©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeted Disruption of the -Adrenergic Receptor Gene (*)

(Received for publication, May 12, 1995; and in revised form, October 6, 1995)

Vedrana S. Susulic (1) Robert C. Frederich (1) Joel Lawitts (2) Effie Tozzo (1) Barbara B. Kahn (1) Mary-Ellen Harper (3) Jean Himms-Hagen (3) Jeffrey S. Flier (1) Bradford B. Lowell (1)(§)

From the  (1)Division of Endocrinology, Department of Medicine, and the (2)Department of Pathology, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215 and the (3)Department of Biochemistry, The University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta(3)-Adrenergic receptors (beta(3)-ARs) are expressed predominantly in white and brown adipose tissue, and beta(3)-selective agonists are potential anti-obesity drugs. However, the role of beta(3)-ARs in normal physiology is unknown. To address this issue, homologous recombination was used to generate mice that lack beta(3)-ARs. This was accomplished by direct injection of a DNA-targeting construct into mouse zygotes. Twenty-three transgenic mice were generated, of which two had targeted disruption of the beta(3)-AR gene. Mice that were homozygous for the disrupted allele had undetectable levels of intact beta(3)-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional beta(3)-ARs, as demonstrated by complete loss of beta(3)-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. beta(3)-AR-deficient mice had modestly increased fat stores (females more than males), indicating that beta(3)-ARs play a role in regulating energy balance. Importantly, beta(1) but not beta(2)-AR mRNA levels up-regulated in white and brown adipose tissue of beta(3)-AR-deficient mice (brown more than white), strongly implying that beta(3)-ARs mediate physiologically relevant signaling under normal conditions and that ``cross-talk'' exists between beta(3)-ARs and beta(1)-AR gene expression. Finally, acute treatment of normal mice with CL 316,243 increased serum levels of free fatty acids (FFAs) (3.2-fold) and insulin (140-fold), increased energy expenditure (2-fold), and reduced food intake (by 45%). These effects were completely absent in beta(3)-AR-deficient mice, proving that the actions of CL are mediated exclusively by beta(3)-ARs. beta(3)-AR-deficient mice should be useful as a means to a better understanding of the physiology and pharmacology of beta(3)-ARs.


INTRODUCTION

``Atypical'' beta-ARs (^1)were originally identified pharmacologically, as beta-AR-like activity that resisted blockade by classical beta-AR antagonists(1, 2) . Atypical beta-AR activity has been observed predominantly in white and brown adipose tissue (1, 2) and the gastrointestinal tract(3, 4) . Further evidence for the existence of atypical beta-ARs has been the synthesis of novel beta-AR agonists that potently stimulate lipolysis, energy expenditure, and gut relaxation, while having little or no effect on beta(1)- or beta(2)-AR-mediated processes(5, 6, 7) . Because atypical beta-AR agonists increase energy expenditure, these compounds are currently being developed as anti-obesity agents(5, 6, 7) .

The gene encoding a third member of the human beta-AR family, designated the beta(3)-AR, has been cloned and characterized (8) . More recently, rat (9, 10) and mouse (11) beta(3)-AR genes have also been isolated. The RNA message encoding the beta(3)-AR is found predominantly in white and brown adipose tissue and the gastrointestinal tract(9, 10, 11) . Pharmacologic characterization of cell lines expressing recombinant receptors has demonstrated that cloned beta(3)-ARs are relatively resistant to blockade by conventional beta-AR antagonists and are potently stimulated by atypical beta-AR agonists(8, 9, 10, 11) . Given that atypical beta-AR activity and cloned beta(3)-ARs have similar tissue distributions and pharmacologic profiles, it is generally accepted that the beta(3)-AR is the atypical beta-AR(2, 12) .

beta(1)-, beta(2)-, and beta(3)-ARs all couple via Galpha to adenylate cyclase, leading to an increase in cAMP. The resulting activation of protein kinase A mediates the major actions of these receptors which include stimulation of lipolysis in white adipocytes and thermogenesis in brown adipocytes (for review, see (13) ). Since adipose tissue expresses all three beta-ARs, it has been difficult to determine the functional significance of each subtype. A distinguishing feature of the beta(3)-AR is that it appears to be relatively resistant to desensitization and down-regulation(14, 15, 16, 17, 18, 19) . This has led to the hypothesis that its primary function might be to maintain signaling during periods of sustained sympathetic stimulation, such as that which occurs in brown adipose tissue during periods of chronic cold exposure or diet-induced thermogenesis.

Murine white adipose tissue expresses beta(1)-, beta(2)-, and beta(3)-AR mRNA transcripts at a ratio of 3:1:150, respectively(20) , with a qualitatively similar relationship observed in brown adipose tissue, suggesting that beta(3)-ARs might play a dominant role. Because of the absence of radioligands capable of detecting beta(3)-ARs, however, it has been difficult to determine the relative number of beta(3)- versus beta(1)- and beta(2)-AR binding sites. Historically, beta-AR subtypes in adipose tissue have been quantified functionally by measuring beta-AR agonist-stimulated adenylate cyclase activity in isolated membrane preparations, followed by the resolution of dose-response curves into low (beta(3)-AR) and high (beta(1)- and beta(2)-ARs) affinity components. Using such an approach it has been estimated that 23% of maximally stimulated adenylate cyclase activity in mouse adipose tissue is mediated by beta(1)- and beta(2)-ARs and that 77% is mediated by beta(3)-ARs(20) . In contrast, using rat adipocyte membranes it has been estimated that 70% of maximal stimulation is due to beta(1)- and beta(2)-ARs, and 30% is due to beta(3)-ARs(14) . Additional complexity is introduced when considering downstream responses such as lipolysis in white adipocytes or thermogenesis in brown adipocytes. Since only modest increases in cAMP are required to produce maximal activation(21, 22, 23) , it is likely that large increases in lipolysis or thermogenesis might be observed with occupancy of only a fraction of available beta-ARs (beta(1), beta(2), or beta(3)). Studies of lipolysis using rat adipocytes have been conflicting, concluding that beta(1)- and beta(2)-ARs play either an important role (24) or a minor role relative to beta(3)-ARs(25) . With respect to isolated brown adipocytes, it has been suggested that beta(3)-ARs are predominantly responsible for mediating norepinephrine-induced thermogenesis(26) .

A number of in vivo studies have provided clues regarding the possible physiologic significance of beta(3)-ARs. In genetically obese fa/fa rats (10) and ob/ob mice(20) , beta(3)-AR mRNA levels are significantly down-regulated, raising the possibility that decreased beta(3)-AR function might contribute to the development of obesity in these animals. Of note, it has been reported recently that a missense mutation of the human beta(3)-AR tends to be associated with obesity, decreased energy expenditure, reduced insulin sensitivity, and earlier onset of non-insulin-dependent diabetes(27, 28, 29) . These observations suggest that beta(3)-ARs might also play an important role in humans as well.

In summary, significant uncertainty exists regarding the physiologic importance of beta(3)-ARs as well as the relative role of beta(3)versus beta(1)- and beta(2)-ARs in mediating beta-AR signal transduction in white and brown adipose tissue. In an attempt to clarify these issues, we have used homologous recombination to generate mice that lack beta(3)-ARs.


EXPERIMENTAL PROCEDURES

Targeting Constructs

Using a mouse 129/SvJ genomic library (Stratagene) and published mouse beta(3)-AR sequence(11) , beta(3)-AR genomic clones were obtained and mapped (Fig. 1). Two targeting constructs were generated: beta(3)-KO+TK (for ES cell studies) and beta(3)-KO (for zygote microinjections) (Fig. 1). To construct the vectors, the 5` side of the targeting vector (from BamHI to XhoI) was directionally subcloned into PGEM-11 (Promega) to generate p5`beta(3)-AR. A segment of beta(3)-AR coding sequence was removed by replacing the 306-bp beta(3)-AR NheI and XhoI fragment (corresponding to beta(3)-AR residue 120, in the middle of the third transmembrane domain, to residue 222, at the COOH-terminal end of the fifth transmembrane domain) with a PGK-NEO-Poly(A) expression cassette, excised from pPGK-NEO-BKS (see below) with XbaI and SalI, to create p5`beta(3)-AR+NEO. The right side of the targeting vector (from XhoI to SalI) was subcloned into PGEM-11 at the XhoI and SalI sites to create p3`beta(3)-AR (p3`beta(3)-AR clones with intact XhoI and SalI sites were selected). The insert was excised from p5`beta(3)-AR+NEO with NotI and SalI and subcloned into p3`beta(3)-AR opened at NotI and XhoI to create the mouse zygote targeting plasmid pbeta(3)-KO. To generate the ES cell targeting plasmid (pbeta(3)-KO+TK), the entire insert was excised from pbeta(3)-KO with NotI and SalI and subcloned into pHSV-TK-PGEM (see below) opened at NotI and XhoI. pPGK-NEO-BKS was prepared by subcloning the PGK-NEO-Poly(A) expression cassette (30) into bluescript KS at the EcoRI and HindIII sites. pHSV-TK-PGEM was prepared by subcloning the HSV-TK expression cassette pIC19R/MC1-TK (31) excised with XhoI and SalI into PGEM-11 at XhoI and SalI (clones with intact XhoI and SalI sites were selected).


Figure 1: The beta(3)-AR gene, targeting vectors, and Southern blot detection schemes. Shown is a partial restriction enzyme map of the beta(3)-AR gene, the beta(3)-KO (mouse zygote) and beta(3)-KO+TK (ES cell) targeting vectors, and the predicted structure of the recombinant allele. The empirically determined map is consistent with a previously reported genomic map (11) . The targeting vectors contain 12 kb of homologous beta(3)-AR genomic DNA, with 5 kb located 5` and 7 kb located 3` of the PGK-NEO-Poly(A) cassette(30) . The PGK-NEO-Poly(A) vector replaces 306 bp of beta(3)-AR coding sequence between NheI and XhoI corresponding to beta(3)-AR residue 120, in the middle of the third transmembrane domain, to residue 222, at the COOH-terminal end of the fifth transmembrane domain. In addition, the beta(3)-KO+TK targeting vector contains the HSV-TK expression cassette, pIC19R/MC1-TK(31) , as well as plasmid sequence on the 3` end. Southern blot probes A and C are located outside of the targeting vector sequence. Boxes refer to exons, the locations of which have been described previously(65, 69) . The translated segments are shown in black. Arrows refers to orientation of transcription. B, BamHI; H, HindIII; K, KpnI; N, NheI; P, PstI; S, SalI; X, XhoI.



ES Cell Culture

J1-ES cells, derived by E. Li and R. Jaenisch from a 129/terSv embryo, were cultured on -irradiated neomycin-resistant primary embryo fibroblasts as described previously(32) . Fifteen million ES cells were electroporated with 20 µg of pbeta(3)-KO+TK (linearized with NotI) and plated on 10 10-cm plates. On the following day, 5 plates received G418 (182 µg/ml active dose), and the other 5 plates received G418 and FIAU (0.2 µM 1-[2-deoxy,2-fluoro-beta-D-arabinofuranosyl]-5-iodouracil; Bristol Meyers). Following 8 days of selection, drug-resistant clones were expanded individually, and genomic DNA was obtained for Southern blot analysis ( Fig. 1and Fig. 2).


Figure 2: Southern blot analysis of ES cell and mouse tail genomic DNA. Genomic DNA was digested with PstI, electrophoresed, blotted, and then hybridized to probe A (Fig. 1). Panel A, ES cell clone DNA. Three G418-resistant ES cell clones are shown. Clones 2 and 3 have targeted disruption of the beta(3)-AR gene. B, mouse tail DNA. Two mice known to be wild type (+/+), two knockout founders (B1-F and B2-F), two heterozygous offspring of the founders (B1-30 and B2-5), and two homozygous null offspring of a cross between B1-30 and B2-F (B1.2 (-/-)) are shown.



Zygote DNA Microinjections

Prior to injection, the beta(3)-KO targeting vector was excised from pbeta(3)-KO using NotI and SalI and then separated from the plasmid sequence using gel purification. Approximately 300-500 copies of the 14-kb beta(3)-KO insert were injected into the male pronucleus of zygotes using standard techniques(33) . Zygotes for injection were generated by mating 129/SvJ females and males (129/SvJ zygotes), FVB/N females and 129/SvJ males (FVB/N [mult] 129/SvJ hybrid zygotes), or FVB/N females and males (FVB/N zygotes). Injected zygotes were transferred to FVB/N pseudopregnant recipients as described previously(33) , and genomic DNA was obtained from live born offspring for Southern blot analysis (Fig. 2). All animals had free access to food (Purina Chow 5008) and water and were handled in accordance with the principles and guidelines established by the National Institutes of Health.

DNA and RNA Analyses

Genomic DNA was isolated from ES cells and mouse tails by sodium dodecyl sulfate/proteinase K digestion followed by salt precipitation(34) . The DNA was analyzed using standard Southern blotting techniques(35) . Total RNA was isolated from interscapular brown adipose tissue, epididymal white adipose tissue, and liver using either the guanidium-cesium chloride approach (Fig. 3) (35) or RNAzol (Fig. 4) (Cinna/Biotecx Laboratory, Houston, TX). beta(1)-, beta(2)-, beta(3)-AR and actin mRNAs were detected using standard Northern blotting techniques and 30 µg of total RNA(35) . Murine beta(1)-(36) , beta(2)-(37) , beta(3)-AR(11) , and beta-actin (38) hybridization probes were generated using random priming from cDNA templates corresponding to codons 88-176 of the beta(1)-AR (263 bp), codons 230-386 of the beta(2)-AR (465 bp), codons 120-222 of the beta(3)-AR (306 bp), and codons 220-303 of beta-actin (250 bp). Note that the 306-bp probe used to detect beta(3)-AR mRNA levels represents the same NheI-XhoI beta(3)-AR fragment deleted during the construction of the targeting vector (Fig. 1). beta(3)-AR and beta-actin mRNAs were also detected using an RNase protection assay(39) . The radioactive beta(3)-AR and actin cRNA probes were transcribed from the fragments described above. RNA samples (20 µg of total RNA) were hybridized with both P-labeled cRNA probes (0.5 ng) at 30 °C for 18 h. Nonhybridized probes were digested with an RNase mixture (40 µg/ml RNase A, 2 µg/ml RNase T1) for 1 h at 37 °C. The protected probes were resolved on a nondenaturing 5% acrylamide gel. The gels were dried and exposed to autoradiographic film.


Figure 3: beta(3)-AR RNase protection assay. beta(3)-AR and actin mRNA levels were determined in brown adipose tissue and liver samples using an RNase protection assay as described under ``Experimental Procedures.'' Samples were obtained from the offspring of a cross between two heterozygous mice. Genotype was determined by Southern blotting.




Figure 4: beta(1)-, beta(2)-, and beta(3)-AR mRNA levels in white and brown adipose tissue by Northern blotting. beta(1)-, beta(2)-, and beta(3)-AR mRNA levels were determined by Northern blotting using 30 µg of total RNA isolated from epididymal white adipose tissue and interscapular brown adipose tissue of 5 control (C) and 5 beta(3)-AR-deficient (K) 12-week-old male mice. Representative lanes from two control and two beta(3)-AR-deficient samples are shown. PhosphorImager analyses of Northern blots were performed using all five control and five beta(3)-AR-deficient samples (PhosphorImager data are described under ``Results''). The blots were also hybridized with a beta-actin probe. Northern blotting and the generation of probes were performed as described under ``Experimental Procedures.'' The approximate sizes (in kb) of the detected signals are as follows: beta(1)-AR, 2.4; beta(2)-AR , 2.2; beta(3)-AR, 2.4; and beta-actin, 2.1.



Body Weight, Perigenital Fat Pad Weights, and Total Body Lipid Analyses

Studies were performed on animals that had free access to water and chow (Purina 5008) and were maintained at 23 °C. All mice were of the following genotype: +/+ or -/- at the beta(3)-AR gene locus on an inbred FVB background. Two separate experiments were performed and the results are shown in Table 2. In experiment 1, male and female control (+/+) and null (-/-) littermates of heterozygous parents were weaned at age 21 days and housed individually. Body weights were obtained at the age of 12 weeks. In experiment 2, control (+/+) offspring derived from wild type parents and null (-/-) offspring derived from null parents were weaned at age 21 days. Only offspring from litters containing 9-11 mice were used; all others were excluded. The males were housed individually, and females of similar genotype were housed two/cage. Body weight, perigenital fat pad weights (male, epididymal fat pads; female, parametrial fat pads), and carcasses were analyzed at the age of 15 weeks. For each individual animal, the paired fat pads were combined and weighed. Total body lipid content was assessed using alcoholic potassium hydroxide digestion with saponification of all fats, neutralization, and then enzymatic determination of glycerol as described previously(40, 41) .



Brown Adipose Tissue and Chronic Cold Exposure

Male control (+/+) and null (-/-) animals, age 10 weeks, were housed individually with free access to water and chow. The animals were divided and maintained at either room temperature (23 °C) or at 4 °C. Three weeks later, the interscapular depot of brown fat was obtained and assessed for the following parameters: wet weight, total protein, DNA and uncoupling protein (UCP) content. Protein and UCP were determined as described previously(41) , and DNA was quantitated using the Hoechst dye 33258 and a Hoefer fluorometer according to the manufacturer's recommended protocol (Hoefer Scientific Instruments, San Francisco, CA).

Lipolysis and Adenylate Cyclase Assays

Adipocytes were isolated from epididymal fat pads of male mice using collagenase digestion as described previously(42, 43) . For analysis of adenylate cyclase activity, membranes were obtained from either isolated white adipocytes (44) or interscapular brown adipose tissue (45) by centrifugation as described previously. Isolated membranes were assayed for adenylate cyclase activity in the presence of varying concentrations of agonists over a 30-min period according to the method of Salomon(46) , modified by the addition of tritiated cAMP to correct for cAMP recovery.

For analysis of lipolysis, isolated white adipocytes (100 µl of a 10% isolated fat cell suspension) were incubated in a final volume of 500 µl, and glycerol release was measured over a 15-min period. Previous studies have demonstrated that glycerol release is linear for at least 15 min of incubation. (^2)Except when noted, the incubation medium consisted of a Krebs-Ringer-Hepes (30 mM) buffer (pH 7.4) supplemented with 2.5% bovine serum albumin (fraction V), 10 µM PIA (N^6-[R-(-)-1-methyl-2-phenyl]adenosine), 1 unit/ml adenosine deaminase, and varying concentrations of agonists. Glycerol content of the incubation medium was determined using a sensitive radiometric assay(47) , and fat cell number was assessed as described previously(48, 49) .

In Vivo Effects of beta-AR Agonists on Serum Levels of FFAs, Glycerol, Glucose, and Insulin

CL, isoproterenol, or saline was injected intraperitoneally into control and beta(3)-AR-deficient mice; 15 min later the animals were quickly sacrificed using a small animal decapitator. Whole blood was collected and analyzed for blood glucose levels (One Touch Blood Glucose Meter, Lifescan, Inc., Milpitas, CA). Serum was then isolated and assayed for FFAs (NEFA C kit, Wako Pure Chemical Industries, Ltd.), glycerol (GPO-Trinder Kit, procedure 337, Sigma), and insulin (rat insulin kit, Linco Research Inc., St. Louis, MO).

In Vivo Effects of beta-AR Agonists on O Consumption

Oxygen consumption was measured as described previously (41) in 12-week-old control and beta(3)-AR-deficient male mice. The animals were conscious for studies using CL and isoproterenol but were anesthetized for studies using norepinephrine.

In Vivo Effects of CL 316,243 on Food Intake

Control and beta(3)-AR-deficient male mice, 8 weeks old, were treated with an intraperitoneal injection of either saline or CL (1 mg/kg) on day 0. To acclimate the mice to injections and handling, saline was injected daily for 3 days preceding day 0 of the study. The mice were housed individually during the study. Food was weighed before injection and 24 h after, and the differences were assumed to represent g of food eaten per day. The cages were inspected for food spillage, and none was noted.

Reagents and Statistics

All reagents, except where noted, were obtained from Sigma and were of the highest reagent grade. CL 316,243 was kindly provided by Dr. Thomas Claus and Dr. Elliot Danforth Jr. (American Cyanamid Co., Pearl River, NY). Tritiated cAMP and [alpha-P]ATP were obtained from ICN (Costa Mesa, CA). All statistical analyses were performed using the unpaired, two-tailed t test.


RESULTS

Targeting in ES Cells

The beta(3)-KO+TK construct (Fig. 1) was electroporated into J1-ES cells and drug-resistant clones were selected. Genomic DNA was isolated and subjected to Southern blot analysis ( Fig. 1and 2A). Approximately 50% of the G418-resistant clones and 75% of the G418, FIAU doubly resistant clones had targeted disruption of the beta(3)-AR gene (Table 1), indicating that homologous recombination events occurred at a frequency that equaled random integration events. Similarly high targeting frequencies have been observed with other genes(50) .



Targeting by Direct Injection of DNA Vectors into Mouse Zygotes

A previous study demonstrated that homologous recombination can occur following microinjection of DNA into mouse zygotes(51) . However, this approach appeared to be inefficient since gene targeting occurred in only 1 of 506 transgenic mice. Encouraged by the high targeting frequency observed in ES cells in the present study, a modified vector (beta(3)-KO, Fig. 1) was then microinjected into the pronucleus of mouse zygotes to determine whether homologous recombination at the beta(3)-AR locus might occur in the one cell embryo. Initially, injections were performed using inbred 129/SvJ mouse zygotes since these would be isogenic with the targeting vector (derived from a 129/SvJ mouse genomic library). However, the poor breeding performance of 129/SvJ mice severely limited these efforts. To circumvent this problem, hybrid zygotes were used (129/SvJ males mated with FVB/N females), and the targeting vector was injected into the peripheral, presumably male (129/SvJ), pronucleus. The targeting vector was also injected into inbred FVB/N zygotes. As shown in Fig. 2B, two transgenic lines with targeted disruption of the beta(3)-AR were generated (B1 and B2). Further analysis revealed that the B1 founder was mosaic for the targeting event, transmitting the disrupted allele to only 2 of 31 offspring and that the B2 founder had a second integration event that was random (as shown below). B2 transmitted the disrupted allele to 17 of 35 offspring but also transmitted DNA encoding the neomycin-resistant cassette to 5 of 11 offspring that were wild type at the beta(3)-AR locus (data not shown). Thus, the B2 founder had a second, unlinked, random integration event. Offspring of B2 which were heterozygous at the beta(3)-AR locus and lacked any unlinked, random integration event as determined by breeding studies and Southern blot analyses, were further expanded and analyzed. An initial cross of two heterozygous mice resulted in the following eight offspring: two wild type mice, four heterozygous mice, and two homozygous ``null'' mice (the latter two null samples are shown in Fig. 2B as B1.2 (-/-)).

Shown in Table 2is a summary of all mouse zygote injections. Using the beta(3)-KO vector, 513 zygotes were injected and transferred into foster mothers resulting in 158 live born mice of which 23 were found to be transgenic. Of these 23 transgenic mice, 2 had targeted disruption of the beta(3)-AR gene (B1 and B2). Of note, the two targeting events occurred in the FVB/N transgenic mice that were not isogenic with the targeting vector (129/SvJ).

beta-AR mRNA Levels by RNase Protection Assay

To determine whether beta(3)-AR null mice lack intact beta(3)-AR mRNA, an RNase protection assay was performed on RNA obtained from brown adipose tissue. The probe used in the protection assay corresponded to the 306-bp segment of beta(3)-AR coding sequence deleted during the construction of the targeting vector (Fig. 1). As is shown in Fig. 3, a protected beta(3)-AR signal was present in wild type and heterozygous mice but was absent in null mice. No signal was observed in liver RNA obtained from wild type mice, a tissue known not to express the beta(3)-AR gene. A control signal for actin was observed in all lanes. Thus, beta(3)-AR null mice do not express detectable amounts of intact beta(3)-AR mRNA.

beta-, beta-, and beta-AR mRNA Levels in White and Brown Adipose Tissue by Northern Blotting

To determine whether beta(1) and/or beta(2)-AR mRNA levels up-regulated in adipose tissue of mice lacking beta(3)-ARs, Northern blotting was performed using total RNA isolated from epididymal white adipose tissue and interscapular brown adipose tissue of control and beta(3)-AR-deficient male mice. As is shown in Fig. 4, intact beta(3)-AR mRNA was not detected in white or brown adipose tissue of knockout mice. Given that no signal was detected using the same beta(3)-AR probe in a sensitive RNase protection assay (Fig. 3), it can be concluded that the extremely faint signal observed in knockout samples by Northern blotting (Fig. 4) represents nonspecific background. beta(2)-AR mRNA levels were unchanged in white and brown adipose tissue of knockout animals (Fig. 4), indicating that compensatory increases in this family member do not occur in beta(3)-AR-deficient mice. In contrast, beta(1)-AR mRNA levels significantly up-regulated in brown adipose tissue of knockout mice, with a smaller increase observed in white adipose tissue (Fig. 4). PhosphorImager analysis (Molecular Dynamics, Image Quant software) of Northern blots containing white and brown adipose tissue RNA derived from five control and five knockout mice indicated that beta(1)-AR mRNA levels were increased by 76% in brown adipose tissue (p < 0.01) and by 42% in white adipose tissue (p < 0.05) of beta(3)-AR-deficient mice.

Phenotype of beta-AR-deficient Mice

In total, 166 offspring of heterozygous male and female mice were analyzed for genotype at the age of 21 days, and the following distribution was observed: +/+, n = 45; +/-, n = 80; and -/-, n = 41. These results closely approximate the expected ratio of 1:2:1, indicating that beta(3)-AR deficiency does not adversely affect pre- or postnatal viability.

Data shown in Table 3indicate that there is a tendency for beta(3)-AR-deficient mice to have greater lipid stores (females more than males). In experiment 1, the body weights of female but not male null mice were increased by 19% (p < 0.05). However, in experiment 2, the mean body weights of male and female null animals were only slightly increased (not statistically significant). In males, total body fat stores were increased slightly by 34% (p < 0.05), although epididymal fat pad weights were not significantly greater. In females, fat stores were more markedly increased, with total body fat stores of null females being 131% greater than controls (p < 0.01) and parametrial fat pads from null animals weighing 99% more than controls (p < 0.05). Blood glucose, serum insulin, and FFA levels, all assessed in the fed state, as well as FFA levels following a 2-day fast, were normal in null animals (data not shown). Food intake was assessed in experiment 1 and was found to be unchanged in beta(3)-AR-deficient mice (data not shown).



Cold exposure-induced hypertrophy of brown adipose tissue is a well documented phenomenon (for review see (52) ) which is thought to be mediated by norepinephrine released from sympathetic nerve terminals. When control (+/+) mice were cold-exposed for 3 weeks, brown fat weight increased by 78%, brown fat protein content by 142%, brown fat DNA content by 66%, and UCP content by 320% (Table 4). Each of these cold exposure-induced responses occurred normally in beta(3)-AR-deficient animals. In addition, colonic temperatures in control and null mice were normal (37 °C) throughout the 3-week study period. Thus, beta(3)-ARs are not required for cold exposure-induced hypertrophy of brown adipose tissue.



Adenylate Cyclase Activity and Lipolysis

To determine the functional consequences of beta(3)-AR deficiency on beta-adrenergic signaling, the effects of beta-adrenergic agonists on adenylate cyclase activity and lipolysis were assessed. For determination of adenylate cyclase activity (Fig. 5), plasma membranes were obtained from isolated white adipocytes or interscapular brown adipose tissue and then incubated in the presence of various agonists. Adenylate cyclase activity was assessed by quantitating the production of cAMP(46) . To assess lipolysis (Fig. 6, A and B), white adipocytes were isolated from epididymal fat pads and then incubated in the presence of various agonists. Rates of lipolysis were determined by quantitating the release of glycerol.


Figure 5: Adenylate cyclase activity in response to CL 316,243 and isoproterenol. Membranes were obtained from isolated white adipocytes and brown adipose tissue of 8-12-week-old male wild type (+/+) and beta(3)-AR-deficient (-/-) littermates and then assayed for adenylate cyclase activity. Panel A, dose-response curves for stimulation by isoproterenol (ISO, a nonselective beta-AR agonist) and CL (a beta(3)-AR selective agonist). Results are expressed as percentage of maximal stimulation by isoproterenol in lean membranes and are the mean (± S.E.) of three replicates. Panel B, adenylate cyclase response to maximally, or near maximally effective doses of CL and isoproterenol. Results are expressed as the mean (± S.E.) of 10 experiments.




Figure 6: Lipolysis in isolated white adipocytes. White adipocytes were isolated from epididymal fat pads of 8-12-week-old male wild type (+/+) and beta(3)-AR-deficient (-/-) littermates and then assayed for glycerol release as an indicator of lipolysis. Previous studies using wild type adipocytes demonstrated that 10 µM CL, 100 µM isoproterenol (ISO), and 10 µM ACTH produced maximal increases in lipolysis. Panel A, lipolysis assays performed in the presence of adenosine deaminase (ADA) and PIA. Average fat cell sizes (ng of lipid/cell) were 216 ± 14 for control cells and 248 ± 22 for null cells. Results are expressed as the mean (± S.E.) of 10 experiments. Panel B, lipolysis assays performed in the presence of adenosine deaminase, with or without PIA. Average fat cell sizes (ng of lipid/cell) were 203 ± 18 for control cells and 167 ± 10 for null cells. Both panels represent results of assays performed on the same day (with or without PIA) and are the mean (± S.E.) of three experiments.



Maximally effective doses of CL, a beta(3)-selective agonist(7) , markedly stimulated adenylate cyclase activity in membranes (Fig. 5) and lipolysis in adipocytes (Fig. 6, A and B) of wild type mice. These responses, however, were completely absent in membranes and cells derived from beta(3)-AR-deficient mice ( Fig. 5and Fig. 6, A and B). Qualitatively similar results were obtained with submaximal doses of CL (data not shown for lipolysis assays). These results demonstrate that beta(3)-AR-deficient mice lack functional beta(3)-ARs and confirm that the stimulatory effect of CL on these processes is mediated exclusively by beta(3)-ARs.

As expected, maximally effective doses of isoproterenol, a nonselective beta-AR agonist, markedly stimulated adenylate cyclase activity in membranes derived from wild type mice (Fig. 5). In membranes obtained from beta(3)-AR-deficient mice, however, the stimulatory effect of isoproterenol on adenylate cyclase activity was reduced (by 70% in membranes derived from brown adipose tissue and by 80% in membranes derived from isolated white adipocytes). Qualitatively similar results were obtained with submaximal doses of isoproterenol. Thus, beta(3)-ARs appear to be responsible for mediating 70-80% of isoproterenol-induced maximally stimulated adenylate cyclase activity. It is likely that beta(1)- and beta(2)-ARs mediate the remaining 20-30%.

Assessment of isoproterenol-stimulated lipolysis in control versus beta(3)-AR-deficient adipocytes revealed additional complexity, which involved an interaction between A(1) adenosine receptors and the relative roles of beta(3)versus beta(1)- and beta(2)-ARs. Adenosine induces an inhibitory effect on adenylate cyclase, mediated by A(1) adenosine receptors and Galpha(i). It has been recognized previously that significant but variable amounts of adenosine are generated during the isolation and incubation of adipocytes and that this can have a confounding influence on lipolysis assays(53, 54, 55) . To circumvent this problem, it has become common practice to remove endogenously generated adenosine through the addition of adenosine deaminase and to add a stable A(1) adenosine receptor agonist (usually PIA). This procedure effectively clamps the influence of adenosine at a fixed level. In the present study, lipolysis assays were initially performed in the presence of adenosine deaminase and PIA (+ADA, +PIA in Fig. 6). As is shown in Fig. 6A, a maximally effective concentration of isoproterenol stimulated lipolysis in wild type adipocytes by about 5-fold. This response, however, was completely absent in beta(3)-AR-deficient adipocytes. The absence of isoproterenol-stimulated lipolysis in beta(3)-AR-deficient adipocytes was again observed in a latter series of experiments performed under identical conditions (+ADA, +PIA in Fig. 6B, left panel). In contrast, when the adenosine agonist was omitted from the assay (+ADA, -PIA in Fig. 6B, right panel) isoproterenol-stimulated lipolysis in beta(3)-AR-deficient adipocytes was only slightly reduced (by 33%). Of note, isoproterenol-stimulated lipolysis in control adipocytes was only minimally inhibited by PIA (Fig. 6B, left panel versus right panel). Thus, when PIA was present during the assay, beta(3)-ARs appeared to be solely responsible for mediating isoproterenol-stimulated lipolysis. When PIA was absent and endogenous adenosine was removed, beta(1)- and/or beta(2)-ARs appeared to mediate the majority of isoproterenol-stimulated lipolysis.

Finally, adrenocorticotropic hormone (ACTH), which also stimulates lipolysis via a seven-transmembrane receptor-Galpha(s)-adenylate cyclase-coupled mechanism(56, 57) , increased lipolysis equally well in adipocytes derived from wild type and beta(3)-AR-deficient mice (Fig. 6A). Similarly, forskolin, a direct activator of adenylate cyclase, stimulated lipolysis equally well in wild type and beta(3)-AR-deficient cells (Fig. 6A). Normal responsiveness to ACTH and forskolin makes it unlikely that impaired responsiveness of beta(3)-AR-deficient cells to isoproterenol results from a direct inhibitory effect of beta(3)-AR deficiency on Galpha(s), adenylate cyclase, or more distal components of the activation pathway for lipolysis.

In Vivo Effects of Adrenergic Agonists on Lipolysis and Thermogenesis

To determine the effect of beta(3)-AR deficiency on beta-adrenergic signaling in vivo, various adrenergic agonists were administered to control and knockout mice, and the effects on serum levels of FFAs and glycerol and thermogenesis were assessed. Treatment of control mice with the beta(3)-selective agonist, CL, produced a 3.2-fold increase in serum FFA levels (Table 5) and a 2-fold increase in oxygen consumption (Fig. 7). Both of these effects were totally absent in beta(3)-AR-deficient mice, indicating that they are mediated exclusively by beta(3)-ARs. When control mice were treated acutely with maximally effective doses of isoproterenol, serum FFA and glycerol levels also increased by 2-3-fold. However, in contrast to the findings with CL, these responses to isoproterenol were normal in beta(3)AR-deficient male mice and only slightly reduced in beta(3)-AR-deficient female mice (Table 5). Similarly, isoproterenol- and norepinephrine-stimulated oxygen consumption was not significantly reduced in beta(3)-AR-deficient male mice (Fig. 7). Overall, these findings demonstrate that under normal conditions, beta(3)-ARs are not required for the stimulation of lipolysis or thermogenesis by exogenously administered isoproterenol or norepinephrine.




Figure 7: Effects of CL, isoproterenol, and norepinephrine on O(2) consumption. Control (+/+) and beta(3)-AR-deficient (-/-) male mice, 10-12 weeks old, were treated with either CL (1.0 mg/kg, subcutaneously), isoproterenol (ISO, 0.3 mg/kg, subcutaneously), or norepinephrine (NE, 0.6 mg/kg subcutaneously), and effects on O(2) consumption were assessed. Mice that received CL and isoproterenol were awake and unrestrained for the study. Mice that received norepinephrine were anesthetized with pentobarbital for the study. The results are expressed as the mean ± S.E. (CL, +/+ = six mice, -/- = five mice; isoproterenol, +/+ = four mice, -/- = four mice; norepinephrine, +/+ = four mice, -/- = three mice).



Acute Effects of a beta-Selective Agonist on Insulin Release and Food Intake

Acute treatment with beta(3)-selective agonists in vivo produces a marked increase in circulating insulin levels (58) and a significant decrease in food intake(59, 60) . However, it has not been possible to exclude the possibility that these potentially important effects are caused by nonspecific interactions with alternative receptors. To assess rigorously the role of beta(3)-ARs in mediating these responses, the beta(3)-selective agonist CL was administered to control and beta(3)-AR-deficient mice, and the effects on insulin concentrations and food intake were determined. Acute treatment with CL produced a 140-fold increase in plasma insulin levels and a 56% reduction in blood glucose in control mice but was completely without effect in beta(3)-AR-deficient mice (Fig. 8). Similarly, acute treatment with CL produced a 45% reduction in food intake in control mice but was completely ineffective in beta(3)-AR-deficient mice (Fig. 9). These results demonstrate that the large effects of acute CL treatment on insulin secretion and food intake are mediated exclusively by beta(3)-ARs.


Figure 8: Acute effect of saline or CL 316,243 on plasma insulin and blood glucose concentrations. Wild type (+/+) and beta(3)-AR-deficient (-/-) female littermates (8 weeks old) were treated with an intraperitoneal injection of either saline (Sal) or CL (1 mg/kg). Blood was obtained from the tail 15 min after injection. The results are expressed as the mean ± S.E. (+/+ = seven mice, -/- = four mice).




Figure 9: Effect of a single dose of CL on food intake. Wild type (+/+) and beta(3)-AR-deficient (-/-) male littermates (8 weeks old) were treated with an intraperitoneal injection of either saline (Sal) or CL (1 mg/kg). The mice were housed individually during the test period, and results for each group are the mean of five animals (± S.E.). Food intake was determined during the 24 h following CL or saline treatment.




DISCUSSION

beta(3)-ARs are found predominantly in white and brown adipose tissue where they have been proposed to play an important role in the regulation of lipolysis, thermogenesis, and energy balance. Pharmacologic activation of beta(3)-ARs results in marked stimulation of energy expenditure, and beta(3)-selective agonists are being developed as potential anti-obesity drugs. However, the role of this receptor in normal physiology and its precise role in mediating the many pharmacologic actions of atypical beta-AR agonists are undefined. To address these issues, we have used gene targeting to create mice that lack beta(3)-ARs.

The mouse genome can be modified in a directed fashion using gene targeting in ES cells(61) . Microinjection of DNA into mouse zygotes is an alternative approach to gene targeting which bypasses the use of ES cells. A previous study concluded that this technique was possible but not efficient since targeting occurred in only 1 of 506 transgenic founders(51) . In the present study, direct injection of a beta(3)-AR targeting vector into mouse zygotes was attempted after high targeting frequencies were observed using ES cells (50%, Table 1). 513 zygotes were injected, resulting in 158 live born mice of which 23 were found to be transgenic. Of these 23 transgenic mice, 2 had targeted disruption of the beta(3)-AR gene. Overall, this study demonstrates that a zygote pronuclear microinjection approach to gene targeting is potentially feasible. However, its use is presently limited to genes that target with high frequencies. If future advances in gene targeting methodology result in increased targeting frequencies, then the direct injection approach could have broad application.

In the present study, a beta(3)-AR null mutation was induced on an FVB inbred background. Null (-/-) animals lack a wild type beta(3)-AR gene, detectable levels of intact beta(3)-AR mRNA, and functional beta(3)-ARs. Based upon analyses presented in this study, the phenotype of beta(3)-AR-deficient mice appears to be relatively mild. Null (-/-) animals, females more than males, tend to demonstrate a modest increase in body fat (Table 3). It is not yet known, however, whether this increase would be more pronounced if the beta(3)-AR null genotype coexisted with a variety of other factors that promote obesity such as advancing age, exposure to high fat diets, or the presence of an obesity-susceptible genetic background(62) . Furthermore, it should be noted that the increase in fat stores observed in beta(3)-AR-deficient mice is relatively small in comparison to other genetic models of rodent obesity(63) , suggesting that either beta(3)-ARs play only a small role in regulating energy balance or that compensatory adaptations occur in response to beta(3)-AR deficiency, thus limiting the development of obesity (discussed in greater detail below).

Recently, it was reported that a missense mutation (W64R) in the human beta(3)-AR gene tends to be associated with obesity, decreased energy expenditure, reduced insulin sensitivity, and earlier onset of non-insulin-dependent diabetes(27, 28, 29) . In many cases, these associations were observed in heterozygotes. Given these observations in humans, one might have expected homozygous beta(3)-AR-deficient mice to be markedly obese. Instead, this was not the case, raising the possibility that development of severe obesity and insulin resistance in response to knockout of the beta(3)-AR gene requires the presence of coexisting genetic and/or environmental factors that are present in many humans but not in FVB inbred mice maintained on a low fat chow diet. Of course, caution must be exercised in extrapolating results from genetically engineered mice to humans (and vice versa).

Earlier studies have demonstrated that beta(3)-ARs, unlike beta(1)- and beta(2)-ARs, are relatively resistant to desensitization and down-regulation (14, 15, 16, 17, 18, 19) . Because of this, it has been proposed that beta(3)-ARs function to maintain sympathetic activation of adipose tissue during periods of chronic stimulation, when signaling via beta(1)- and beta(2)-ARs is expected to be reduced. Chronic cold exposure-induced hypertrophy of brown fat is such a situation in that it results from sustained, intense sympathetic stimulation and is mediated, in large part, by activation of beta-ARs (for review see (52) ). Although proliferation of precursor cells is thought to be mediated by beta(1)-ARs(64) , cellular hypertrophy and proliferation of mitochondria are thought to be mediated by beta(3)-ARs(60) . Therefore, one might predict that beta(3)-AR-deficient mice would be impaired in their brown fat response to cold exposure. However, this was not the case. beta(3)-AR-deficient mice responded normally to chronic cold exposure, demonstrating the expected increases in brown fat weight, protein, DNA, and UCP content. Thus, adaptation of brown fat to chronic cold exposure does not require the presence of beta(3)-ARs.

Given that body lipid stores in beta(3)-AR-deficient mice are only modestly elevated and that brown fat responds normally to cold exposure, the following two possibilities are plausible. Either beta(3)-ARs contribute minimally to the regulation of brown fat thermogenesis and energy balance in the absence of pharmacologic stimulation, or, alternatively, compensatory mechanisms operate in response to beta(3)-AR deficiency to maintain brown fat function, thus limiting the development of obesity. Concerning this later point, previous gene targeting experiments have noted compensatory effects on related family members. For example, up-regulation of Myf-5, a muscle determination gene, was observed following knockout of the functionally related gene, MyoD, thus limiting adverse effects on muscle development(66, 67) . With respect to beta-AR signaling, it is conceivable that beta(1)- and/or beta(2)-AR gene expression could increase to compensate for the loss of beta(3)-ARs. Like beta(3)-ARs, they are also expressed in adipose tissue, albeit at significantly lower levels(20) , and both receptors respond to norepinephrine, resulting in the production of cAMP. As is shown in Fig. 4, beta(1)- but not beta(2)-AR mRNA levels significantly up-regulated in white and brown adipose tissue of mice lacking beta(3)-ARs. Up-regulation of beta(1)-AR gene expression, in response to primary loss of beta(3)-ARs, strongly implies that beta(3)-ARs transduce physiologically relevant beta-AR signaling and that abrogation of this signaling in adipocytes is detected and responded to, resulting in compensatory increases in beta(1)-AR gene expression. Importantly, the observed increase in beta(1)-AR mRNA levels was more pronounced in brown fat, which is distinguished by its dense sympathetic innervation, raising the possibility that beta(3)-ARs transduce a greater percentage of beta-AR signaling in brown versus white adipose tissue. At present, the regulatory pathways that mediate cross-talk between beta(3)-ARs and beta(1)-AR gene expression and the reasons why such cross-talk does not exist between beta(3)-ARs and beta(2)-AR gene expression remain to be determined.

The precise relationship between receptors defined pharmacologically versus genetically by nucleotide sequence has sometimes been difficult to establish(68) . With respect to the atypical beta-AR of adipose tissue, a likely candidate has been cloned and is referred to as the beta(3)-AR. Using cell lines expressing beta(3)-ARs, it has been demonstrated that the recombinant receptor is pharmacologically similar to atypical receptor activity found in adipose tissue(8, 9, 10, 11) . Thus, prevailing data indicate that the cloned beta(3)-AR is the atypical beta-AR(2, 12) . Through the use of gene targeting to create mice lacking a given receptor, it is possible to establish unequivocally the relationship between cloned receptors and pharmacologically defined activities. We have used this approach for beta(3)-AR. Mice homozygous for the disrupted beta(3)-AR totally failed to respond to the atypical beta-AR agonist, CL 316,243. In control mice, CL increased adenylate cyclase activity and lipolysis in adipose tissue by about 4-5-fold. These responses were completely absent in beta(3)-AR-deficient mice, indicating that CL-induced stimulation of adenylate cyclase activity and lipolysis is mediated exclusively by beta(3)-ARs. Thus, the present study agrees with previous reports (8, 9, 10, 11) and establishes the beta(3)-AR as the atypical beta-AR of murine adipose tissue.

Acute treatment of normal animals with beta(3)-selective agonists such as CL leads to a number of diverse responses including increased serum FFA and insulin levels, increased whole body energy expenditure, and decreased food intake (58-60 and this study). Although a role for beta(3)-ARs in mediating these responses has been suspected, uncertainty has existed since it is known that drugs can often interact with additional receptors, especially when injected in vivo, to produce a variety of nonspecific responses. However, when CL was administered to beta(3)-AR-deficient mice, each of these effects was completely absent, indicating that each of these responses to CL is mediated exclusively by beta(3)-ARs. Of particular interest are the large effects of acute CL treatment on insulin secretion and food intake. At present, there is no evidence that beta(3)-ARs are expressed on insulin-secreting beta-cells of the pancreas or brain centers involved in appetite regulation. Thus, further investigation will be required to determine the site(s) through which stimulation of beta(3)-ARs causes these potentially important effects.

Controversy exists regarding the relative importance of beta(3)- versus beta(1)- and beta(2)-ARs in mediating beta-adrenergic signaling in fat. The inability to resolve this point is due, in part, to the lack of beta(3)-selective antagonists. To address this issue, we have assessed beta-adrenergic signaling in adipose tissue of mice lacking beta(3)-ARs. The ability of isoproterenol, a nonselective beta-agonist, to stimulate adenylate cyclase activity maximally, was markedly impaired in membranes derived from beta(3)-AR-deficient mice. Specifically, maximally stimulated adenylate cyclase activity was decreased by 80% in isolated white adipocyte membranes and 70% in brown adipose tissue membranes. These findings are in agreement with a previous study that concluded, using an alternative approach, that 77% of maximally stimulated adenylate cyclase activity in mouse adipose tissue is mediated by beta(3)-ARs and that only 23% is mediated by beta(1)- and beta(2)-ARs(20) .

With regards to regulation of lipolysis, however, the situation appears to be more complex. In the present study, lipolysis assays were performed in the presence of adenosine deaminase, which removes endogenously generated adenosine, and with or without PIA, an A(1) adenosine receptor agonist. A(1) adenosine receptors couple negatively with adenylate cyclase. When PIA was present, isoproterenol-stimulated lipolysis was completely absent in beta(3)-AR-deficient adipocytes. However, when PIA was omitted, isoproterenol-stimulated lipolysis in beta(3)-AR-deficient adipocytes was only modestly reduced (by 33%). Since it can be inferred that beta(1)- and/or beta(2)-ARs mediate isoproterenol-stimulated lipolysis in beta(3)-AR-deficient adipocytes, these findings indicate that maximal activation of lipolysis by beta(1)- and/or beta(2)-ARs, but not beta(3)-ARs, is extremely sensitive to inhibition of adenylate cyclase by PIA. This finding most likely relates to the observation that in mouse white adipocytes, beta(1) and beta(2) mRNA transcripts are ^1/ to ^1/ as abundant as beta(3)-AR mRNA transcripts(20) , implying that white adipocytes possess many more beta(3)-ARs than beta(1)- and beta(2)-ARs. Consequently, maximal activation of beta(1)- and/or beta(2)-ARs, in contrast to maximal activation of beta(3)-ARs, produces only a small increase in adenylate cyclase activity ( Fig. 5and (20) ). Because of this, the stimulatory effect of beta(1)- and/or beta(2)-ARs on a downstream process such as lipolysis can be inhibited readily by negative influences on adenylate cyclase, such as that induced by PIA treatment. Overall, our results with incubated mouse adipocytes suggest that beta-adrenergic-mediated lipolysis is either entirely or minimally dependent on beta(3)-ARs, depending upon the integrated effects of other receptors such as the A(1) adenosine receptor on adenylate cyclase activity.

In an attempt to assess the status of adipose tissue beta-adrenergic signaling in vivo, control and beta(3)-AR-deficient mice were treated with various agonists, and effects on lipolysis (serum FFA and glycerol levels) and thermogenesis (whole body oxygen uptake) were determined. It should be noted, however, that such studies represent imprecise assessments of white or brown adipocyte adrenergic signaling, since the observed effects could be mediated through ARs located in sites other than adipose tissue. For example, increases in energy expenditure following treatment with isoproterenol or norepinephrine could result from effects on liver or skeletal muscle, and increases in serum FFA and glycerol levels could be caused by alterations in blood flow to adipose tissue. Nevertheless, the following observations have been made. Isoproterenol-induced increases in serum FFA and glycerol levels were normal in male beta(3)-AR-deficient mice and were only slightly reduced in beta(3)-AR-deficient female mice. isoproterenol- and norepinephrine-induced increases in thermogenesis were also normal in male beta(3)-AR-deficient mice (not studied in female mice). Thus, the abilities of isoproterenol to increase serum FFAs and glycerol, and isoproterenol and norepinephrine to increase thermogenesis, do not require the presence of beta(3)-ARs. These normal or nearly normal in vivo responses to injected isoproterenol or norepinephrine in beta(3)-AR-deficient mice are possibly mediated by up-regulated beta(1)-AR mRNA levels and are in agreement with the normal cold exposure-induced hypertrophy of brown fat observed in beta(3)-AR-deficient mice.

In summary, mice with targeted disruption of the beta(3)-AR gene were used to define the physiology and pharmacology of beta(3)-ARs. The major conclusions are as follows. Mice lacking beta(3)-ARs have a modest increase in body fat indicating that beta(3)-ARs play a role in regulating energy balance. beta(1) but not beta(2)-AR mRNA levels up-regulate in white and brown adipose tissue of beta(3)-AR-deficient mice, implying that beta(3)-ARs mediate physiologically relevant beta-AR signaling and that cross-talk exists between beta(3)-ARs and beta(1)-AR gene expression. The ability of CL 316,243, a beta(3)-selective agonist, to increase adipocyte adenylate cyclase activity and lipolysis, serum insulin levels, and whole body energy expenditure and to reduce food intake is mediated exclusively by beta(3)-ARs. This study demonstrates the potential value of gene knockout technology as a means of defining the precise relationship between drugs and the gene or genes that are thought to encode their receptors.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grants DK02119, DK49569, and DK4600 (to B. B. L.), DK28082 (to J. S. F.), and DK43051 (to B. B. K.). 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.

§
Harcourt General Researcher supported by the Harcourt General Charitable Foundation. To whom correspondence should be addressed: Dept. of Medicine, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. blowell@bih.harvard.edu.

(^1)
The abbreviations used are: beta-AR(s), beta-adrenergic receptor(s); ACTH, adrenocorticotropic hormone; bp, base pair(s); CL, CL 316,243; ES cells, embryonic stem cells; FFA, free fatty acid; FIAU, 1-[2-deoxy,2-fluoro-beta-D-arabinofuranosyl]-5-iodouracil; kb, kilobase pair(s); NEO, neomycin resistance; PIA, N^6-[R-(-)-1-methyl-2-phenyl]adenosine; UCP, uncoupling protein.

(^2)
E. Tozzo and B. Kahn, unpublished data.


ACKNOWLEDGEMENTS

We thank J. Gossen for helpful discussions regarding gene targeting by pronuclear injections, M. Jakubowski for demonstrating RNase protection assay methodologies, A. Greenberg for discussions regarding adenylate cyclase and lipolysis studies, T. Claus and E. Danforth Jr. for providing CL 316,243, E. Li and R. Jaenisch for providing J1-ES cells, C. Adra for providing the PGK-NEO-Poly(A) construct, K. Thomas and M. Capecchi for providing the pIC19R/MC1-TK construct, A. Hamann for setting up the beta(3)-AR mRNA RNase protection assay, and K. Herzberg for help in the generation of transgenic mice.


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