Unidad de Endocrinología Molecular, Instituto de Investigaciones Biomédicas, Centro Mixto "Alberto Sols," Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28029 Madrid, Spain
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ABSTRACT |
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Type II 5'-iodothyronine deiodinase
(D2), produces triiodothyronine (T3) and is
stimulated by cold exposure via norepinephrine (NE) release in brown
adipose tissue. Cultured rat brown adipocytes require T3
for the adrenergic stimulation of D2 activity. D2 mRNA expression in
cultured brown adipocytes is undetectable with the use of basal
conditions or NE without T3. Full D2 expression is achieved
using NE + T3, especially after prolonged
T3 exposure. 3-Adrenergic agonists mimic the
NE action, whereas cAMP analogs do not. Prolonged exposure to
T3 alone increases D2 mRNA. High T3 doses (500 nM) inhibit the adrenergic stimulation of D2 activity while increasing
D2 mRNA. The effects obtained with NE + T3 or T3 alone are suppressed by actinomycin, but not by
cycloheximide, which leads to accumulation of short D2 mRNA
transcripts. Prolonged or short exposure to T3 did not
change D2 mRNA half-life, but T3 seemed to elongate it. In
conclusion, T3 is an absolute requirement for the
adrenergic stimulation of D2 mRNA in brown adipocytes. T3
upregulates D2 mRNA, an effect that might involve stimulation of
factors required for transcription or for stabilization of D2 mRNA.
norepinephrine; uncoupling protein-1
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INTRODUCTION |
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BROWN ADIPOSE TISSUE (BAT) is a thermogenic tissue specialized in the production of heat in demanding situations such as cold exposure, during arousal from hibernation, or by the cold experienced after birth. This process is called facultative thermogenesis. The production of heat is accomplished by a mitochondrial protein, called uncoupling protein-1 (UCP1), the specific marker of BAT. Adrenergic stimulation is the main effector for the activation of UCP1, and thyroid hormones, specifically triiodothyronine (T3), have been described as being necessary for the full expression of UCP1 in rats (3, 4). Moreover, T3 in BAT is locally produced from thyroxine (T4) via a deiodinase that plays an important role generating the T3 required for UCP1 expression (5).
The deiodinases are enzymes that regulate thyroid hormone availability in peripheral tissues. Three different deiodinases have been described that catalyze outer- and inner-ring deiodination. Outer-ring deiodination is a key pathway of thyroid hormone metabolism that leads to the production of T3, whereas inner-ring deiodination results in the formation of inactive compounds. Most of the T3 present in tissues is produced from T4 via outer ring 5'-deiodination. Two isoenzymes catalyze this activating pathway: type I and type II 5'-iodothyronine deiodinases (D1 and D2). D1 and D2 differ in their kinetic characteristics, sensitivity to inhibition by 6-N-propyl-2-thiouracil (PTU), tissular distribution, and response to thyroid status (23). Different from D1, D2 is insensitive to PTU, it prefers T4 as substrate, and its Michaelis-Menten constant is in the nanomolar range. D2 activity is upregulated in hypothyroidism and inhibited by T4 (26, 47, 48). D2 activity is localized in brain, adenohypophysis, BAT, pineal gland, and the maternal side of the placenta, among other tissues (24, 26, 39, 44, 53). D2 activity is regulated in rat tissues, glial cells, or brown adipocytes by a number of factors such as thyroid hormones (26, 27, 47), adrenergic agents (19, 22, 32, 35, 38, 39, 44), cAMP (10, 17, 22), growth factors (11), and insulin (29, 46).
The cDNAs coding for the three deiodinases have been isolated in rat, human, and other species (2, 12, 13). Sequence analysis has demonstrated that all of them contain an in-frame TGA codon that is translated as selenocysteine due to the presence of a specific structure, named selenocysteine insertion sequence (SECIS), in the 3' untranslated region of their mRNAs (2, 6). Analysis of D2 expression in human tissues has shown that D2 is also present in human heart, skeletal muscle, and thyroid (12, 42).
In rat BAT, D2 activity is stimulated by hypothyroidism
(27) and by cold exposure, norepinephrine (NE), and
adrenergic stimuli (44). T3-induced
hyperthyroidism augmented the response to NE (47). These
situations also increase D2 mRNA levels (12). As already
mentioned, D2 activity produces most of the T3 found in BAT
(45), which saturates nuclear T3 receptors,
and the T3 produced is necessary for a complete thermogenic
function, namely the full expression of the uncoupling protein UCP1,
specific marker of BAT (5). Besides, BAT D2 activity is
upregulated by insulin (46), as shown after insulin
injection, or in diabetic rats, as well as in floating rat brown
adipocytes (29). In these cells, D2 activity is stimulated
by adrenergic agents, and a synergy between 1- and
-adrenergic pathways has been described (32, 38),
whereas in cultures of mouse brown adipocytes the main pathway is
-adrenergic (35).
Using primary cultures of rat brown adipocytes, we have previously
shown that D2 activity is poorly stimulated by NE or cAMP analogs and
that T3 is required for and amplifies the adrenergic response 10- to 20-fold (19). The effect of T3
is exerted when NE or 3-adrenergic agents are used and
is only poorly mimicked when cAMP-elevating agents are used. The
stimulatory effect of T3 on the adrenergic responses of D2
activity overcomes the inhibition caused by T4, and such
increases are proportional to the time of preexposure to T3
(19). The T3 + NE effect requires de novo protein synthesis and is fully inhibited by actinomycin.
In the present study, we have studied the regulation of D2 mRNA expression by adrenergic stimulation and by T3 by use of primary cultures of rat brown adipocytes differentiated in culture from preadipocytes. Our results disclose a novel role of T3 in the regulation of D2 mRNA levels, both in cooperation with NE and by itself, indicating the importance of T3 for the brown adipocyte.
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MATERIALS AND METHODS |
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Materials. The source of most of the reagents used has been previously described (21). Newborn calf serum (NCS) was purchased from Flow (Paisley, Scotland) or from GIBCO Life Technologies (Uxbridge, UK). Isoproterenol (ISO) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) were obtained from Sigma Chemical (St. Louis, MO). BRL-37344 was kindly provided by SmithKline Beecham Pharmaceuticals (Welwyn, UK). Ion exchange resins AG1-X8 and AG50W-X2 were obtained from Bio-Rad (Richmond, CA). Oligo(dT) cellulose was obtained from New England Biolabs (Beverly, MA), TRIzol from GIBCO-BRL Life Technologies, AmpliTaq Gold DNA polymerase from Roche Molecular Systems (Branchburg, NJ), and AMV reverse transcriptase (RT) from Promega (Madison, WI).
Cultures of brown adipocytes. Precursor cells were obtained from the interscapular BAT of 20-day-old rats (Sprague-Dawley), isolated according to the method described by Néchad et al. (30) by using collagenase digestion (0.2%) in DMEM + 1.5% BSA at 37°C and filtered through 250-µm silk filters. Mature cells were allowed to float, and the infranatant was filtered through 25-µm silk filters and centrifuged. Precursor cells were seeded in 25-cm2 culture flasks or 145-cm2 plates at a density of 1,500-2,000 cells/cm2 on day 1 and grown in DMEM supplemented with 10% NCS, 3 nM insulin, 10 mM HEPES, 50 IU of penicillin, and 50 µg of streptomycin/ml and 15 µM ascorbic acid. Culture medium was changed on day 1 and every 2-3 days until the experiment was performed. Precursor cells proliferate actively under these conditions, reach confluence on the 4th or 5th day after seeding (40,000-60,000 cells/cm2), and then differentiate into mature brown adipocytes. Most studies and treatments were done in fully differentiated brown adipocytes (on the 9th day after seeding). For deiodinase activity, 25-cm2 flasks were used, and for mRNA studies, 2-3 plates (145 cm2), seeded at the same cellular density, were pooled.
Both NCS and hypothyroid (Hypo) serum were used for culture. The latter was obtained by depleting NCS of thyroid hormones with the anion exchange resin AG1-X8, as described (43). Hypo serum contained ~10% or less of the original amount of thyroid hormones, as assessed by RIA (36). In NCS, concentrations of T4 and T3 were 77 and 1.3 nM, respectively, before its dilution with medium. These levels decreased to 2.2 nM T4 and 0.13 nM T3 in Hypo serum. In some experiments, T3 and T4 concentrations were measured in medium and cells (after purification of the extracts) by means of specific RIAs (36). Free T3 concentrations were measured by ultrafiltration as described (36). Free T3 concentration was 2 or 4% of the total T3 when 10 or 5% serum, respectively, (either NCS or Hypo serum) was used. The actual free T3 concentrations were 5-10 pM when 10% NCS was used and 200-500 pM T3 when 10 nM T3 was added to the culture medium supplemented with 10 or 5% Hypo serum, respectively.RNA preparation and Northern blot analysis.
For the isolation of poly(A)+ RNA, cells were collected and
mRNA isolated using oligo(dT) cellulose as described
(52). For Northern analysis, poly(A)+
(5-7 µg) was denatured and electrophoresed on a 2.2 M
formaldehyde/1% agarose gel in 1× MOPS buffer (pH 7.0) and
transferred to nylon membranes (Nytran) as described (18,
21). A 1.988-kb fragment of the rat D2 cDNA clone
(12), corresponding to the entire coding region of the rat
D2, 580 nucleotides from the 5' region and 30 nucleotides of the 3'
end, was used as a probe by labeling with [-32P]dCTP
with the use of random primers (>108 cpm/µg DNA).
Filters were hybridized for 20 h at 50°C [40% formamide, 20 µg/ml salmon sperm DNA, 50 µg poly(A), and 50 µg poly(C)/ml, 5×
saline sodium citrate (SSC), 2× Denhardt's, 0.1% SDS] and washed four times in 2× SSC/0.2% SDS at room temperature for 15 min and then
twice in 0.1× SSC/0.2% SDS at 65°C for 20 min. Autoradiograms were
obtained from the filters and quantified by laser
computer-assisted densitometry (Molecular Dynamics,
Sunnyvale, CA). The filters were hybridized with cyclophilin as a
control to correct for differences between lanes in the amount of
poly(A)+ mRNA (14). All of the experiments
were repeated 2-3 times, using Northern blot analysis in all. The
more complete or representative experiments are shown in the figures.
RT-PCR assay.
One microgram of total RNA (TRIzol, GIBCO) was used for RT-PCR
amplification under the conditions previously described
(12). We used the same RNA samples without RT reaction as
controls. The PCR reaction conditions were 94°C for 5 min, and 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and
a final 7-min period at 72°C, using AmpliTaq Gold DNA polymerase. The specific oligonucleotides used for amplification of a 590-bp product were: sense, 5'-ACT CGG TCA TTC TGC TCA AG-3' and antisense, 5'-TTC AAA
GGC TAC CCC ATA AG-3', as previously described (12). After gel electrophoresis in 1% agarose, the PCR products were transferred to a filter and hybridized with a radiolabeled, nested oligonucleotide rat D2 probe (AAT GCC ACC TTC TTG ACT TT), using
[-32P]ATP, as previously described (12).
The optimal conditions were chosen to avoid the presence of DNA in the
samples, unspecific annealing in the PCR reaction, etc.
Determination of D2 activity. Cells were scraped, collected in buffer A [0.32 M sucrose, 10 mM HEPES, 10 mM dithiothreitol (DTT), pH 7.0] and homogenized. D2 activities were determined in homogenates by measuring the release of iodide as described (27), with modifications (19, 33), using as final concentrations 2 nM T4 (50.000 cpm [125I]T4), 1 µM T3, 50 mM DTT, 1 mM PTU, 80-100 µg protein/100 µl of total volume, pH 7.0, during 1 h at 37°C. In these conditions, >95% of the activity was insensitive to inhibition with PTU. Each cell homogenate was tested in triplicate using 2-3 culture flasks per treatment. Protein content was determined by the method of Lowry (28) after precipitation of the homogenates with trichloroacetic acid to avoid interference of DTT in the colorimetric reaction (19). Results were expressed in femtomoles per hour per milligram of protein.
The high specific activity [125I]T4 used was obtained in our laboratory (>3,000 µCi/µg) with the use of chloramine T and T3 as substrate (19, 33). Before each assay, [125I]T4 was purified by paper electrophoresis to separate the contaminating iodide, using ammonium acetate 0.05 M, pH 6.8. The amount of iodide in the blanks assay was routinely less than 1% of the total radioactivity. Preliminary experiments were performed to validate the assay: 1) the production of equimolar amounts of iodide and T3, 2) the linear production of iodide by use of increasing amounts of protein, and 3) the within-assay coefficient of variation that was <5%.Statistical analysis. Mean values ± SE are given. One-way analysis of variance (ANOVA) was done, after homogeneity of variance was ensured, using square roots or logarithmic transformations if the homogeneity was not found with the raw data. Significance of differences between groups was assessed using the protected least significant difference test. All calculations were done as described (49).
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RESULTS |
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We have previously shown that, in primary cultures of rat brown adipocytes, the adrenergic stimulation of D2 activity requires the presence of T3 (19). This T3 requirement is similar to the one observed for the adrenergic stimulation of UCP1 mRNA in the same cell model (20).
D2 mRNA induction by NE and T3.
In rat BAT, D2 mRNA presents several bands at 7.5, 4.0, 2.5, and 2.0 kb, the first being the most abundant. D2 mRNA expression is
clearly stimulated in the BAT of cold-exposed rats (12). In the present study, we used Northern blot analysis of
poly(A)+-enriched mRNA from differentiated brown
adipocytes. D2 mRNA expression was undetectable using regular culture
conditions no matter the amount or type of serum used (not shown) or
when the cells were treated with NE alone (Fig.
1A, lanes 1 and 4). The combined addition of NE and T3
led to increases in D2 mRNA expression (lane 3), in parallel
with the findings for the adrenergic stimulation of D2 activity
(19), where T3 was a requirement for NE to
become effective whereas NE or T3 separately had no effect.
Moreover, in the absence of adrenergic stimulation, prolonged exposure
(40 h) to T3 increased D2 mRNA levels (2-fold) (Fig.
1A, lane 2), whereas short exposures to
T3 did not increase D2 mRNA (not shown).
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Induction of D2 activity by NE and T3.
In parallel, we examined the adrenergic stimulation of D2 activity
using different exposure times to T3 (Fig.
2). The presence of T3 per se
(or NE alone, Fig. 2, dashed line) did not increase D2 activity.
Adrenergic increases of D2 activity were larger in cells exposed to
T3 from confluence (72 h) than in those exposed for a
shorter time (24 h) to T3. The effect of T3 was
also examined in the absence of insulin, which increases D2 activity in
BAT, and the results were similar, although lower, indicating that the
T3 effect is independent of insulin. The increases observed in D2 activity after prolonged exposure to T3 were modest
compared with those observed at the mRNA level.
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Effect of high doses of T3.
We then examined whether high doses of T3 (100 nM) inhibit
D2 mRNA in brown adipocytes, as described in pituitary cells
(25). Figure 3A
shows that D2 mRNA expression is not suppressed by a 100 nM dose of
T3 in either the presence or absence of NE. We further
tested in the presence of NE a larger T3 dose (500 nM), which did not inhibit D2 expression but induced further increases in D2
mRNA (2.5-fold vs. 10 nM T3 + NE). T3
concentrations were very high, as measured by specific RIAs (50- and
250-fold increases in the medium and cells, when 100 and 500 nM
T3, respectively, were used vs. 10 nM T3). In
parallel, we tested whether these T3 doses inhibited D2
activity (Fig. 3B). Increasing amounts of T3
inhibited the adrenergic stimulation of D2 activity, effects that were
detectable from 20 nM T3, and maximal inhibition was observed when using 100 nM T3.
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Effect of adrenergic agonists.
We have previously shown (19) that, in cultured brown
adipocytes, the adrenergic responses of D2 activities were mediated mainly by NE, whereas cAMP analogs did not reproduce the NE effect. The
thermogenic responses in BAT have been shown to be mediated via
3-adrenergic receptors (55). We tested the
effect of the
3-adrenergic agonist BRL-37344, compared
with NE and ISO, on D2 activity and D2 mRNA expression (Fig.
4, A and B). Using
dose-response curves, we observed that BRL-37344 is more potent than NE
and ISO at the dose of 0.1 µM (P < 0.05) in the
stimulation of D2 activities (half-maximal concentrations were 0.05 µM for BRL-37344 vs. 0.2 µM for NE and ISO). A plateau was reached
at higher concentrations.
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D2 mRNA inductions are blocked by actinomycin D and increased by
cycloheximide.
We studied whether D2 mRNA adrenergic inductions were dependent on
increases in gene transcription and required de novo protein synthesis.
Cells were exposed for 1 or 3 days to T3 and to NE for
7 h, in the presence or absence of either actinomycin D (Act) or
cycloheximide (CHX) (both added 15 min before NE). We had previously tested the complete inhibition of protein synthesis using these doses
of CHX in culture (20). Addition of Act or CHX for periods longer than 20 h was toxic for the cells. Figure
5A shows that Act inhibits the
induction of D2 mRNA, suggesting that the adrenergic response requires
increases in gene transcription, whereas CHX increases D2 mRNA levels,
indicating that inhibition of de novo protein synthesis for 7 h
results in accumulation of the D2 mRNA, especially of a short mRNA
species (~1.9 kb). Similar results were obtained when T3
was added for a short or a long time (+NE) (Fig. 5A). When
both inhibitors were added to T3 alone (in the absence of
NE), the results were similar to those using NE + T3 (see Fig. 5B). Addition of CHX to cells in basal conditions
or using NE alone did not result in accumulation of mRNA (data not shown), indicating that such an accumulation is found only in the
presence of T3.
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D2 mRNA stability. We explored whether the effect of T3 could be due to stabilization of D2 mRNA transcripts leading to increases in its half-life, as this effect of T3 is observed in other T3-dependent genes in brown adipocytes, such as UCP1, malic enzyme, or S14 (3, 18, 20, 37).
To measure the stability of the D2 mRNA, brown adipocytes were cultured in the presence of T3 for 1 or 3 days and exposed to NE (5 µM) during the last 10 h to stimulate D2 mRNA expression. Thereafter, Act was added to some of the flasks, and cells were harvested at different times for mRNA preparation. Because D2 mRNA expression was decreasing during this time, the appropriate controls were run in parallel. Figure 6 shows first that, in the absence of Act, a decrease in D2 mRNA expression occurred during the period studied, as the effect of NE decreased between 10 and 18 h, so D2 mRNA expression decreased by 50% after 7 h, both when T3 was added for long or short times (1 or 3 days). Act induced a further reduction in D2 mRNA, but no significant changes were observed between exposure to T3 for 1 or 3 days. D2 mRNA half-life was ~6 h, so the presence of T3 for short or long times does not seem to play a role in increasing D2 mRNA stability. Nevertheless, as we cannot measure D2 mRNA half-life in the absence of T3, it is likely that T3 is playing a role in the stabilization of D2 mRNA transcripts.
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DISCUSSION |
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The main regulator of D2 activity in BAT is cold exposure, via the adrenergic stimulation (44), a situation in which D2 provides the T3 required for the full expression of UCP1, the specific marker of BAT (5). T3 plays other important roles in brown adipocytes, increasing mitocondriogenesis, the expression of lipogenic markers (18, 37), and favoring differentiation programs. The action of T3 on D2 expression does not seem to be an indirect effect linked to the differentiation program, but rather an action required for the NE action.
NE.
D2 is stimulated adrenergically in floating adipocytes
(32), and a synergy between the - and
1-adrenergic pathways was found (38), the
adrenergic stimulation of D2 being mostly
-adrenergic in mouse brown
adipocytes (35). We have shown that T3
is an absolute requirement for the adrenergic stimulation of D2 and amplifies this response (19).
T3 and NE.
The adrenergic stimulation of D2 mRNA in cultured brown adipocytes
requires the presence of T3. This finding is similar to what is found for the adrenergic stimulation of UCP1 mRNA, where a
synergy between NE and T3 is required, postulated to be
exerted via CREs. In the UCP1 gene, an enhancer region has been found that has several thyroid response elements, retinoic acid response elements, and possibly peroxisome proliferator-activated response elements that synergized with the response of NE via cAMP. The cooperative interaction between the T3 and NE actions is
firmly stablished (3, 4), but the specific proteins
involved (coactivators) have not yet been identified. The recruitment
of coactivators would increase the affinity or phosphorylation of
CCAAT/enhancer-binding protein/p300 to increase transcription. Another
possible candidate is PGC-1, the coactivator 1 for peroxisome
proliferator-activated receptor- (PPAR
), induced in BAT upon cold
exposure. The participation of these proteins in the synergistic
action of NE and T3 is not elucidated. Recent reports using
mice with targeted disruption of the D2 gene suggest that the
T3 derived from D2 is essential for the thyroid-sympathetic
synergy required for adaptative thermogenesis (15).
T3. In addition, we have demonstrated that T3 by itself increases D2 mRNA expression. Although the extent of this increase may seem modest, it is an unexpected finding. The increased D2 mRNA expression observed with the use of T3 could be due to direct stimulation of transcription (as Act blunts it), stimulation of factors required for transcription, or stabilization of the preexisting mRNA, as the T3 effect requires long times to be observed. T3 could also stimulate basal D2 expression by increasing the basal adenylyl cyclase activity, as T3 reduces Gi levels (9). Nevertheless, basal cAMP levels are not modified by addition of T3 in our cells. Of note, RT-PCR is able to amplify D2 transcripts after 24 h of T3 treatment alone, a fact not observed when NE is used alone.
This effect of T3 is in contrast to what is found in vivo in other tissues such as brain (7, 12, 26) or pituitary cells (25), where T3 inhibits D2 mRNA by 50%, suggesting specific modulation of D2 in each cell. The inhibition of D2 activity by T3 is much lower than that of T4 and reverse T3 (47, 48). In BAT, T3 has little ability, if any, in inhibiting D2 activity and even 2-fold increases were reported (47). We found that constant infusion of T3 into rats stimulated D2 activity in BAT and brain from fetal and adult rats (16, 31). In the present study, we observe clear inhibition of D2 activity when using 20 nM T3 or higher, but at the mRNA level, we do not observe decreases in D2 expression, even when very high T3 doses are used, and further increases in D2 mRNA are observed with the use of 500 nM T3 (a T3 dose that increase T3 concentrationsMechanisms. The experiments done using CHX and Act indicate that D2 mRNA induction using NE + T3 or T3 is activated at the transcriptional level and that the inhibition of de novo protein synthesis results in accumulation of mRNA, especially of a shorter mRNA species. CHX treatment of T3-exposed cells produces a marked increase in a 1.9-kb mRNA species that was barely detectable in control cells (Fig. 5). This transcript might be a short-lived product generated by alternative mRNA processing. The nature of this mRNA is unknown, but it might correspond to a 1.9-kb D2 cDNA isolated from brown fat (12) that does not code for an active protein because it is lacking a putative SECIS element. If this is the case, expression of this shorter mRNA might be an important regulatory mechanism of expression of active D2 enzyme. The accumulation of D2 mRNA with the use of CHX has been reported in pituitary cells (25) (mRNAs sizes were not described) and in pineal glands, where a short band is also observed (22). Of note, CHX per se, or in the presence of NE without T3, is unable to accumulate shorter or larger mRNA species (results not shown).
The studies performed to determine whether a prolonged exposure to T3 contributes to increases in D2 mRNA half-life suggest that this is not the case. Nevertheless, we do not exclude the possibility that T3 is inducing a stabilization of the D2 mRNA transcripts, as we were unable to measure D2 mRNA half-life in the absence of T3. A much shorter D2 mRNA half-life (2 h instead of the 6 h we find) has been reported in pituitary cells (25), suggesting that the presence of T3 might increase D2 mRNA half-life. In summary, we have demonstrated that T3 is an absolute requirement for the adrenergic stimulation of D2 mRNA (and activity) in brown adipocytes. This stimulation occurs mainly via ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. D. St. Germain and J. G. Sutcliffe for the D2 and cyclophilin cDNAs, respectively. We thank SmithKline Beecham Pharmaceuticals for the gift of the BRL-37344.
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FOOTNOTES |
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This work was supported by research grants PB 95-0097 from Dirección General de Investigación Científica y Técnica, FISS 94/0274 and 99/0813 from Fondo de Investigaciones Sanitarias (FIS), and CAM 08.6/0030/1998 from Comunidad de Madrid (CAM) (Spain). R. Martinez-DeMena was supported by research grants FISS 94/0274 (predoctoral studies) and CAM 08.6/0030/1998 (as postdoctoral).
Address for reprint requests and other correspondence: M. J. Obregón, Instituto Investigaciones Biomédicas "Alberto Sols" (CSIC), Arturo Duperier, 4, 28029 Madrid, Spain (E-mail: mjobregon{at}iib.uam.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.
10.1152/ajpendo.00433.2001
Received 26 September 2001; accepted in final form 6 January 2002.
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