Down-regulation of beta3 adrenoreceptor gene expression in brown fat cells is transient and recovery is dependent upon a short-lived protein factor.

The regulation of the expression of the β3 adrenoreceptor gene was examined in the brown adipose tissue of intact mice and in murine brown fat primary cell cultures. Both in vivo and in vitro, high levels of β3 receptor mRNA were observed. Acute cold exposure of mice resulted in a marked and rapid down-regulation of β3 gene expression; this down-regulation was, however, transient. Similarly, in brown fat cell cultures, norepinephrine addition led to down-regulation of β3 gene expression, with a lag phase of 30 min and with an apparent half-life of β3 mRNA of ~30 min. This down-regulation was stimulated via the β3 receptors themselves and mediated via cAMP; the apparent affinity of norepinephrine was extremely high (<1 nM). The degradation rate after actinomycin was identical to that after norepinephrine and was not affected by the presence of norepinephrine; thus, the down-regulation was due to cessation of transcription but not to an increased rate of degradation. Notably, inhibition of protein synthesis by cycloheximide also led to down-regulation. The norepinephrine-induced down-regulation was transient; spontaneous recovery occurred after ~18 h and was not due to depletion of adrenergic agent. Recovery did not occur in the presence of cycloheximide. After recovery, the cells showed a functional desensitization of the down-regulation process itself (EC50 now ~10 nM). It is concluded that a down-regulated state cannot explain the functional desensitization of β3 adrenergic responsiveness observed in brown fat cells isolated from cold-acclimated animals (i.e. physiologically chronically adrenergically stimulated brown fat cells); since the β3 receptor is not subject to desensitization via phosphorylation processes, no satisfactory explanation for the functional desensitization exists as yet. A model is presented for the down-regulation/recovery process, involving the participation of a phosphorylatable short-lived transcription factor.

Exposure of cells to hormones to which they are responsive often leads to a desensitization of the receptor system for that hormone (1,2). This homologous desensitization can occur through receptor phosphorylation, through receptor internalization, or through a down-regulation in the steady-state levels of the mRNA for the receptor, resulting in a decrease in receptor density (2)(3)(4). The physiological role of such desensitization is believed to be a modulation (attenuation) of the sensitivity of a responsive tissue to chronic stimulation.
Brown adipose tissue is richly sympathetically innervated, and release of norepinephrine, which occurs e.g. during cold exposure of a mammal, stimulates the brown adipocytes to combust fat and to generate heat (5). This thermogenic process is known to continue as long as the mammal requires extra heat production. Thus, in order to continue to fulfill its physiological function, the tissue must be chronically adrenergically stimulated, and there is evidence that this stimulation indeed continues unabated during prolonged demands for thermogenesis (6). This should imply from the above that the adrenergic receptors in the tissue could become desensitized. Nonetheless, for homeostatic reasons, the heat production must remain elevated. There is thus good reason to postulate that the tissue should remain fully responsive to norepinephrine, despite uninterrupted stimulation by the agonist.
Nonetheless, we and others have shown that the brown adipocytes do indeed show physiologically induced functional desensitization. Thus, brown fat cells isolated from animals that have been exposed to cold for a prolonged period show a decreased responsiveness to norepinephrine in vitro, both with respect to the extent of maximal stimulation of oxygen consumption (heat production) and, more importantly, with respect to the EC 50 for norepinephrine, which is shifted significantly to higher concentrations (7)(8)(9)(10)(11)(12)(13). The molecular mechanism behind this functional desensitization is currently not clarified. The decreased sensitivity to norepinephrine can in part be a postreceptor effect, e.g. resulting from increased phosphodiesterase activity, which would decrease the final level of cyclic AMP generated (12), or a transduction effect, resulting from a decreased content of G s ␣-subunits, which would attenuate the coupling between the receptor and the adenylyl cyclase system (13). However, it is doubtful that these postreceptor mechanisms can fully explain the reported desensitization. It may therefore be suggested that also the receptor itself is involved in the desensitization process, either by being desensitized or by being down-regulated in brown adipocytes.
Pharmacological studies indicate that the ␤-receptor subtype responsible for the stimulation of oxygen consumption is exclusively the ␤ 3 subtype (14). The ␤ 3 receptor lacks most of the serine/threonine residues that are phosphorylated by the ␤-adrenergic receptor kinase and by protein kinase A in the classical process of desensitization (15,16), and the ␤ 3 receptor should consequently lack this fundamental process (3,17,18). An alternative explanation for the functional desensitization could therefore be that the ␤ 3 receptor is down-regulated, i.e. that a decreased amount of ␤ 3 receptors could be found. Such a process can be observed in certain types of transfected cells, e.g. murine L cells, where a decreased amount of ␤ 3 receptors is found after adrenergic stimulation (19). In other transfected cells (hamster CHW cells), little effect is observed (18). Not least for technical reasons, it is unknown whether such a downregulation process occurs under physiological conditions. Since no selective high affinity antagonist has been available for the ␤ 3 receptor, there has been no suitable radioligand for determination of receptor density in direct radioligand binding studies, and this means that the situation is difficult to analyze when several ␤-receptor subtypes may be found within one tissue, as is the case in brown adipose tissue (20,21). Some studies of ␤ 3 receptor densities in brown adipose tissue have been performed with agonists, but these studies have not addressed the question whether ␤ 3 receptor down-regulation occurs in animals when the tissue is exposed to a chronic adrenergic influence during physiological stimulation (22)(23)(24)(25)(26).
However, it is possible to investigate directly at the level of gene expression whether a down-regulation occurs. In shortterm studies in intact animals, steady-state levels of mRNA for the ␤ 3 adrenergic receptor have previously been determined. It has been found that brief cold exposure or acute treatment with norepinephrine or ␤ 3 selective agonists decreases the mRNA level for the ␤ 3 receptor (23,27). However, it has not been investigated whether this process could explain the long term desensitization referred to above.
In an attempt to elucidate what changes would be anticipated in the ␤ 3 adrenergic receptor levels after chronic cold exposure or agonist stimulation, we have here analyzed steadystate mRNA levels for the ␤ 3 adrenergic receptor both in intact animals and in primary cultures of brown adipocytes, exposed acutely and chronically to adrenergic agents. Our results indicate that a ␤ 3 mediated process indeed causes a decrease in ␤ 3 mRNA levels in these cells, where the presence of the ␤ 3 receptor is due to endogenous expression and not to transfection and vector-driven expression in a cell line. Thus, the down-regulation is apparently a physiologically relevant phenomenon. However, we observed that despite continuous stimulation, the ␤ 3 mRNA level gradually spontaneously recovered, to reach control levels within less than 24 h; this recovery process was dependent on protein synthesis. We here forward a model for these events and discuss the complex situation that brown fat cells become functionally desensitized to ␤ 3 adrenergic stimulation despite the fact that the ␤ 3 receptor lacks the molecular prerequisites for desensitization and despite the absence of a persistent down-regulation process.

MATERIALS AND METHODS
Animals-Six-week-old male mice (NMRI strain, Eklunds, Stockholm) were kept at ϩ28°C for at least 7 days with free access to food and water. The animals were thereafter either transferred to ϩ4°C (cold exposure) or handled in the same way and returned to ϩ28°C (control) for different times. The animals were killed by CO 2 , followed by decapitation, and the interscapular brown adipose tissue was removed. Homogenization with Ultraspec (Biotecx) was performed with a Potter-Elvehjem homogenizer with a tightly fitting Teflon pestle (10 -15 strokes), and total RNA was isolated according to the manufacturer's total RNA isolation method.
Cell Culture-Brown fat precursor cells were isolated in principle as described previously (28) from 3-week-old mice that had been kept at the institute at 22°C for 2-3 days before dissection. The mice were of the same NMRI outbred strain as above. The interscapular, axillary, and cervical brown adipose tissue depots were dissected under sterile conditions. The tissue was carefully minced and transferred to the Hepes-buffered solution (pH 7.4) detailed by Néchad et al. (29), contain-ing 0.2% (w/v) crude collagenase type II (Sigma). Routinely, pooled tissue from six mice was digested in 10 ml of the Hepes-buffered solution. The tissue was digested for 30 min at 37°C and vortexed every 5 min. The digest was poured through a 250-m silk filter into sterile tubes. The solution was then put on ice for 15 min to allow the mature brown fat cells and lipid droplets to float. The infranatant was filtered through a 25-m silk filter and collected in 10-ml sterile tubes. The precursor cells were collected by centrifugation for 10 min at 700 ϫ g, resuspended in Dulbecco's modified Eagle's medium, and recentrifuged. The pellet was resuspended in a volume corresponding to 0.5 ml of cell culture medium for each mouse dissected.
The cell culture medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum (Flow), 4 nM insulin, 10 mM Hepes and 50 IU of penicillin, 50 g of streptomycin, and 25 g of sodium ascorbate per ml (30). Aliquots of 0.5 ml of cell suspension were cultivated in 25-cm 2 tissue culture flasks (Bibby) with 4.5 ml of cell culture medium, or 0.2 ml of cell suspension were cultivated in six-well culture dishes (Corning) with 1.8 ml of cell culture medium in each well. The cultures were placed at 37°C in a water-saturated atmosphere of 8% CO 2 in air, in a Heraeus CO 2 -auto-zero B5061 incubator. On days 1 and 3, the cells were washed with prewarmed Dulbecco's modified Eagle's medium, and the medium was changed. Most experiments were performed with cells after 6 -7 days in culture, i.e. at confluence. Detailed protocols are found in the description of each experiment.
RNA Isolation-At the end of each experiment, the medium was discarded, the cells were dissolved in 800 l of an Ultraspec (Biotecx) solution, and the manufacturer's procedure for RNA isolation was followed. The final pellet was suspended in 75 l of 10 mM EDTA, and the RNA was extracted at 70°C for 15 min with vortexing every second min. RNA concentration was measured, and the absence of protein contamination was checked on a Beckman DU 50 spectrophotometer with readings at 260 and 280 nm. The ratio of 260/280 nm was routinely higher than 1.7.
Northern Blot-The solution containing RNA and 10 mM EDTA was lyophilized in a SpeedVac. The RNA was then dissolved in 17 l of RNA mixture consisting of 50% (v/v) formamide, 0.02 M MOPS, 1 and 9% (v/v) formaldehyde and 3 l of loading buffer consisting of 50% (w/v) glycerol and 0.1 mg/ml bromphenol blue. The solution was incubated for 5 min at 70°C and then chilled on ice. The samples were loaded on a gel (1.25% agarose, 20 mM MOPS, 6.2% (v/v) formaldehyde, and 15 l of 1 mg/ml ethidium bromide). The gel was run in 20 mM MOPS buffer for 2-3 h at 4 -5 V/cm. After electrophoresis, it was verified under UV light from the intensity of the 18 to 28 S rRNA bands that all samples were equally loaded and that no degradation was observable.
The RNA was blotted from the gel to a Hybond-N membrane overnight in 20 ϫ SSC. Three sheets of Whatman 3MM soaked in 20 ϫ SSC were placed on top of the Hybond-N membrane. The gel and the Hybond-N membrane were examined under UV light. The RNA was crosslinked to the Hybond-N membrane (UV Stratalinker 1800 (Stratagene) with the auto-cross-link program).
The Hybond-N membrane was prehybridized with 10 ml of a solution containing 5 ϫ SSC, 5 ϫ Denhardt's solution, 0.5% SDS, 50 mM sodium phosphate, 50% formamide, and 100 g/ml of degraded DNA from herring sperm (Sigma) in a hybridization oven (Hybaid) at 45°C for 3 h. After prehybridization, the Hybond-N membrane was transferred to a similar solution containing the denatured, labeled probe at a concentration of 1-3 ϫ 10 6 cpm/ml. The hybridization was carried out for at least 16 h at 45°C. The Hybond-N membrane was then washed twice in 2 ϫ SSC, 0.2% SDS at 30°C for 30 min each and then twice in 0.1 ϫ SSC, 0.1% SDS at 50°C for 45 min. The membrane was sealed in a plastic envelope and exposed to a PhosphorImager screen. The screens were analyzed on a Molecular Dynamics PhosphorImager with the ImageQuant program.
␤ 3 Adrenoreceptor cDNA Probe-The probe originated from the A43 probe earlier characterized (16). A fragment of the mouse ␤ 3 adrenoreceptor gene was subcloned in pUC18 at the XbaI site. This genomic DNA fragment has a length of 300 base pairs and corresponds to the 5Ј coding region of the ␤ 3 adrenoreceptor from ATG to the second transmembranal loop (TM2). To generate the ␤ 3 adrenoreceptor probe used here, the plasmid was cut with the restriction enzymes BamHI and SalI to a length of 0.5 kilobase pairs.
The probe was labeled with a DNA labeling kit (Boehringer Mannheim). Probes were labeled to an activity of 10.000 -60.000 cpm/l.
and collagenase (type II) were obtained from Sigma. BRL-37344 was a gift from SmithKline Beecham Pharmaceuticals, and CGP-12177 was a gift from Ciba-Geigy. ICI-89406 and ICI-118551 were gifts from Imperial Chemical Industries Zeneca. All adrenergic agents were freshly dissolved in water. Norepinephrine was dissolved in 0.125 mM sodium ascorbate (as in medium).

Physiological Regulation of ␤ 3 Adrenoreceptor Gene Expression in Intact
Animals-In order to investigate whether the functional desensitization observed in brown adipocytes from cold-acclimated animals involved a down-regulation of the expression of the ␤ 3 adrenoreceptor gene, the effects of acute and chronic cold exposure on ␤ 3 mRNA levels in brown adipose tissue were investigated.
Acute exposure of mice to cold led to a decrease in the levels of mRNA for the ␤ 3 adrenergic receptor in brown adipose tissue (Fig. 1). The level was reduced by as much as 50% after 12 h. This reduction occurs at the same time as a large increase in the level of mRNA for the uncoupling protein can be observed in these animals (31), and the reduction is therefore not the result of a stress effect, diminishing gene expression in general. The decreased level of ␤ 3 mRNA in cold-exposed mice is principally in agreement with the results of earlier work performed with rats (27), and these acute results (i.e. the 12-h result), taken alone, could be taken as evidence that the functional desensitization to norepinephrine earlier observed may be due to down-regulation of ␤ 3 gene expression. However, we observed that after longer exposure to cold, the mRNA levels returned toward control levels: already after 24 h, the level was not significantly different from that found in control mice (Fig.  1). In this respect, there may be species differences between mice and rats, since a more prolonged down-regulation was observed in rats (27). The transient nature of the down-regulation observed in mice was further confirmed in independent long term experiments. After mice had been 10 weeks at 4°C, the ␤ 3 receptor mRNA levels were 80 Ϯ 18% of the initial level (n ϭ 4), i.e. again not significantly different from the initial levels.
Thus, the down-regulation that may be observed immediately after cold exposure is transient. As the increased level of adrenergic stimulation persists unabated during prolonged exposure to cold (6), the recovery observed should not be due to a lower concentration of norepinephrine in the synaptic areas. Further, since there is no persistent decrease in the level of ␤ 3 mRNA, receptor down-regulation cannot explain the functional desensitization observed in brown fat cells isolated from coldacclimated animals.
Down-regulation of ␤ 3 Gene Expression in Cell Cultures-In order to study the mechanism behind this transient downregulation and the nature of the recovery process of ␤ 3 receptor gene expression, we performed detailed experiments in primary cultures of mouse brown adipocytes. In these cell cultures, isolated undifferentiated brown fat cell precursors grow to reach confluence; at the time of confluence (day 5-6 in culture), the cells have differentiated and contain multilocular lipid droplets, and they have the ability to show norepinephrine-induced expression of the gene for the tissue-specific mitochondrial uncoupling protein thermogenin (28,32). We have elsewhere reported in preliminary form (33,34) that the ␤ 3 receptor in these cells is well expressed after day 4 in culture and is coupled to adenylyl cyclase.
Norepinephrine-induced Down-regulation in Cultured Brown Adipocytes-The decrease in ␤ 3 gene expression ( Fig. 1) was observed in intact animals under experimental conditions where a chronic adrenergic stimulation of the tissue should occur. We therefore investigated whether the inhibition of ␤ 3 gene expression could be explained by a direct effect of norepinephrine on the brown fat cells themselves. Cultured cells (day 6) were treated with norepinephrine for various times, and the level of ␤ 3 receptor mRNA in the total RNA was determined by Northern blot analysis. The results are exemplified in the Northern blot in Fig. 2A. It is seen in the control lane that the size of the most abundant mRNA species was about 2.4 kilobases, with a minor band of slightly larger size. The sizes of these ␤ 3 mRNA species from the mouse cell culture system are thus in good agreement with what has been observed in RNA isolated from brown adipose tissue of mice and from the 3T3-F442A cell line of murine origin (16,35) but different from that observed in rats (36,37) and humans (15,38).
It can be seen on the Northern blot that norepinephrine rapidly decreased the ␤ 3 mRNA level. Already after 1 h, a marked reduction in mRNA level had occurred, and an almost total absence of ␤ 3 mRNA was observed after ϳ2 h; the level was maintained depressed for at least 6 h.
In Fig. 2B, quantitative analyses of the results from a series of similar experiments are depicted. It is clear that there was a lag phase of approximately 30 min, after which there was a very rapid decrease in ␤ 3 mRNA levels. In Fig. 2C, the relevant data from Fig. 2B are plotted on a semilogarithmic scale. The disappearance reasonably followed first-order kinetics; the half-life of ␤ 3 mRNA after norepinephrine stimulation of the cells was as short as 33 Ϯ 2 min.
Effect of Norepinephrine Dose on ␤ 3 Receptor mRNA Levels-A dose-response curve for the norepinephrine-stimulated decrease in ␤ 3 mRNA levels is depicted in Fig. 3. The cells were treated for 2 h with different concentrations of norepinephrine and then harvested. It is clear from this curve that already FIG. 1. Levels of ␤ 3 -adrenergic receptor mRNA in brown adipose tissue of mice exposed to cold. Total RNA was isolated from the brown adipose tissue of 6-week-old mice that had been preacclimated to 28°C for 7 days and were then maintained at 28°C or were placed at 4°C for the indicated times. 20 g of RNA was analyzed by Northern blot analysis. The mean value at 28°C was set to 100%, and the other values are expressed relative to this. Each point represents the mean Ϯ S.E. from two to four mice. nanomolar concentrations of norepinephrine were effective in inducing the down-regulation. The calculated EC 50 value was remarkably low, 0.7 nM. No other reported action of norepinephrine on brown fat cells has demonstrated such a high apparent affinity; the EC 50 for stimulation of the expression of the gene for the uncoupling protein in these cells under identical conditions was ϳ10 nM (28), and that for norepinephrine stimulation of cell proliferation was ϳ20 nM (39). Heat production (80 nM (14)) and cAMP accumulation (1000 nM (8,12)) in isolated mature brown fat cells show much higher EC 50 values for norepinephrine.
Characterization of the Adrenergic Receptor Involved-The nature of the adrenergic receptor responsible for the rapid decrease in ␤ 3 receptor mRNA levels was studied with various adrenergic agonists. As seen in Table I, norepinephrine and epinephrine were equally effective in effecting the down-regulation, as was the subtype-nonselective ␤-agonist isoprenaline. The subtype-nonselective ␣-agonist phenylephrine failed to induce a decrease in mRNA. It may also be noted that the subtype-selective ␣-adrenergic antagonists prazosin (␣ 1 ) and yohimbine (␣ 2 ) failed to prevent the norepinephrine-induced down-regulation. Thus, the norepinephrine-induced decrease in ␤ 3 gene expression is clearly mediated via ␤-adrenergic receptors.
Also, the subtype-selective ␤-adrenergic antagonists ICI-89406 (␤ 1 ) and ICI-118551 (␤ 2 ) failed to prevent the norepinephrine-induced down-regulation. However, the ␤ 3 -selective agonist BRL-37344 mimicked the effect of norepinephrine, and CGP-12177 (an absolute ␤ 3 agonist (40) in that it is an antagonist on ␤ 1 and ␤ 2 receptors) also induced the downregulation. Thus, it was clear that stimulation of the ␤ 3 subtype could induce the down-regulation, and it is unlikely that the ␤ 1 receptor had this ability (the possibility that it could induce down-regulation cannot be fully excluded from the experiments performed, but ␤ 3 stimulation is clearly sufficient for a full response).
It may be observed that the subtype-nonselective ␤-adrenergic antagonist propranolol failed to prevent the norepinephrine action. Although propranolol has antagonist action on ␤ 3 receptors (pA 2 ϳ5-6), its pA 2 values on ␤ 1 and ␤ 2 receptors are ϳ3 orders of magnitude higher (41). In view of the fact that the sensitivity to norepinephrine of the down-regulation response is so high, this failure of propranolol to prevent the norepinephrine effect is in accordance with the norepinephrine-induced decrease in ␤ 3 receptor mRNA levels being fully a ␤ 3 receptormediated process. Under the experimental conditions of Table  I and assuming an apparent affinity of norepinephrine of 0.7 nM and a pA 2 of 5 for propranolol, the receptor would still be 98% stimulated if it adheres to simple Michaelis-Menten kinetics.
Thus, the combined adrenergic agonist and antagonist studies indicate that the ␤ 3 receptor down-regulation is mediated via the ␤ 3 receptor itself.
Concerning the intracellular mediation of the adrenergic signal, it is seen that the Ca 2ϩ ionophore A23187 failed to induce a decrease in ␤ 3 mRNA. However, the adenylyl cyclase activator forskolin, which markedly increases cAMP levels in in these brown adipocyte primary cultures (42), repressed ␤ 3 receptor gene expression. Thus, the down-regulation is presumably brought about by increases in intracellular cAMP levels. The implication of these experiments is that the downregulation process is not dependent on the ␤-receptor as such (or on receptor occupancy), but only on an increase in cAMP levels.
Effect of Inhibition of mRNA Synthesis-The down-regulation induced by norepinephrine could be due to an increase in the rate of degradation of the ␤ 3 mRNA, an inhibition of the transcription of the ␤ 3 gene, or both. In order to investigate this, the effects of norepinephrine alone were compared with those of the RNA synthesis inhibitor actinomycin D (with or without norepinephrine). Actinomycin was used at a concentration previously shown not to be detrimental to the cells during short term treatment (43,44). The results of these experiments are shown in Fig. 4. As seen in Fig. 4A, the half-life of the ␤ 3 mRNA after norepinephrine addition was in this series 35 min (uncertainty interval of 28 -42 min). The half-life after treatment with actinomycin alone was also 35 min (uncertainty interval of 32-38 min) (Fig. 4B); this would in itself indicate that the down-regulation was mainly due to cessation of transcription. This was confirmed by the observation (Fig. 4B) that the presence of norepinephrine did not significantly alter the rate of down-regulation of ␤ 3 mRNA (the half-life was then 41 min; uncertainty interval of 40 -42 min); i.e. an increased rate of ␤ 3 mRNA degradation was not induced by norepinephrine. The most likely interpretation is thus that the effect of norepinephrine is fully due to suppression of transcription of the ␤ 3 gene.
Effect of Inhibition of Protein Synthesis-In many instances, inhibition of protein synthesis has been shown to lead to increases in mRNA levels (so-called "superinduction"). We have shown this in the brown adipocyte cultures for the c-fos protooncogene (42). Also for some receptors, such an adrenergic up-regulation has been reported (45,46). However, when the brown adipocytes were treated with cycloheximide in the present study (Fig. 4C), the mRNA levels declined markedly (55min half-life, uncertainty range of 45-65 min). This is in contrast to that which has been observed after cycloheximide treatment of 3T3-F442A cells (47). Thus, in these primary cultures of brown adipocytes, which express this receptor endogenously, synthesis of a protein of very short half-life is apparently required for maintenance of mRNA levels of the ␤ 3 receptor.

Spontaneous Recovery of ␤ 3 Gene Expression in Cell
Cultures-When ␤ 3 mRNA levels were followed in cultures that were chronically exposed to norepinephrine, it was found that the dramatic down-regulation seen in Fig. 2 was transient. This is exemplified in Fig. 5A. As seen in Fig. 5B, after 10 h of treatment, the ␤ 3 mRNA level started to recover, and by 18 h the level had returned to the level of untreated, control cultures of the same age. The control level was thereafter maintained in the treated cultures for at least a further 20 h. This result was principally in accordance with the results of chronic cold exposure in vivo shown in Fig. 1B.
The transient nature of the down-regulation prompted an investigation as to whether the recovery was a consequence of an artefactual or physiological removal of the stimulus or whether recovery would occur although the stimulus was maintained.
Action of Long-lived Agonists-When the initial down-regulation was initiated by 0.1 M CGP-12177 or by 1 M forskolin, both of which are anticipated to be more long-lived in their action than norepinephrine, the ␤ 3 gene expression was again down-regulated, and the up-regulation occurred with approximately the same kinetics as with 0.1 M norepinephrine (Fig.  6). An additional dose of forskolin added at the start of the recovery phase delayed recovery somewhat, but it nonetheless occurred. Thus, the recovery phenomenon would not seem to be due to agonist depletion.
This could be further confirmed by the following experiment (Fig. 7A). Cells were treated with 0.1 M CGP-12177 for 24 h. At this time point, recovery had been attained. The medium from these cells was collected and added to a culture that had not previously been exposed to CGP-12177. Down-regulation was observed in these fresh cultures after 2 h and was of the same magnitude as that seen in the original culture (Fig. 7A). Similar results were obtained with norepinephrine (not shown). Thus, again, the recovery process does not seem to be a result of full depletion of agonist. When norepinephrine was readded during the recovery phase, a down-regulation similar to the first one was observed, with respect to both rate and extent (Fig. 7B). This downregulation apparently occurred in the presence of surviving agonist. Taken together with the observation that surviving agonist could induce down-regulation in naive cells (Fig. 7A) these results would be understandable if some depletion in agonist level had occurred, provided that a desensitization of the adrenergic receptor system had also occurred.
Adrenergically Treated Cells Are Desensitized-To investigate this putative desensitization, cell cultures were pretreated for 18 h with 0.1 M norepinephrine. Thereafter a dose-response curve for norepinephrine-induced down-regulation was made and compared with one obtained with control cells (Fig.  8). It is evident that the pretreatment resulted in cells that were clearly desensitized with respect to their ability to show norepinephrine-induced down-regulation of ␤ 3 mRNA levels; in the naive cells, the EC 50 value was 0.9 nM, whereas in the pretreated cells the EC 50 value had increased to 7.2 nM. This sensitivity is in the order earlier observed in these cells for other responses (28,39).
Recovery Requires Protein Synthesis-Self-evidently, the recovery phase was completely prevented in the presence of actinomycin (not shown). In addition, the recovery was also completely prevented in the presence of cycloheximide, demonstrating a requirement for ongoing protein synthesis (Fig. 9). This is in agreement with the finding shown above (Fig. 4C) that the addition of cycloheximide resulted in a rapid decline in mRNA levels. Since no recovery could occur, a protein of short half-life is apparently required for gene transcription to proceed. This could be a short-lived transcription factor, the nature of which is currently unknown.

FIG. 4. Influence of inhibition of RNA synthesis (B) and protein synthesis (C) on ␤ 3 -receptor mRNA levels in cultures of brown adipocytes.
A, confluent cultures of brown adipocytes (day 6 in culture) were treated with with 0.1 M norepinephrine (NE) (q--q) or treated only with vehicle (E-E). The cells were harvested at the indicated times, and total RNA was isolated and analyzed as described in the legend to Fig. 2. The values are the means Ϯ S.E. of four experiments performed in duplicate. In each experiment, the initial level was set to 100. ␤ 3 mRNA half-life was analyzed as in Fig. 2C; the 30-min lag phase was not included in these analyses. B, cultures were treated with 1 g/ml actinomycin (Ⅺ--Ⅺ) or with 0.1 M norepinephrine ϩ1 g/ml actinomycin (f--f). Data were from the same series as A and were similarly analyzed. C, cultures were treated with 50 M cycloheximide (छ--छ). Data were from three of the four series in A.

DISCUSSION
In an attempt to address the question of the molecular basis for the physiologically induced functional desensitization of the ␤ 3 adrenergically mediated thermogenic response in brown fat cells, we have examined in the present investigation the effects of physiological stimulation in vivo and of adrenergic stimulation in vitro on ␤ 3 adrenoreceptor gene expression. Although our results concerning the acute effects agree with those of some others in showing down-regulation of the ␤ 3 gene expression, we have observed, both in vivo and in vitro, that a spontaneous recovery occurs in the maintained presence of physiological or pharmacological stimulation. Thus, the observed acute down-regulation cannot explain the functional desensitization earlier reported.
In order to further study the phenomenon of this transient down-regulation, we have used primary cultures of brown adipocytes from mouse. These cells have previously been demonstrated to develop into genuine brown adipocytes, both with respect to their ability to express the uncoupling protein gene upon adrenergic stimulation (28,32) and to express the ␤ 3 receptor well, observed as ␤ 3 mRNA (34), as ␤ 3 -induced increases in cAMP levels (34,39) and as functional effects of ␤ 3 stimulation (28).
The Decrease in ␤ 3 Adrenoreceptor Gene Expression-As anticipated from the in vivo experiments in mice, adrenergic stimulation rapidly and dramatically decreased mRNA levels for the ␤ 3 adrenergic receptor in cultured murine brown fat cells; this decrease was clearly mediated through the ␤ 3 receptors themselves and through an elevation in cAMP levels ( Fig.  2 and Table I).
The observation that acute adrenergic stimulation can decrease ␤ 3 gene expression is in principle in agreement with recent observations by Klaus and co-workers (26) working with brown fat cells from Siberian hamsters, and also with earlier observations in murine L cells transfected with the human ␤ 3 receptor gene (19) and in the white adipocyte-like cell line 3T3-F442A (37,47). However, Thomas et al. (48) reported the opposite: an elevation of ␤ 3 gene expression by adrenergic stimulation in the same 3T3-F442A cell line. It has also been suggested (18, 37) that the down-regulation response is cell type-and species-specific; when transfected into CHW cells (19) or SK-N-MC cells (37), the human ␤ 3 gene showed little or no agonist-induced down-regulation. Thus, it has been of importance to verify in a well studied physiological system (murine brown fat primary cultures) whether or not the gene of the physiologically predominant receptor in that system, the ␤ 3 receptor, is down-regulated by adrenergic stimulation. This was clearly the case. Further analysis of the down-regulation process showed several interesting characteristics.
The down-regulation had an extremely high sensitivity to norepinephrine (0.7 nM), the highest reported for any adrenergic system in brown adipose tissue and also a much higher sensitivity than any other earlier reported for ␤ 3 -mediated responses. Thus, although certain types of experiments have been interpreted to indicate that ␤ 3 receptors are only stimulated at high norepinephrine concentrations (and ␤ 1 receptors at lower norepinephrine concentrations) (49), it is clearly not the case that the ␤ 3 receptors are inherently relatively insensitive to norepinephrine. Further, the very high sensitivity implies that occupancy of only a fraction of the ␤ 3 receptors by norepinephrine would be sufficient to suppress gene transcription. This high sensitivity is not seen for adrenergic stimulation of the gene expression of the uncoupling protein thermogenin in these cells and is therefore specific for the down-regulation phenomenon. We have currently no explanation for the exceptionally high sensitivity. The down-regulation was due to an increase in the level of the second messenger cAMP (and not directly related to receptor occupancy), as evidenced by the fact that forskolin could also induce down-regulation. This is in agreement with other similar studies (50,51), and, as a consequence of the high sensitivity, the down-regulation process must be exquisitely sensitive to very low cAMP concentrations.
The down-regulation showed a distinct lag phase (ϳ30 min) (Fig. 2B). After the lag phase, the down-regulation was very rapid, with a half-life of only ϳ30 min. This is somewhat more rapid than that reported in other similar systems: about 2 h for the ␤ 3 receptor mRNA when down-regulated by isoprenaline in 3T3-F442A cells (37), about 4 h for the same receptor mRNA down-regulated by ␤-agonists in Siberian hamster brown adipocytes (26), 90 min when down-regulated by insulin in 3T3-F442A cells (52), 60 min when down-regulated by isoprenaline in murine L cells (19), and 50 min for the ␤ 2 receptor mRNA when down-regulated by epinephrine in DDT 1 MF-2 cells (53).
A decrease in ␤ 3 mRNA was of course also induced by transcriptional inhibition with actinomycin. The degradation rate was identical to that observed after norepinephrine. Thus, an inhibition of transcription would be sufficient to explain the down-regulation; a decreased stability of the ␤ 3 mRNA was not induced by norepinephrine.
Interestingly and unexpectedly, down-regulation was also induced by the protein synthesis inhibitor cycloheximide, with a half-life (ϳ55 min) longer than that observed after norepinephrine stimulation. This could indicate that a protein (possibly a transcription factor) with a rapid turnover is necessary for continued transcription of the ␤ 3 gene in this cellular environment.
The Transient Nature of the Down-regulation-In our murine brown fat cell cultures, the rapid down-regulation induced by norepinephrine proved to be only transient: after about 18 h, the levels of ␤ 3 receptor mRNA had returned to control levels (Fig. 5). This phenomenon was in agreement with our observations in mice in vivo (Fig. 1). In both cell cultures and in the animals, we found no agonist-induced increase above the control levels of ␤ 3 mRNA, even when the agonist was present for up to about 40 h, although such a long term up-regulation has been reported to occur in 3T3-F442A cells (48).
Although this observation of spontaneous recovery of gene expression is important for the analysis of the ␤ 3 desensitization phenomenon, and although similar data indicating an almost total disappearance of ␤ 3 transcript and full spontaneous recovery have not been reported earlier for the ␤ 3 receptor, the observation of such recovery of receptor expression after down-regulation is not unique. With respect to adrenergic receptors, a similar transient decrease was reported for ␣ 1B adrenergic receptor mRNA in smooth muscle cells stimulated by norepinephrine via ␣ 1 receptors (51, 54). Hough and Chuang (51) also reported a transient decrease in ␤ 2 receptor mRNA levels. Similarly, mRNA levels for both the angiotensin II receptor and the novel vascular smooth muscle receptor decrease and then subsequently increase following a challenge by cyclic AMP elevating agents (55,56). Also endothelin-B receptor mRNA levels decrease only transiently in response to stimulation of the relevant second messenger pathways (Ca/protein kinase C) (57).
We have demonstrated that the recovery is not caused by depletion of agonist (Fig. 7). Rather, the recovery process could perhaps be understood as being an obligatory event, inevitably occurring after down-regulation (see below). The recovery proc- q---q, with norepinephrine alone; ⅜---⅜, control. ess was completely prevented by cycloheximide and thus required protein synthesis. This is most easily interpreted as indicating a requirement for synthesis of an obligatory transcription factor.
The Down-regulation/Recovery Cycle-In order to interpret the events taking place after the addition of agonist to naive cells (and those taking place in an animal suddenly exposed to cold), we propose the following hypothetical scheme.
During the maturation process, the brown fat cells "spontaneously" acquire the ability to express ␤ 3 receptors. The expression of the ␤ 3 receptor in these cells would be under acute positive control of a transcription factor with a rapid turnover (thus protein synthesis inhibition leads to cessation of ␤ 3 gene expression). When these cells are exposed to norepinephrine for the first time, the ensuing increase in cAMP would lead to phosphorylation of this putative transcription factor. This transcription factor should be active only in the dephosphorylated form (a novel member of the cAMP response element-binding protein family having this property has been reported by Kwast-Welfeld and co-workers (58)). Since the presence of the nonphosphorylated form of this transcription factor is supposed to be essential for the transcription of the ␤ 3 gene, downregulation would occur due to the increase in cAMP. In the subsequent complete absence of ␤ 3 mRNA (Fig. 2), the synthesis of the ␤ 3 receptor ceases, and the existing ␤ 3 receptors would disappear. The rate with which this would occur is not presently known. We have attempted to establish the half-life of ␤ 3 receptors in this system with the use of antibodies to the ␤ 3 receptor, but the quantity of plasma membranes needed for such studies greatly exceeds that which can feasibly be obtained from these cultures. However, the reported half-lives of ␤ 3 receptors in murine L cells (19) and ␤ 2 receptors in DDT 1 MF-2 cells (53) are about 12 h. If the ␤ 3 receptor half-life in the present system is somewhat shorter, it is feasible to think that practically all ␤ 3 receptors would have disappeared during about 12 h. However, with no receptors present, no cAMP would be produced, even in the continued presence of agonist (the experiments with forskolin may indicate that also post-cAMP processes may become desensitized). Therefore, in the absence of cAMP, the transcription factor would become dephosphorylated, and ␤ 3 gene transcription would be reinitiated. However, it would take some time to regain a full ␤ 3 receptor complement, and during this time a higher fraction of ␤ 3 receptors would have to be stimulated in order to elevate cAMP levels sufficient to reinduce ␤ 3 down-regulation. Thus, higher concentrations of norepinephrine would be necessary to reinduce down-regulation during the recovery phase. Even shortly after apparent full recovery of gene expression, when the mRNA would have fully returned to the initial level, the complement of ␤ 3 receptors still would not have reached the initial level (in a transient recovery process, the protein level necessarily lags behind the mRNA level (59)), and this would be evidenced as the desensitization seen in Fig. 8.
Although a model of this type may explain the events occurring in vitro reported here, it still fails to explain the functional desensitization observed in brown fat cells isolated from fully cold-acclimated animals, where ␤ 3 gene expression has apparently fully recovered.
The ␤ 3 Receptor and Functional Desensitization-Cell lines and transfected cells have earlier provided information on the regulation of the expression of the ␤ 3 adrenergic receptor (18,19,37,47,48,60). It is, however, evident from the results presented above that it is of importance to complement these studies with studies performed in systems where the ␤ 3 receptor is expressed in its natural background in nonimmortalized cells.
Physiologically, a possible "advantage" of the ␤ 3 receptor was early suggested to be its probable resistance to short term desensitization because of the particular sequences of the third intracellular loop and C-terminal sequence (18,19,37,47,48,60). However, this resistance to one process of functional desensitization was apparently nullified by the down-regulation reported to take place in experiments of an acute nature (27). Although this down-regulation could apparently explain the functional desensitization observed in cells from long term cold-acclimated animals, it removed the teleological basis for the desensitization-resistance advantage of the ␤ 3 receptor. The present experiments indicate that this down-regulation is transient in nature and that spontaneous recovery occurs. Thus, the ␤ 3 receptor still seems to possess properties making it well suited to mediate prolonged adrenergic stimulation, but it would seem unlikely that ␤ 3 receptor-related mechanisms contribute to the functional desensitization observed after physiologically induced chronic adrenergic stimulation of brown fat cells in situ (7)(8)(9)(10)(11)(12)(13).