(Received for publication, May 12, 1995; and in revised form, October 6, 1995)
From the
-Adrenergic receptors (
-ARs)
are expressed predominantly in white and brown adipose tissue, and
-selective agonists are potential anti-obesity drugs.
However, the role of
-ARs in normal physiology is
unknown. To address this issue, homologous recombination was used to
generate mice that lack
-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
-AR gene. Mice that were homozygous
for the disrupted allele had undetectable levels of intact
-AR mRNA, as assessed by RNase protection assay and
Northern blotting, and lacked functional
-ARs, as
demonstrated by complete loss of
-agonist (CL
316,243)-induced stimulation of adenylate cyclase activity and
lipolysis.
-AR-deficient mice had modestly increased
fat stores (females more than males), indicating that
-ARs play a role in regulating energy balance.
Importantly,
but not
-AR mRNA levels
up-regulated in white and brown adipose tissue of
-AR-deficient mice (brown more than white), strongly
implying that
-ARs mediate physiologically relevant
signaling under normal conditions and that ``cross-talk''
exists between
-ARs and
-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
-AR-deficient mice, proving that the actions of CL are
mediated exclusively by
-ARs.
-AR-deficient mice should be useful as a means to a
better understanding of the physiology and pharmacology of
-ARs.
``Atypical'' -ARs (
)were originally
identified pharmacologically, as
-AR-like activity that resisted
blockade by classical
-AR antagonists(1, 2) .
Atypical
-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
-ARs has been the synthesis of novel
-AR agonists
that potently stimulate lipolysis, energy expenditure, and gut
relaxation, while having little or no effect on
- or
-AR-mediated
processes(5, 6, 7) . Because atypical
-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 -AR family, designated the
-AR, has been cloned and characterized (8) .
More recently, rat (9, 10) and mouse (11)
-AR genes have also been isolated. The
RNA message encoding the
-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
-ARs are relatively resistant
to blockade by conventional
-AR antagonists and are potently
stimulated by atypical
-AR
agonists(8, 9, 10, 11) . Given that
atypical
-AR activity and cloned
-ARs have
similar tissue distributions and pharmacologic profiles, it is
generally accepted that the
-AR is the atypical
-AR(2, 12) .
-,
-, and
-ARs all couple via G
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
-ARs, it has been
difficult to determine the functional significance of each subtype. A
distinguishing feature of the
-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 -,
-, and
-AR mRNA transcripts at a ratio of 3:1:150,
respectively(20) , with a qualitatively similar relationship
observed in brown adipose tissue, suggesting that
-ARs
might play a dominant role. Because of the absence of radioligands
capable of detecting
-ARs, however, it has been
difficult to determine the relative number of
- versus
- and
-AR binding
sites. Historically,
-AR subtypes in adipose tissue have been
quantified functionally by measuring
-AR agonist-stimulated
adenylate cyclase activity in isolated membrane preparations, followed
by the resolution of dose-response curves into low
(
-AR) and high (
- and
-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
- and
-ARs and that 77% is mediated by
-ARs(20) . In contrast, using rat adipocyte
membranes it has been estimated that 70% of maximal stimulation is due
to
- and
-ARs, and 30% is due to
-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
-ARs (
,
, or
). Studies of lipolysis using
rat adipocytes have been conflicting, concluding that
- and
-ARs play either an important
role (24) or a minor role relative to
-ARs(25) . With respect to isolated brown
adipocytes, it has been suggested that
-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
-ARs. In genetically obese fa/fa rats (10) and ob/ob mice(20) ,
-AR mRNA levels are significantly down-regulated,
raising the possibility that decreased
-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
-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
-ARs might also play an important role in
humans as well.
In summary, significant uncertainty exists regarding
the physiologic importance of -ARs as well as the
relative role of
versus
-
and
-ARs in mediating
-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
-ARs.
Figure 1:
The
-AR gene, targeting vectors, and Southern blot
detection schemes. Shown is a partial restriction enzyme map of the
-AR gene, the
-KO (mouse zygote) and
-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
-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
-AR coding sequence between NheI and XhoI corresponding to
-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
-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.
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 -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.
Figure 3:
-AR RNase protection
assay.
-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:
-,
-,
and
-AR mRNA levels in white and brown adipose tissue
by Northern blotting.
-,
-, and
-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
-AR-deficient (K) 12-week-old male
mice. Representative lanes from two control and two
-AR-deficient samples are shown. PhosphorImager
analyses of Northern blots were performed using all five control and
five
-AR-deficient samples (PhosphorImager data are
described under ``Results''). The blots were also hybridized
with a
-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:
-AR,
2.4;
-AR ,
2.2;
-AR,
2.4; and
-actin,
2.1.
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. ()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
-[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) .
Shown
in Table 2is a summary of all mouse zygote injections. Using the
-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
-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).
Data shown in Table 3indicate that there is a tendency for
-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
-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 -AR-deficient animals. In addition,
colonic temperatures in control and null mice were normal (37 °C)
throughout the 3-week study period. Thus,
-ARs are not
required for cold exposure-induced hypertrophy of brown adipose tissue.
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 -AR-deficient
(-/-) littermates and then assayed for adenylate cyclase
activity. Panel A, dose-response curves for stimulation by
isoproterenol (ISO, a nonselective
-AR agonist) and CL (a
-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
-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 -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
-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
-AR-deficient mice lack
functional
-ARs and confirm that the stimulatory
effect of CL on these processes is mediated exclusively by
-ARs.
As expected, maximally effective doses of
isoproterenol, a nonselective -AR agonist, markedly stimulated
adenylate cyclase activity in membranes derived from wild type mice (Fig. 5). In membranes obtained from
-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,
-ARs appear to be responsible for mediating
70-80% of isoproterenol-induced maximally stimulated adenylate
cyclase activity. It is likely that
- and
-ARs mediate the remaining 20-30%.
Assessment
of isoproterenol-stimulated lipolysis in control versus -AR-deficient adipocytes revealed additional
complexity, which involved an interaction between A
adenosine receptors and the relative roles of
versus
- and
-ARs.
Adenosine induces an inhibitory effect on adenylate cyclase, mediated
by A
adenosine receptors and G
. 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
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
-AR-deficient adipocytes. The absence of
isoproterenol-stimulated lipolysis in
-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
-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,
-ARs
appeared to be solely responsible for mediating
isoproterenol-stimulated lipolysis. When PIA was absent and endogenous
adenosine was removed,
- and/or
-ARs
appeared to mediate the majority of isoproterenol-stimulated lipolysis.
Finally, adrenocorticotropic hormone (ACTH), which also stimulates
lipolysis via a seven-transmembrane
receptor-G-adenylate cyclase-coupled
mechanism(56, 57) , increased lipolysis equally well
in adipocytes derived from wild type and
-AR-deficient
mice (Fig. 6A). Similarly, forskolin, a direct
activator of adenylate cyclase, stimulated lipolysis equally well in
wild type and
-AR-deficient cells (Fig. 6A). Normal responsiveness to ACTH and forskolin
makes it unlikely that impaired responsiveness of
-AR-deficient cells to isoproterenol results from a
direct inhibitory effect of
-AR deficiency on
G
, adenylate cyclase, or more distal components of the
activation pathway for lipolysis.
Figure 7:
Effects of CL, isoproterenol, and
norepinephrine on O consumption. Control (+/+)
and
-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
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).
Figure 8:
Acute
effect of saline or CL 316,243 on plasma insulin and blood glucose
concentrations. Wild type (+/+) and
-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 -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.
-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
-ARs results in marked
stimulation of energy expenditure, and
-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
-AR agonists
are undefined. To address these issues, we have used gene targeting to
create mice that lack
-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
-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
-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
-AR null mutation was induced on an FVB inbred
background. Null (-/-) animals lack a wild type
-AR gene, detectable levels of intact
-AR mRNA, and functional
-ARs. Based
upon analyses presented in this study, the phenotype of
-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
-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
-AR-deficient mice
is relatively small in comparison to other genetic models of rodent
obesity(63) , suggesting that either
-ARs play
only a small role in regulating energy balance or that compensatory
adaptations occur in response to
-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 -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
-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
-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 -ARs, unlike
- and
-ARs, are relatively resistant to desensitization and
down-regulation (14, 15, 16, 17, 18, 19) .
Because of this, it has been proposed that
-ARs
function to maintain sympathetic activation of adipose tissue during
periods of chronic stimulation, when signaling via
-
and
-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
-ARs (for review see (52) ). Although proliferation of precursor cells is thought to
be mediated by
-ARs(64) , cellular hypertrophy
and proliferation of mitochondria are thought to be mediated by
-ARs(60) . Therefore, one might predict that
-AR-deficient mice would be impaired in their brown
fat response to cold exposure. However, this was not the case.
-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
-ARs.
Given that body lipid stores in -AR-deficient mice
are only modestly elevated and that brown fat responds normally to cold
exposure, the following two possibilities are plausible. Either
-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
-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
-AR
signaling, it is conceivable that
- and/or
-AR gene expression could increase to compensate for
the loss of
-ARs. Like
-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,
- but not
-AR mRNA levels
significantly up-regulated in white and brown adipose tissue of mice
lacking
-ARs. Up-regulation of
-AR
gene expression, in response to primary loss of
-ARs,
strongly implies that
-ARs transduce physiologically
relevant
-AR signaling and that abrogation of this signaling in
adipocytes is detected and responded to, resulting in compensatory
increases in
-AR gene expression. Importantly, the
observed increase in
-AR mRNA levels was more
pronounced in brown fat, which is distinguished by its dense
sympathetic innervation, raising the possibility that
-ARs transduce a greater percentage of
-AR
signaling in brown versus white adipose tissue. At present,
the regulatory pathways that mediate cross-talk between
-ARs and
-AR gene expression and the
reasons why such cross-talk does not exist between
-ARs and
-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 -AR of adipose tissue, a likely candidate has been
cloned and is referred to as the
-AR. Using cell lines
expressing
-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
-AR is the
atypical
-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
-AR. Mice homozygous for the disrupted
-AR totally failed to respond to the atypical
-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
-AR-deficient
mice, indicating that CL-induced stimulation of adenylate cyclase
activity and lipolysis is mediated exclusively by
-ARs. Thus, the present study agrees with previous
reports (8, 9, 10, 11) and
establishes the
-AR as the atypical
-AR of murine
adipose tissue.
Acute treatment of normal animals with
-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
-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
-AR-deficient mice, each of these effects was
completely absent, indicating that each of these responses to CL is
mediated exclusively by
-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
-ARs are expressed on insulin-secreting
-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
-ARs causes these potentially
important effects.
Controversy exists regarding the relative
importance of - versus
- and
-ARs in mediating
-adrenergic signaling in fat.
The inability to resolve this point is due, in part, to the lack of
-selective antagonists. To address this issue, we have
assessed
-adrenergic signaling in adipose tissue of mice lacking
-ARs. The ability of isoproterenol, a nonselective
-agonist, to stimulate adenylate cyclase activity maximally, was
markedly impaired in membranes derived from
-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
-ARs and that only 23% is mediated by
- and
-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 adenosine receptor agonist. A
adenosine receptors
couple negatively with adenylate cyclase. When PIA was present,
isoproterenol-stimulated lipolysis was completely absent in
-AR-deficient adipocytes. However, when PIA was
omitted, isoproterenol-stimulated lipolysis in
-AR-deficient adipocytes was only modestly reduced (by
33%). Since it can be inferred that
- and/or
-ARs mediate isoproterenol-stimulated lipolysis in
-AR-deficient adipocytes, these findings indicate that
maximal activation of lipolysis by
- and/or
-ARs, but not
-ARs, is extremely
sensitive to inhibition of adenylate cyclase by PIA. This finding most
likely relates to the observation that in mouse white adipocytes,
and
mRNA transcripts are
/
to
/
as abundant
as
-AR mRNA transcripts(20) , implying that
white adipocytes possess many more
-ARs than
- and
-ARs. Consequently, maximal
activation of
- and/or
-ARs, in
contrast to maximal activation of
-ARs, produces only
a small increase in adenylate cyclase activity ( Fig. 5and (20) ). Because of this, the stimulatory effect of
- and/or
-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
-adrenergic-mediated lipolysis is either entirely or minimally
dependent on
-ARs, depending upon the integrated
effects of other receptors such as the A
adenosine receptor
on adenylate cyclase activity.
In an attempt to assess the status of
adipose tissue -adrenergic signaling in vivo, control and
-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
-AR-deficient mice and were only
slightly reduced in
-AR-deficient female mice.
isoproterenol- and norepinephrine-induced increases in thermogenesis
were also normal in male
-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
-ARs. These normal or nearly normal in vivo responses to injected isoproterenol or norepinephrine in
-AR-deficient mice are possibly mediated by
up-regulated
-AR mRNA levels and are in agreement with
the normal cold exposure-induced hypertrophy of brown fat observed in
-AR-deficient mice.
In summary, mice with targeted
disruption of the -AR gene were used to define the
physiology and pharmacology of
-ARs. The major
conclusions are as follows. Mice lacking
-ARs have a
modest increase in body fat indicating that
-ARs play
a role in regulating energy balance.
but not
-AR mRNA levels up-regulate in white and brown adipose
tissue of
-AR-deficient mice, implying that
-ARs mediate physiologically relevant
-AR
signaling and that cross-talk exists between
-ARs and
-AR gene expression. The ability of CL 316,243, a
-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
-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.