although we cannot discard that the hormonal exposure time is not enough to see this coordinated effect mediated on NRFs by PGC-1{alpha}. To this extent, it has been recently discovered that this coactivator can interact with androgen and estrogen receptors (17), suggesting that sex hormones can exert a role in the PGC-1{alpha} function in addittion to transcriptional mechanisms. Curiously, although pgc1{alpha} mRNA is strongly induced by beta-adrenergic agonists (13, 30) and also by physiological conditions known to increase ATP or heat demand, such as exercise and cold exposure, our study failed to show any modulation by NE at the concentration or time exposure used, although these conditions were enough to stimulate UCP1 transcription in a similar model (34). Nevertheless, NE reverted the downregulation of pgc1{alpha} under testosterone treatment.

Similarly to 17beta-estradiol, a high dosage of testosterone induced a downregulation of pten mRNA expression. Since testosterone, but not dihydrotestosterone, was used in this experiment, we cannot discard that the testosterone effect may in fact have been due to the conversion of testosterone to estradiol by means of aromatase (2). PTEN has been demonstrated to be physiologically downregulated under cold exposure, a situation where beta-adrenergic signaling is activated. However, in our study, no regulation of pten mRNA under NE treatment was reached except in combination with testosterone, where an upregulation of pten mRNA was observed. Further research is needed to clarify this point.

In summary, we have demonstrated that 17beta-estradiol, testosterone, and progesterone play a role in the control of mitochondrial biogenesis by modifying the mRNA expression of several mitochondrial transcription factors and other upstream links of specific signaling pathways. To date, it is tempting to speculate that these changes could, at least in part, be responsible for the sex differences in the mitochondrial recruitment process. The characterization of these signaling pathways is a challenging work that will fuel more research in the study of mitochondrion biology.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Fondo de Investigaciones Sanitarias of the Spanish Government (PI 021339, PI 042294, PI 042377) and Comunitat Autònoma de les Illes Balears (PRDIB-2002GC4-24). S. Rodríguez-Cuenca was funded by a grant from the University of the Balearic Islands.


   ACKNOWLEDGMENTS
 
Current affiliation of S. Rodríguez-Cuenca: Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Roca, Departament de Biologia Fonamental i Ciències de la Salut, Ed. Guillem Colom. Universitat de les Illes Balears, Cra. Valldemossa, Km 7.5, 07122-Palma de Mallorca, Spain (e-mail: pilar.roca{at}uib.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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