©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Uncoupling Protein Gene ( Ucp) by , , and -Adrenergic Receptor Subtypes in Immortalized Brown Adipose Cell Lines (*)

Elizabeth M. Rohlfs (1), Kiefer W. Daniel (2) (3), Richard T. Premont (4), Leslie P. Kozak (5), Sheila Collins (2) (3)(§)

From the (1) Departments of Medicine, (2) Psychiatry, and Behavioral Sciences, (3) The Sarah W. Stedman Center for Nutritional Studies, and the (4) Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 and the (5) Jackson Laboratory, Bar Harbor, Maine 04609

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three -adrenergic receptor subtypes, including -adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective -adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). -, -, and -adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antagonists CGP20712A and I-cyanopindolol showed the proportions of AR and AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% AR, 65% AR; brown adipocytes from tissue: AR 41%, 59% AR). Expression of brown fat-specific mitochondrial uncoupling protein ( Ucp) was stimulated by -adrenergic agonists in two of the four cell lines. The ability of individual AR subtypes to regulate Ucp expression was examined with combinations of selective -adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the -adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three -adrenergic receptor subtypes simultaneously (100 µM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 µM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of AR/AR, plus AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that -adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of -adrenergic receptor expression and function, and the control of Ucp expression and activity.


INTRODUCTION

The major function of the white adipocyte in mammals is to serve as a storage depot for excess metabolic energy that can be retrieved during periods of net caloric insufficiency. In addition to white adipocytes, brown adipose tissue contains a specialized adipocyte which stores triglycerides as an energy source dedicated to fueling nonshivering thermogenesis, a process important for regulating body temperature and composition (lean versus fat mass) (1, 2) . Catecholamines play a major role in regulating both of these processes. In both white and brown adipocytes, catecholamine activation of -adrenergic receptors (ARs)() 1 and stimulation of the intracellular cAMP pathway mediate this metabolic regulation (3) . -Agonist stimulation of brown adipocyte thermogenesis occurs in response to cold exposure (4) or excess caloric intake (5) . Brown adipocytes contain a large number of mitochondria and express, in response to -adrenergic stimulation, a unique mitochondrial proton channel, the ``uncoupling protein'' ( Ucp) (6) . This channel uncouples mitochondrial respiration from oxidative phosphorylation to dissipate energy as heat instead of synthesizing ATP. All three AR subtypes, including the adipocyte-specific AR, are expressed in white and brown adipocytes (7, 8, 9, 10, 11) , and each is coupled to the stimulation of adenylyl cyclase with the consequent elevation of intracellular cAMP levels. -Adrenergic agonists have been shown to stimulate lipolysis (5, 12, 13, 14) , Ucp expression (6, 15, 16) , and proliferation (17, 18) of brown adipocytes. In particular, AR and AR have been implicated in cell proliferation and Ucp induction, respectively (15, 18, 19, 20, 21, 22) , but the relationships between the expression and function of AR subtypes, and their roles in brown adipocytes capable of proliferation versus those that are developmentally mature and able to express Ucp, are not understood. Moreover, factors that modulate AR expression such as -adrenergic agonists and corticosteroids (23, 24) , which may consequently affect Ucp expression, have not been investigated in brown adipose tissue.

The lack of adequate cell culture models has hindered progress in examining these biochemical aspects of brown adipocyte physiology. Several short term brown adipocyte primary culture systems have been developed (15, 16, 18, 25, 26, 27) . However, these cultures show only transient expression of Ucp, and attempts to establish cell lines from normal stromal vascular cells derived from BAT have failed. In addition, since some of these cultures were reported to require glucocorticoids in order to differentiate (26, 28) , and these steroids by themselves have been shown to modulate AR expression (23, 29, 30) , cell lines dependent upon these agents may be unsuitable for studies of AR function in adipocytes.

Recently, immortalized mouse brown adipocyte cell lines have been established which appear to retain tissue-specific characteristics of brown adipose tissue, including norepinephrine-induced Ucp expression and cell proliferation (31) . In these studies we have characterized the ARs and their signal transduction properties in four of these clonal cell lines by Northern blotting and -agonist-stimulated adenylyl cyclase activity and compared these data to results obtained from mouse brown adipocytes derived from interscapular BAT. Two of the four clones examined express all three AR subtypes and stimulate Ucp transcription in response to -agonists. Thus, these cell lines provide a new tool for examining the mechanisms involved in -adrenergic regulation of brown adipocyte differentiation and Ucp expression, and the interactions among the three AR subtypes coupled to the same second messenger pathway.


MATERIALS AND METHODS

Animals

Genetically lean (+/+) C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). They were housed at the Duke University Vivarium at 20-22 °C and fed Purina mouse chow and water ad libitum. Animals were killed at 11-12 weeks of age by intraperitoneal nembutal injection (5 mg in 0.1 ml), and interscapular brown adipose tissue was collected for the isolation of total RNA and preparation of plasma membranes. All procedures were conducted in accordance with principles and guidelines established by the National Institutes of Health for the care and use of laboratory animals.

Cell Culture

Brown adipocyte cell lines were derived from a mouse brown adipose tumor as described previously (20, 31) . Fig. 1shows a phase contrast photograph of clones B3 ( A) and B7 ( B). The cells were grown in Dulbecco's modified Eagle's medium supplemented with D-biotin (33 µM), ascorbate (100 µM), calcium pantothenate (17 µM), fetal bovine serum (10%), penicillin (10 units/ml), streptomycin (0.1 mg/ml), and fungizone (0.125 mg/ml) (Life Technologies, Inc.) in a humidified atmosphere of 5.5% CO, 94.5% air. Cells maintained in this standard medium contain multilocular lipid droplets, lipid synthesizing enzymes, and all AR subtypes. The adipogenic characteristics of the cells can be enhanced by the addition of insulin (50 nM) and T3 (0.5 nM) (31) .


Figure 1: Photomicrographs of B3 and B7 brown adipocyte cells, showing the accumulation of lipid droplets and the morphology of the cells. A, B3 cells; B, B7 cells.



Isolation and Analysis of RNA

Total cellular RNA was prepared by the cesium chloride gradient method as previously detailed (32) . For Northern blot hybridization, RNA was denatured by the glyoxal procedure, fractionated through 1.2% agarose gels, and blotted onto Biotrans nylon membranes (ICN, Irvine, CA) as described previously (23) . DNA fragments that were used as hybridization probes were obtained from the following sources. For the three AR subtypes, fragments specific for each receptor were prepared by polymerase chain reaction as described previously (11) . The cDNA fragment for ADD1, a transcription factor associated with adipocyte differentiation and highly expressed in BAT, was generously provided by Dr. Bruce M. Spiegelman (33) . For the mitochondrial Ucp, a 300-base pair BglI fragment was used (34) . The probe for the lipogenic enzyme glycerol-3-phosphate dehydrogenase was a 0.8-kilobase pair HindIII fragment from exon 2 of the Gdc-1 gene (35) . The cDNA fragment for the ubiquitous glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase was obtained from Clontech (Palo Alto, CA). DNA fragments specific for individual isoforms of adenylyl cyclase were generated by PCR using domain 2 primers as described (36) . Radiolabeled probes were prepared by nick translation of the purified DNA fragments in the presence of [-P]deoxy-CTP to a specific activity of between 3 and 7 10 dpm/µg DNA. Blots were hybridized and washed as described (11, 23) .

Reverse transcriptase-PCR analyses were performed using a GeneAmp RNA PCR Kit from Perkin-Elmer according to the manufacturer's instructions. Moloney murine leukemia virus reverse transcriptase was used to synthesize the cDNA for amplification. The oligonucleotide primers were designed according to Nahmias et al.(7) . For the AR, the sense strand primer (5`-GCTCCGTGGCCTCACAG-3`) and antisense primer (5`-CTCGGCATCTGCCCCTA-3`) correspond to nucleotides 572-588 and nucleotides 1098-1114, respectively, of the mouse AR gene, as reported by Nahmias et al.(7) and van Spronsen et al.(37) . Prior to amplification total cellular RNA from mouse brown adipocyte cell lines and mouse brown adipose tissue was incubated in a 20-µl reaction containing 50 mM Tris, 10 mM MgCl, and 1 unit of RNase-free DNase (U. S. Biochemical Corp.) for 30 min at 37 °C. The DNase was denatured by heating to 95 °C for 5 min. The RNA (250 ng) was then annealed to the antisense primer in a 20-µl reaction containing 5 mM MgCl and reverse-transcribed at 42 °C for 15 min, followed by incubation at 99 °C for 5 min to denature the enzyme. After addition of the sense strand primer, 2.5 units of AmpliTaq polymerase and 10 PCR buffer (final concentrations: 10 mM Tris, 50 mM KCl, 1.5 mM MgCl), the PCR was initiated (95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1.5 min; 40 cycles). Amplification reactions without the initial reverse transcriptase step were included as controls for all samples. Mouse genomic DNA (500 ng) was amplified as a positive control, and water was amplified as a control for reagent contamination. Ten percent of each PCR reaction mixture was applied to a 3% (w/v) MetaPhor agarose gel (FMC, Rockland, ME) and stained with ethidium bromide (1 µg/ml). For reverse transcriptase-PCR analysis of adenylyl cyclase isoforms, primers were prepared to sequences in domain 2 that are conserved among all the isoforms and amplified as described (36) . Amplified products were isolated by gel electrophoresis and subcloned into PCRII TA cloning vector (Invitrogen; San Diego, CA). Individual clones were selected, grown, and plasmid DNA was prepared and sequenced from vector primers (T7 and SP6) using [-P]dATP and Sequenase (U. S. Biochemical Corp.).

Preparation of Plasma Membranes and Adenylyl Cyclase Assay

Brown adipocytes were isolated from interscapular brown adipose tissue by collagenase digestion as described by Rodbell (38) with slight modifications. The tissue was minced and then incubated in Dulbecco's modified Eagle's medium supplemented with 20 mg of bovine serum albumin/ml and 0.24 mg/ml collagenase type I (Worthington Biochemical, Freehold, NJ) for 30-45 min with shaking at 37 °C. Plasma membranes were prepared from the purified brown adipocytes and from the brown adipocyte cell lines as described in earlier studies (11) . Adenylyl cyclase activity was assessed in these plasma membrane preparations by measuring the cAMP formed by radioimmunoassay (39) using a polyclonal antiserum produced as described (40) . (-)-Norepinephrine, (-)-isoproterenol, salbutamol, and (-)-propranolol were obtained from Sigma. CGP20712A was a gift from Ciba-Geigy (Summit, NJ), BRL37344 was a gift from Smith-Kline Beecham (Surrey, United Kingdom), and CL316,243 was a gift from American Cyanamid Company (Pearl River, NY). ICI118,551 was purchased from Cambridge Research Biochemicals (Wilmington, DE). Protein concentrations were determined by the method of Bradford (41) .

Competition Binding Assays to Determine Proportion of AR and AR

Binding assays were performed on membranes prepared from purified interscapular brown adipocytes and brown adipocyte cell lines according to established methods (11, 23) . The 25 mM Hepes-buffered incubation media contained 30 µg of membrane protein, 30 pMI-cyanopindolol (CYP) (DuPont NEN), 5 µM CL316,243, 10 mM MgCl, and indicated concentrations of various unlabeled competing ligands. The AR-selective agonist CL316,243 was included to block I-CYP binding to a small, but significant, percentage of AR (see legend to Fig. 6). Following incubation at 37 °C for 1 h, the suspensions were filtered through Whatman GF/C filters and rapidly washed four times with ice-cold 0.5 phosphate-buffered saline. The radioactivity remaining on the filters was counted.


Figure 6: Competition binding analysis to determine proportions of AR versus AR in membranes from purified interscapular BA and B7 adipocytes. Plasma membranes prepared from B7 adipocytes or purified mouse brown adipocytes were incubated for 1 h at 37 °C with 30 pMI-CYP and increasing concentrations of unlabeled (-)-propranolol () or CGP20712A (). Five µM CL316,243 was also included in all tubes to eliminate I-CYP binding to a small percentage of AR (, inset). Membrane-bound I-CYP was collected on filters and counted. Data are expressed as B/ B, the fraction of total counts bound in the presence of the competing ligand.



Data Analysis

Adenylyl cyclase dose-response curves and competition binding curves were analyzed by least squares nonlinear regression and assessed for best fit to a 1-component or 2-component model (Graphpad Inplot, San Diego, CA and LIGAND, National Institutes of Health, Bethesda, MD). Test of the adequacy of the 1- versus 2-component model was by an F-test. Comparisons between data sets were made by analysis of variance. A value of p < 0.05 was considered significant.


RESULTS

The expression of individual AR subtypes in four brown adipocyte cell lines was evaluated by Northern blot hybridization (Fig. 2). Comparable levels of AR mRNA transcripts were detected in all four cell lines (B13 not shown) as well as from interscapular brown adipose tissue (IBAT). The estimated size of the AR transcript (2.6 kb) is similar to previous reports in a variety of tissues including adipocytes (11, 30, 42) . AR mRNA was expressed in all the cells and brown adipocytes derived from fresh tissue. The presence of two transcripts for AR, a major 2.4-kb and a minor 2.0-kb species, is similar to our earlier observations (11, 23) . Since these data are from clonal cell lines and purified adipocytes isolated from tissues, it is apparent that the presence of AR mRNA is not simply due to contamination from surrounding vasculature and neurons. The expression of AR mRNA, the AR subtype that is expressed predominantly in adipose tissue (7, 8, 9, 11, 13) , varied the most among the cell lines. By Northern blotting we detected AR mRNA in the B3 and B7 cells, but not in B9 or B13 cells. Fig. 2shows that AR mRNA is most abundant in the B7 cell line and, as we observed previously in white and brown adipose tissue (11) , consists of three distinct AR transcripts: a major species of 2.1 kb and minor transcripts of 2.6 and 3.5 kb. Although we did not detect AR mRNA in the B9 and B13 cells by Northern blotting, AR-specific amplification products were identified in all four cell lines by reverse transcriptase-PCR (Fig. 3). Since B9 and B13 cells also generated a modest response to a highly selective AR agonist (see below), these data together suggest that all four cell lines express AR, with the highest levels of functional AR in B3 and B7 cells.


Figure 2: Northern blot of brown adipocyte cell lines (B3, B7, B9) and IBAT. Forty µg of total cellular RNA from IBAT or adipocytes that had been incubated in the absence (-) or presence (+) of 10 M norepinephrine ( NE) were fractionated through 1.2% agarose gels and blotted onto nylon membranes as described under ``Materials and Methods.'' Blots were hybridized with -P-labeled DNA probes (3.0 10 dpm/ml) for the mouse -adrenergic receptor subtypes (AR, AR, and AR) and selected adipose tissue gene products such as uncoupling protein ( Ucp), IBAT transcription factor ADD1, and glycerol-3-phosphate dehydrogenase ( GPDH). Blots were also hybridized with the ubiquitous glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) as an internal control for the amount of RNA applied to the gel. Final washing conditions were 0.1 SSC, 0.1% SDS at 55 °C.




Figure 3: Identification of AR transcript by reverse transcriptase-PCR. Total cellular RNA (250 ng) from brown adipocyte lines (B3, B7, B9, B13) and IBAT was annealed to the antisense primer and reverse transcribed with Moloney murine leukemia virus reverse transcriptase. The cDNA was amplified by PCR in the presence of 1.5 mM MgCl as described under ``Materials and Methods'' ( + lanes). Control PCR reactions containing 500 ng of mouse genomic DNA ( DNA lane) or RNA without the initial reverse transcriptase step ( - lanes), were included as positive and negative controls, respectively. A water blank was also reverse-transcribed and amplified as a control for reagent contamination ( H 0 lane). The arrow indicates the predicted fragment size of 542 base pairs. Lane M contains HaeIII-digested X174 DNA molecular weight markers ranging from 1353 to 271 base pairs.



We next examined these cell lines for the expression of gene products characteristic of adipocytes and of brown adipocytes in particular. As reported in the initial characterization of the original brown fat tumor cells from which these lines were derived (20, 31) , the brown fat-specific uncoupling protein ( Ucp) was expressed only in response to AR agonists. The lower portion of Fig. 2shows that Ucp expression was readily induced in the B7 and B9 cells by 10 M norepinephrine for 4 h ( ``+'' lanes). By contrast, Ucp was undetectable in the B3 or B13 cells, at least by Northern blot analysis. The level of Ucp expression was greater in the B7 than the B9 cells but was still significantly less than in IBAT. For example, although equal amounts of RNA were analyzed for each sample, the IBAT lane was exposed to film for 8 h and the adipocyte lanes were exposed for 5 days. All four cell lines also contained abundant levels of mRNA for ADD1, a transcription factor preferentially expressed in brown adipose tissue (33) . Expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase (GPDH) was also present, with similar levels of GPDH mRNA in the cell lines and IBAT. Although the expression of these markers was not significantly changed by the presence of 10 M norepinephrine for 4 h, it is interesting to note that the expression of the AR transcript was slightly elevated by norepinephrine in B7 cells ( n = 2). On the other hand, norepinephrine also appeared to decrease the expression of the - and AR transcripts in the B3 and B7 adipocytes ( n = 3).

The functional activity of the ARs in the brown adipocyte lines was assessed by the ability of -agonists to stimulate adenylyl cyclase activity and compared with that observed in brown adipocytes (BA) purified from IBAT (Fig. 4). Since preliminary experiments performed in intact cells and plasma membranes gave essentially identical results, we chose to use plasma membranes in these experiments for greater sampling consistency. The stimulation of adenylyl cyclase activity in BA membranes by norepinephrine, the predominant endogenous ligand for ARs in brown adipose tissue, was complex and best fit to a two-site curve. The high-affinity component of the curve, with a K of 5.2 10 M, accounted for 15.2% of the adenylyl cyclase activity, with the remaining 84.8% from a lower affinity component ( K = 4.2 10 M). Dissection of the norepinephrine dose-response curves into components which could be attributed to all three AR subtypes is difficult, since there is less than a 10-fold difference in the reported K for norepinephrine to stimulate adenylyl cyclase by the individual ARs (43) . The brown adipocyte cell lines responded to norepinephrine with similar maximal levels of stimulation (ranging from 72.8 to 89.6 pmol of cAMP/mg of membrane protein/min). Among the four cell lines, the greatest maximal response was achieved in the B7 and B3 cells and was slightly less than that in the BA membranes. The K of the adipocyte lines ranged from 1.6 10 M to 5.1 10 M. Although the results from B7 cells appeared to fit a two-site model as in the BA from tissue, they were not statistically significant ( p = 0.075). The data from cell lines B3, B9, and B13 were best fit to a single component. Interestingly, the BA consistently displayed a significantly higher basal adenylyl cyclase activity than the BAT cell lines. However, although BA was capable of a greater maximal stimulation, the cell lines actually produced a higher degree of agonist stimulation over basal activity (B3, 14.4-fold; B7, 17.6-fold; B9, 11.3-fold; B13, 10.4-fold) than the BA membranes (3.8-fold). The reason for this difference is not immediately clear.


Figure 4: Norepinephrine-stimulated adenylyl cyclase activity in membranes from purified interscapular BA and brown adipocyte cell lines (B3, B7, B9, B13). The assays contained 10-12 µg of adipocyte membrane protein and were incubated at 37 °C for 10 min. The cAMP produced was measured by radioimmunoassay as described under ``Materials and Methods.'' The data are expressed as picomoles of cAMP produced per mg of membrane protein/min of incubation.



To evaluate more specifically the functional activity of the AR expressed in the brown adipocyte cell lines, we used the highly selective AR agonist CL316,243 (44) to stimulate adenylyl cyclase and compared the results to parallel experiments performed in BA. The BA, B3, and B7 dose-response curves displayed good stimulation (Fig. 5) and were best fit to a single component, as would be expected for an agonist selective for a single receptor subtype. The K of B3 and B7 were comparable with that of the BA membranes (). Once again, the basal adenylyl cyclase activity was much higher in the BA membranes than in the cell lines, whereas the effective -fold stimulation over basal activity was greater in the cell lines (B3, 13.3-fold; B7, 11.7-fold) than the BA membranes (3.5-fold). The dose-reponse curves for the B9 and B13 cells were considerably weaker, only 2.7- and 1.9-fold over basal, respectively. These results in the B9 and B13 lines are consistent with the finding that only a small amount of AR is present in these cells and indicate that the amount of functional AR in these cells is proportional to the level of the AR mRNA.


Figure 5: Stimulation of adenylyl cyclase activity by the AR-selective agonist CL316,243 in membranes from purified interscapular BA and brown adipocyte cell lines (B3, B7, B9, B13). The assays contained 10-12 µg of adipocyte membrane protein and were incubated at 37 °C for 10 min. The cAMP produced was measured by radioimmunoassay as described under ``Materials and Methods.'' The data are expressed as picomoles of cAMP produced per mg of membrane protein/min of incubation.



The B7 cell line is most similar to IBAT according to the characterization of the cell lines by Northern blot analysis and adenylyl cyclase responses using AR agonists. Thus, this cell line was chosen for further characterization. Our data on functional activity and Northern blots indicate the presence of all three AR subtypes. Although a high-affinity radioligand capable of quantitating AR in tissue preparations is not available, we determined the relative proportions of - and AR that comprise the high-affinity component of adenylyl cyclase stimulation (11) . For this purpose competition binding assays were performed using the radioligand antagonist I-CYP (30 pM) and the AR-selective antagonist CGP20712A, which displays 1000-fold selectivity for AR over AR (45) . In these experiments, shown in Fig. 6 , 5 µM CL316,243 was included in all tubes to eliminate binding of I-CYP to a small but significant fraction of AR sites (see insets to Fig. 6 ). The nonselective /AR antagonist (-)-propranolol inhibited I-CYP binding with similar potency in the B7 and BA brown adipocyte plasma membranes (3.9 10 M and 6.9 10 M, respectively). In both BA and B7 membranes the AR-selective antagonist CGP20712A produced distinctly biphasic curves. The high-affinity component of the curve, defined as AR, accounted for 35% of the binding sites in the B7 plasma membranes with an IC of 8.0 10 M. The remaining sites (65%) comprised a low affinity component (IC 10.5 10 M); this 1000-fold weaker affinity for CGP20712A identifies this second component as the AR (45) . The relative proportions of - and AR and the IC values for the brown adipocyte plasma membranes were similar to that of the B7 cells. The high-affinity AR component of the curve had an IC of 5.9 10 M and accounted for 41% of the binding sites, and there was a slightly smaller proportion of AR in the brown adipocytes (59%) with an IC of 15.5 10 M.

Since this immortalized brown adipocyte cell line contains all three AR subtypes, like brown adipocytes derived from fresh tissue, we investigated the functional significance of these three receptor subtypes for the regulation of adenylyl cyclase stimulation and Ucp expression. Cyclic AMP production mediated through the AR is distinguishable from that generated by activation of - and AR with the highly selective AR agonist CL316,243. We examined whether stimulation of individual AR subtypes alone or in combinations produced additive or nonadditive responses. To determine the contribution of individual ARs to total adenylyl cyclase activity, BA and B7 membranes were stimulated by norepinephrine alone, CL316,243 alone, or by norepinephrine in the presence of a saturating concentration (5 µM) of CL316,243. In both BA and the B7 cell line, norepinephrine stimulated adenylyl cyclase activity to a greater extent than the AR agonist CL316,243 (Fig. 7). These results are essentially identical to the data in Figs. 4 and 5. The data indicate that stimulation of all three AR subtypes with norepinephrine leads to a greater activation of adenylyl cyclase activity than stimulation of AR alone with CL316,243. Interestingly, in both BA and the B7 cell line, when 5 µM CL316,243 was included in all tubes, thus raising the base-line activity to the maximal stimulation achieved with CL316,243 alone, the maximal response to norepinephrine under these conditions was essentially identical to that of norepinephrine alone. These data suggest that adenylyl cyclase stimulation through multiple AR subtypes is additive, in the sense that the same maximum level is reached by any route. The ability of AR, together with AR and AR, to produce this additive cAMP response, in both cell lines and brown adipose tissue, suggests that the contribution of the AR receptor to adenylyl cyclase activation is distinct from that attributed to AR or AR. Alternatively, the AR may be less efficiently coupled to this pathway. Finally, our data show that a significant proportion of the functional response to norepinephrine in either BA derived from IBAT or B7 cells can be attributed to - and/or AR.


Figure 7: Subtype-selective stimulation of adenylyl cyclase in membranes from purified brown adipocytes from IBAT or B7 cells is additive. -Adrenergic agonist stimulated adenylyl cyclase activity in membranes from purified interscapular BA and B7 adipocytes. The assays contained 10-12 µg of adipocyte membrane protein and were incubated at 37 °C for 10 min. The cAMP produced was measured by radioimmunoassay as described under ``Materials and Methods.'' The data are expressed as picomoles of cAMP produced per mg of membrane protein/min of incubation. , CL316,243; , norepinephrine; , norepinephrine + 5 µM CL316,243.



One mechanism whereby individual receptor subtypes within the same cell may modulate the extent of adenylyl cyclase activity is through their differential coupling to particular forms of adenylyl cyclase. There are at least 10 distinct genes for adenylyl cyclase; some show complex regulation by G protein or subunits, others are sensitive to intracellular calcium concentrations or calmodulin (46, 47) . We screened brown and white adipose tissues and cell lines for the expression of adenylyl cyclases by Northern blotting (Fig. 8) and reverse transcriptase-PCR coupled with DNA sequencing. Although it is generally difficult to detect adenylyl cyclase transcripts by Northern blot we could identify types 5, 6, and 10 in white and brown adipose tissue and in the B7 brown adipocyte cell line by this method (Fig. 8). Types 6 and 10 were also detected in differentiated 3T3-F442A white adipocytes. Fig. 8presents two different exposures of the blots for types 5 and 6. These two isoforms appear to be more abundant in WAT than in BAT. The sizes of the transcripts identified for type 5 (8.3 and 6.9 kb) and type 6 (6.0 kb) are consistent with previous estimates (48, 49) . Adenylyl cyclase type 10 (8.3 kb), a newly identified form that has a wide tissue distribution() was also identified in both white and brown adipose tissue and cell lines and appears to be more abundant in BAT than WAT. These Northern blot results were further confirmed by reverse transcriptase-PCR analysis of B7 cells and brown adipocytes (BA) isolated from tissue and sequencing of the PCR products. The majority of clones analyzed from both B7 and BA encoded type 10 (14 of 18 for B7; 13 of 14 for BA). The remaining clones were identified as types 3, 6, and 7.


Figure 8: Expression of adenylyl cyclase isoenzymes in white and brown adipose tissues and cell lines. Total cellular RNA was isolated from interscapular BAT and gonadal WAT isolated from C57BL/6J male mice, B7 brown adipocytes, and differentiated 3T3-F442 cells. Forty µg of RNA were fractionated through 1.2% agarose gels and blotted onto nylon membranes as described under ``Materials and Methods.'' Blots were hybridized with -P-labeled mouse DNA probes (3.0 10 dpm/ml) for adenylyl cyclase types V, VI, and X. The fragments correspond to unique regions of each isozyme as detailed in Ref. 36. Final washing conditions were 0.1 SSC, 0.1% SDS at 55 °C. For cyclase types V and VI two exposures of the same blot are shown to clarify the presence of weaker hybridizing species.



Using a variety of selective agonists and antagonists we evaluated the efficacy of individual AR subtypes to activate Ucp transcription. In the first set of experiments (Fig. 9 A), B7 cells were incubated with norepinephrine (10 M). A significant stimulation of Ucp mRNA was observed ( lane 2). This result is essentially identical to that shown in Fig. 2. The highly selective AR agonist CL316,243 was also able to promote Ucp expression ( lane 3), to a slightly greater extent than norepinephrine. However, the most robust induction of Ucp was observed with isoproterenol at a concentration (10 M) which activates all three AR subtypes ( lane 4). Since norepinephrine at a concentration of 10 M would be expected to primarily activate AR (42, 43) , these results suggest that Ucp expression can be induced through either - or AR individually or through the combined activity of all three subtypes. To explore this issue in greater detail and examine specifically the ability of AR and AR to stimulate Ucp expression, we chose to use selective agonists and antagonists for these two AR subtypes. Fig. 9 B shows that isoproterenol, CL316,243, and norepinephrine at 1 µM could stimulate Ucp gene expression with about equal potency. The nonselective AR-/AR-antagonist propranolol significantly reduced the ability of norepinephrine to induce Ucp, whereas either the -selective antagonist CGP20712A or the AR-antagonist ICI118,551 partially inhibited this response. The AR-selective agonist salbutamol was also able to increase Ucp mRNA levels, and this induction was blocked by ICI118,551. Epinephrine (1 µM) was also able to increase Ucp mRNA levels (not shown). Moreover, confirming conclusions of other investigators that Ucp induction is mediated largely by intracellular cAMP (15, 16) , we found in other experiments that the membrane-permeable analogue of cAMP, dibutyryl cAMP (1 mM), and forskolin (100 µM) stimulated Ucp mRNA levels with equal or greater potency as isoproterenol (2.8- and 7.2-fold over isoproterenol, respectively). Together these data suggest that both AR and AR, in addition to AR, are capable of mediating Ucp induction in brown adipocytes.


Figure 9:Ucp mRNA levels in B7 adipocytes exposed to -adrenergic agonists. The cells were either untreated or exposed to various -adrenergic agonists at the indicated concentrations for 4 h. Forty µg of total cellular RNA from the adipocytes were fractionated through 1.2% agarose gels and blotted as described under ``Materials and Methods.'' Blots were hybridized with -P-labeled DNA probes (3.0 10 dpm/ml) for Ucp or the ubiquitous glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase ( G3PDH), to correct for the amount of RNA applied to the gel. Final washing conditions were 0.1 SSC, 0.1% SDS at 55 °C. A, representative Northern blot of B7 cell RNA. Lane 1, untreated; lane 2, 10 M norepinephrine; lane 3, 10 M CL316,243; lane 4, 10 M isoproterenol. B, analysis of ability of AR and AR activation to induce Ucp expression. Cells were treated with the indicated agonists and antagonists for 4 h, and Ucp expression was analyzed by Northern blotting . Iso, isoproterenol (1 µM); CL, CL316,243 (1 µM); NE, norepinephrine (1 µM); Pro, propranolol (0.1 µM); CGP, CGP20712A (0.5 µM); ICI, ICI118,551 (0.5 µM); Sal, salbutamol (1 µM). The data are expressed as the percentage of the induction observed with isoproterenol (1 µM) and are the average of three to four experiments, except salbutamol, which was performed once. Asterisk, significantly different from isoproterenol-treated cells ( p 0.05).




DISCUSSION

We have characterized -adrenergic-mediated signal transduction and Ucp gene expression in four clonal brown adipocyte cell lines derived from a mouse interscapular brown adipose tumor. By RNA analysis and functional assays our results demonstrate that all three AR subtypes are expressed, similar to brown adipocytes isolated from rodent and hamster brown adipose tissue. We found transcripts for - and AR to be present at equivalent levels in all four cell lines, whereas expression of the AR was variable. In the B7 and B3 cell lines, AR transcripts were readily detected by Northern blot hybridization, whereas reverse transcriptase-PCR was required to detect AR mRNA in B9 and B13 cells. Consistent with these findings, stimulation of adenylyl cyclase activity with the AR-selective agonist CL316,243 produced a robust response in B3 and B7 cells and a reduced level of stimulation in B9 and B13 cells. We also determined by competition binding that both B7 cells and brown adipocytes from tissue possess comparable proportions of AR and AR. As we and others have previously discussed (9, 11, 50) , the lack of a high-affinity radioligand capable of detecting the AR in tissue preparations precludes quantitation of this receptor subtype.

We have also shown in these studies that at least three different forms of adenylyl cyclase are expressed in white and brown adipocyte cell lines and tissues. These include adenylyl cyclase types 5 and 6, and the recently identified type 10 enzyme. This new member of the adenylyl cyclase gene family shows a broad mRNA tissue distribution. Relevant to our studies, type 10 appears to be the most abundant form present in white and brown adipose tissue, as well as in the 3T3-F442A and B7 cell lines. It should be noted that while types 5 and 6 are structurally and functionally similar (36, 46) , the type 10 enzyme represents a new subclass of adenylyl cyclase, whose regulatory features have not yet been characterized. Therefore it is possible that differential coupling of receptors to these adenylyl cyclases will regulate distinct physiological responses. This is the first description of the adenylyl cyclases isoforms present in adipocytes, and it will now be important to examine the biochemical and regulatory significance of these isoforms in AR-mediated adipocyte metabolism.

A key characteristic of these brown adipocyte cell lines is that they have retained the ability to express the brown adipocyte-specific Ucp in response to -adrenergic stimulation. They also display patterns of adipocyte-specific gene expression similar to normal brown fat. Remarkably, even though the cell lines have been derived from a hibernoma induced by the ectopic expression of SV40 T-antigen and have the mark of an immortalized, transformed cell, they nevertheless have retained the ability to proliferate in response to stimulation by norepinephrine (31) . Based upon a number of biochemical and functional criteria the B7 cell line, in particular, appears to closely resemble brown adipocytes derived from tissue, thus making it a good model for in vitro studies. For several years adipocyte cell lines such as the 3T3-L1, 3T3-F442A, and ob17 preadipocytes have been useful for investigating the role of hormones on gene expression during white adipose tissue adipogenesis (51) . Similarly, these brown adipocyte cell lines now provide the opportunity to extend the molecular analysis to genes critical for brown adipocyte physiology in a manner that is not possible in vivo. An unusual and valuable feature of these cells that distinguishes them from other white or brown preadipocyte lines is that they display ``differentiated'' characteristics independent of cell density and do not require treatment with glucocorticoids or agents that affect cAMP levels, such as isobutylmethylxanthine, in order to differentiate. The importance of this difference is that these drugs have been shown to directly affect expression of the ARs (23, 24, 29, 30, 53) . Therefore, results from such cultures that purport to examine adrenergic control of adipocyte function must be interpreted with caution. Also unlike our clonal lines, a brown adipocyte line (HIB 1B) recently established by the laboratories of Spiegelman and Ricquier (28, 52) requires a period of differentiation from a preadipocyte state before expressing either Ucp or AR. It is interesting that while the adipogenic morphology of our brown adipocyte clonal lines has been found to be quite stable (31) , the highest degree of phenotypic variability is associated with AR and Ucp expression, and these differences do not seem to vary in tandem. For example, the B3 cell line contains readily detectable AR, but Ucp induction is essentially absent, whereas the converse is true for the B9 clone. These findings imply that some function besides the ability of catecholamines to stimulate the cAMP pathway is critical for Ucp expression. Such elements might include tissue-specific transcription factors or other downstream effectors that are targets of the cAMP-dependent protein kinase.

The -adrenergic signal transduction system in adipocytes is complex. The tissue contains three known AR subtypes that are each coupled to the stimulation of adenylyl cyclase. Moreover, as we have shown, there are at least three adenylyl cyclase isozymes expressed in adipocytes. Activation of the cAMP pathway, in turn, regulates lipolysis in the white adipocyte. However, in the brown adipocyte cAMP also regulates oxygen consumption, Ucp induction, mitochondriogenesis, and cell proliferation: all processes required for nonshivering thermogenesis. Very little is understood about the coupling mechanism between the receptors and these physiological functions. Because of this complexity, it has been unclear whether the individual AR subtypes in brown adipocytes stimulate a common pool of adenylyl cyclase and whether their synergistic activation is required. For example, we do not know whether a specific AR subtype is coupled to a specific adenylyl cyclase to modulate a specific function. Alternatively, the regulation of thermogenesis may be too important to be left to only one receptor subtype, and the signal transduction pathways in brown fat may be very redundant. In fact, one explanation proposed for the existence of three AR subtypes in adipocytes is the potential to circumvent the development of tachyphylaxis, or ``desensitization,'' in order to maintain thermogenic competence (54) . Biochemical evidence suggests that the three ARs display differences in their ability to be phosphorylated by regulatory kinases. Phosphorylation is associated with the rapid phase of receptor desensitization (55) , and the AR has been reported to be the most resistant to this desensitization (54, 56) . However, given the fact that several different adenylyl cyclase isoforms with various types of regulatory modulation (36, 46) are present in white and brown adipocytes, it is also possible that each AR subtype may be coupled to one or more specific adenylyl cyclase isoforms, leading to compartmentalization of the functional adrenergic responses. In this regard, we note that Hollenga et al.(57) reported that the AR-selective agonist BRL37344 was substantially weaker than isoproterenol to raise intracellular cAMP levels in rat white adipocytes, but BRL stimulated lipolysis more efficiently. One interpretation of their finding is that AR might activate some other signal transduction pathway in addition to adenylyl cyclase. However, it is also possible that the AR is coupled to a particular adenylyl cyclase isoform that generates a more effective, functional, second messenger response. In our studies using both subtype-selective and nonselective -agonists, our data are consistent with a model whereby AR-stimulated adenylyl cyclase activity is distinct from that of - and/or AR. In both B7 cells and brown adipocytes derived from mouse tissue, there is a maximum adenylyl cyclase response that can be reached by stimulating all three subtypes simultaneously, and the individual subtypes appear to contribute discrete proportions to the overall response. We do not discount the possibility that the AR is coupled to more than one signal transduction system; clearly this issue must be explored in greater detail in future studies, and the immortalized brown adipocytes that we describe offer an opportunity to delineate these coupling pathways.

It has been reported that the AR regulates brown adipocyte proliferation while the AR plays no role in this process (18) . However, the experiments that have addressed these properties in the past have not fully excluded the possibility that the AR may have been the only receptor expressed on the cells under study. Perhaps a cell capable of proliferating would have responded to a AR agonist if that receptor subtype had been present. One way to address this problem is to have a readily available population of brown fat cells which retain their physiological functions, AR subtypes, and cyclases and are capable of being cloned in vitro to minimize phenotypic variability. These immortalized clonal cell lines satisfy these criteria and provide a model in which to investigate the control of brown adipocyte physiology by the AR system.

The first major physiological function of brown adipocytes that we have examined with regard to adrenergic regulation is Ucp induction. Our experiments demonstrate that norepinephrine at a concentration expected to activate primarily AR (0.1 µM) can induce Ucp expression in both the B7 and B3 cell lines. When Ucp induction was analyzed in more detail in the B7 cell line, we found that Ucp mRNA levels were induced by the AR-selective agonist CL316,243 and also by a concentration of isoproterenol (100 µM) capable of stimulating all three AR subtypes simultaneously. Moreover, directly increasing intracellular cAMP levels with dibutyryl cAMP or forskolin potently stimulates Ucp expression in B7 cells, in agreement with results obtained by other investigators studying primary cultures of brown adipocytes (15, 16) . Finally, using several selective antagonists for AR or AR we also find that activation of either of these two receptors can lead to stimulation of Ucp expression. From these data it would appear that elevation of cAMP levels, stimulated by any of the AR subtypes, can enhance expression of Ucp, but that the greatest response was achieved by stimulation of all three AR subtypes simultaneously. While this work was in progress, similar conclusions were also obtained by Klaus et al.(52) using the HIB 1B mouse hibernoma cell line. In contrast, Zhao et al.(58) , using brown fat cells isolated from normal Syrian hamster, reported that only the AR is capable of stimulating thermogenesis as measured by changes in oxygen consumption. In order to evaluate whether the differences obtained from the various laboratories reflect differences in the experimental cell system or species, it would have been valuable if Zhao et al.(58) had provided direct evidence for the existence of AR and AR, in addition to AR, in the cells they used by some functional criteria such as adenylyl cyclase activation. The pharmacological data that were presented do not fully address this point.

From our studies we conclude that each of the ARs coexisting in a population of brown adipocytes is capable of contributing to Ucp induction. It is not possible to extrapolate from the present results to know whether other processes such as thermogenesis, cell proliferation, and mitochondriogenesis will also be regulated by each of the ARs present in these brown fat cell lines; however, future experiments will address these issues.

  
Table: Kinetic parameters of adenylyl cyclase stimulation by CL316,243

The dose-response curves shown in Fig. 5 were analyzed by nonlinear regression for best fit to a one- or two-site model as described previously (11). K is the activation constant, expressed in molar concentrations, for half-maximal stimulation of adenylyl cyclase by CL316,243. The values in brackets indicate the 95% confidence intervals associated with the estimates. In all cases the analysis indicated best fit to a single class of sites.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK46793 (to S. C.), HD08431 (to L. P. K.), and T32-DK07568 (to E. M. R.) and by the Stanback Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Mal Tyor Junior Faculty Scholar Fund of Duke University Medical Center. To whom correspondence should be addressed: Box 3557, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8991; Fax: 919-684-3071.

The abbreviations used are: AR, -adrenergic receptor; BA, brown adipocyte; CYP, cyanopindolol; BAT, brown adipose tissue; IBAT, interscapular brown adipose tissue; PCR, polymerase chain reaction; Ucp, mitochondrial uncoupling protein; WAT, white adipose tissue; kb, kilobase(s).

R. T. Premont, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Thomas Gettys for the gift of antisera to cAMP, Dr. Thomas Claus for the gift of CL316,243, and Drs. Marc Caron and Robert Lefkowitz for advice and helpful discussions.


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