Transgenic Mice Overexpressing the ß1-Adrenergic Receptor in Adipose Tissue Are Resistant to Obesity
Veronica Soloveva,
Reed A. Graves,
Mark M. Rasenick,
Bruce M. Spiegelman and
Susan R. Ross
Department of Microbiology/Cancer Center (V.S.,
S.R.R.) University of Pennsylvania Philadelphia, Pennsylvania
19104
Department of Biochemistry (V.S.) Department of
Physiology and Biophysics (M.M.R.) University of Illinois School of
Medicine Chicago, Illinois 60612
Department of Medicine
(R.A.G.) University of Chicago Medical School Chicago, Illinois
60637
Dana Farber Cancer Institute and Department of Cell
Biology (B.M.S.) Harvard Medical School Boston, Massachusetts
02115
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ABSTRACT
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The ratio of
- to ß-receptors is thought to
regulate the lipolytic index of adipose depots. To determine whether
increasing the activity of the ß1-adrenergic
receptor (AR) in adipose tissue would affect the lipolytic rate or the
development of this tissue, we used the enhancer-promoter region of the
adipocyte lipid-binding protein (aP2) gene to direct expression of the
human ß1AR cDNA to adipose tissue. Expression
of the transgene was seen only in brown and white adipose tissue.
Adipocytes from transgenic mice were more responsive to ßAR agonists
than were adipocytes from nontransgenic mice, both in terms of cAMP
production and lipolytic rates. Transgenic animals were partially
resistant to diet-induced obesity. They had smaller adipose tissue
depots than their nontransgenic littermates, reflecting decreased lipid
accumulation in their adipocytes. In addition to increasing the
lipolytic rate, overexpression of the ß1AR
induced the abundant appearance of brown fat cells in subcutaneous
white adipose tissue. These results demonstrate that the
ß1AR is involved in both stimulation of
lipolysis and the proliferation of brown fat cells in the context of
the whole organism. Moreover, it appears that it is the overall ßAR
activity, rather than the particular subtype, that controls these
phenomena.
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INTRODUCTION
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Adrenergic receptors (AR) are known to participate in the
regulation of adipose tissue development and metabolism. There are two
major classes of ARs,
and ß. Stimulation of the ßARs in white
adipocytes leads to increased lipolysis, primarily through the
production of cAMP and the activation of hormone-sensitive lipase and
other pathways, whereas stimulation of the
2AR leads to
increased lipid storage, through the inhibition of cAMP production
(reviewed in Ref.1). There is a large body of evidence that indicates
that the ratio of
2/ß ARs in different adipose tissue
depots affects the lipolytic rate, with depots containing higher levels
of
2ARs more prone to lipogenesis. Thus, the ratio of
functional
2- and ß-receptors present in adipose
tissue may determine whether fat storage or release is activated by
catecholamines.
In addition to differences in receptor numbers, the presence of
particular ß-receptor subtypes is thought to affect lipolytic rates
in adipose tissue. There are three pharmacologically distinct subtypes
of ßARs (ß1, ß2, and ß3)
found in adipocytes. While the ß1- and
ß2-receptors are found in a number of tissues, the
atypical ß3AR is predominantly in fat (2, 3, 4). The ratio
of ß1/ß2/ß3 mRNA in mouse
adipose tissue is approximately 3:1:150 (5). Because of its high level
of expression, it has been proposed that the ß3AR is the
major regulator of lipolysis in mouse adipose tissue. However, mice
with targeted mutagenesis of the ß3AR gene show only a
modest tendency to become obese relative to normal mice, a somewhat
surprising finding if the ß3AR was the major regulator of
lipolysis (6). Thus, it is possible that the ß1- or
ß2-receptors also regulate lipolysis in mice. Moreover,
other species, including humans, have higher levels of ß1
and ß2 in adipose tissue (1).
Activation of ß1ARs affects fat cell proliferation as
well as adipose tissue metabolism. Specifically, it has been shown in
ex vivo explants that ß1-agonists can
stimulate the proliferation of brown fat cells (7). Thermogenesis in
brown adipose tissue (BAT) functions to dissipate energy in the form of
heat through the action of a unique mitochondrial proton transporter,
the uncoupling protein (UCP). UCP expression is stimulated by
catecholamines released by the sympathetic nervous system, and agonist
activation of the ß3AR in BAT leads to thermogenesis
(8, 9, 10). In most mammals, BAT is highly localized, with the largest BAT
depot found in the interscapular region. However, small numbers of
brown fat cells can occasionally be found in white adipose tissue (WAT)
depots, especially in cold-exposed rodents (11).
To determine directly whether shifting the balance between the
- and
ß-subtypes in vivo would affect the metabolism or anatomy
of adipose tissue, we produced transgenic mice that expressed the human
ß1AR in adipose tissue. White adipocytes from these
transgenic mice were highly sensitive to the ß1-selective
agonist dobutamine and had higher rates of lipolysis than those from
control mice. Moreover, transgenic mice overexpressing the
ß1AR gained weight more slowly and had smaller white
adipose depots than their nontransgenic littermates, especially in
response to a high fat diet. Finally, UCP-expressing brown adipocytes
appeared in the subcutaneous white adipose depots of transgenic males.
These results indicate that shifting the ratio between different ARs
has profound effects on adipose tissue metabolism and morphology,
resulting in altered physiological responses to high fat diets.
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RESULTS
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To direct high level expression of the ß1AR to
adipocytes, we created a transgene in which the human
ß1AR cDNA was under the control of the regulatory region
of the aP2 lipid-binding protein gene. The aP2-regulatory region has
been shown to direct expression of several transgenes to WAT and BAT
(12, 13, 14, 15). Four independent founder animals bearing the
aP2-ß1AR transgene were produced and used to generate
pedigrees. Transgene expression in the G1 generation was
analyzed by Northern blot analysis and was detected at high levels in
only one of these strains. Large amounts of transgene-specific RNA were
detected in adipose (Fig. 1A
) but not in other tissues
of this mouse strain (Fig. 1B
). Transgene RNA levels were lower in the
gonadal fat pad than in other depots, which may be due to transgene
integration site effects, the metabolic state of this fat pad, or to
the transgene sequences; we previously showed that some ap2
promoter-driven transgenes showed variable expression in different
adipose depots (14). Expression of the endogenous mouse
ß1AR gene was detectable in the depots at about 50-fold
lower levels (not shown), as has been previously reported for adult
mice (5). Moreover, the ß1AR transgene had no apparent
effect on the endogenous ß3AR RNA levels (Fig. 9
). Under
similar conditions of hybridization and exposure, no ß2AR
RNA was detected (not shown); as described above, the RNA levels of
this receptor are about 1/150th that of the ß3AR in mouse
adipose tissue (5). Assuming that the hybridization efficiencies for
the different probes are the same, the ß1AR transgene RNA
levels were similar to (in gonadal fat) or higher than (in subcutaneous
and brown fat) the ß3AR levels (Figs. 1
and 9
). Thus, the
total level of ß-receptors in the adipose tissues was increased at
minimum 2-fold, at least at the RNA level.

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Figure 1. Expression of the ß1AR Transgene
A, Representative Northern blot of 20 µg total RNA isolated from
gonadal white (epididymal) (WGF), subcutaneous white (WScF), and
interscapular brown (BF) fat depots from a transgenic (T) or
nontransgenic (NT) mouse. The probe used (1 x 109
cpm/µg specific activity) was specific for the SV40 region of the
transgene. Hybridization to ß-actin probe was done as a control for
RNA integrity. B, Northern blot analysis of 20 µg total RNA isolated
from the following organs of a male transgenic mouse: brain (Br), heart
(Hr), kidney (Kd), liver (Li), lung (Lu), muscles (Mu), salivary gland
(SG), spleen (Sp), and testis (Ts). The hybridization to skeletal
muscle RNA is due to the presence of WScF in this tissue, since a probe
to aP2 RNA also hybridizes to this lane (not shown; Ref. 12). Exposure
times for panels A and B were 16 to 18 h.
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Figure 9. RNA Analysis of Gonadal, Subcutaneous, and BAT
Twenty micrograms of total RNA from the epididymal (GF), inguinal
(SQF), and interscapular brown adipose fat pad (BF) of two different
age-matched transgenic (T) and nontransgenic (NT) males each were
analyzed on Northern blots. The blots were hybridized to the probes
(specific activity = 1 x 109 cpm/µg) to the
right of the panels; all exposures were 1618 h. The
multiple bands seen after hybridization to the ß3AR probe
are due to alternative splicing, variable transcript initiation, and
alternative polyadenylylation (41, 42).
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To determine whether functional ß1AR protein was made in
these mice, we measured cAMP production by adipocytes isolated from
gonadal fat pads in response to the ßAR agonist, isoproterenol.
Figure 2
shows the results from such an experiment. As
can be seen, adipocytes from both transgenic and nontransgenic mice
produced cAMP in response to increasing concentrations of the agonist.
However, there was increased ß-adrenergic responsiveness in the
ß1AR transgenic adipocytes; the potency of the response
with transgenic adipocytes was about 20% higher at doses of
isoproterenol between 10-7 M and
10-6 M. Thus, the transgenic animals had more
functional ß-receptors than their nontransgenic littermates.

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Figure 2. Adenylyl Cyclase Activity Is Increased in Adipose
Tissue from the Transgenic Mice
Production of cAMP was measured with crude membrane extracts prepared
from the pooled peritoneal WAT of 15 transgenic (T) and nontransgenic
(NT) female mice in the presence of increasing concentrations of
isoproterenol. The results are the mean and variation of two
experiments with different sets of mice; the SD for most
points was smaller than the size of the point.
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Stimulation of the ß1AR by agonist is known to induce
lipolysis in adipocytes. If the transgenic mice were expressing a
greater number of total ßARs than nontransgenic animals, the rate of
lipolysis should be increased in their fat cells. Mature adipocytes
were isolated from gonadal fat, and lipolytic rates were measured
in vitro in response to isoproterenol and to the
ß1-selective agonist, dobutamine. As shown in Fig. 3A
, adipocytes isolated from the transgenic mice had
higher lipolytic activity in response to isoproterenol, and there was a
striking shift in the EC50 of this response for both males
(7.5 nM vs. 300 nM for transgenic
and nontransgenic males, respectively) and females (1 nM
vs. 15 nM for transgenic and nontransgenic
females, respectively). The EC50 of the response to
dobutamine was also decreased for the transgenic adipocytes (7
nM for males, 9 nM for females) compared with
nontransgenic adipocytes (200 nM for males, 400
nM for females), showing that the altered lipolytic
response was specific to the ß1AR produced by the
transgene (Fig. 3B
). Adipocytes from nontransgenic females had higher
lipolytic rates and lower EC50 values on average than those
from nontransgenic males, especially when stimulated with isoproterenol
(Fig. 3A
).

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Figure 3. Lipolysis in Isolated White Adipocytes
White adipocytes were isolated from the epididymal or periovarian fat
pads of transgenic and nontransgenic males and females. Glycerol
release was assayed after incubation with increasing concentrations of
isoproterenol (A) or dobutamine (B). For panel A, adipocytes were
purified from five animals from each group, and the assays were
performed in duplicate. The data from three independent experiments
were averaged (±SE). For panel B, the adipocytes were
pooled from the fat pads of six animals in each group, and the assays
were performed in duplicate. The basal levels of glycerol release in
the absence of inducer were 11.3 ± 1.5 and 11.0 ± 1.1
nmol/105 cells/h for the transgenic and nontransgenic
males, respectively, and 10.4 ± 0.5 and 10.7 ± 0.8
nmol/105 cells/h for the transgenic and nontransgenic
females, respectively.
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If the ß1AR transgene was functioning in vivo
to increase the lipolytic rate, the transgenic mice might have
decreased lipid accumulation and smaller fat pads than their
nontransgenic littermates. Therefore, total body and fat pad weights of
the transgenic mice and their nontransgenic littermates were analyzed.
The data for 14-week-old mice are shown in Fig. 4
. These
results showed that there was a small decrease in the total body weight
and a statistically significant difference in the WAT (as a percentage
of total body weight) between transgenic and nontransgenic mice of the
same sex (Fig. 4B
). Moreover, the average size of the adipocytes in the
transgenic mice was significantly smaller than that of the
nontransgenic mice (Table 1
).

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Figure 4. Body and Fat Pad Weight of the ß1AR
Transgenic Mice
A, Two groups of 12 mice for each set (transgenic males, transgenic
females, nontransgenic males, and nontransgenic females) were analyzed
at the age of 1314 weeks. The bar graphs represent the
mean weight ± SE. B, Gonadal (GF), subcutaneous
(ScF), and interscapular brown (BF) fat depots were individually
weighed. The data are shown as the mean of the ratio of fat pad to
total body weight for each animal ± SE. *,
P < 0.025; **, P < 0.05; ***,
P < 0.005.
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In general, mice do not accumulate large amounts of body fat when fed
normal mouse chow (4% fat by weight), especially when they are young.
It was therefore not surprising that there was not a highly significant
difference in total body or fat pad weight between the
ß1AR transgenic and nontransgenic mice. In contrast to
regular chow-fed mice, those placed on high-fat diets (40% by weight)
rapidly become obese because of increased lipid storage in adipose
tissue (16). To determine whether expression of the ß1AR
would protect the transgenic mice against this diet-induced obesity,
the mice were placed on high-fat diets, starting at 4 weeks after
birth. Males were kept on this diet for 90 days and females for 120
days.
As shown in Fig. 5
, the transgenic males on the high-fat
diet gained weight more slowly than their nontransgenic littermates. In
contrast, smaller differences in body weight gain were seen with the
transgenic and nontransgenic female mice. In general, the female mice
showed a much greater variability in body weight than did male mice,
which may account for the smaller statistical differences.

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Figure 5. The ß1AR Transgenic Mice on a
High-Fat Diet Gain Weight More Slowly Than Their Nontransgenic
Littermates
Groups of transgenic (T) and nontransgenic (NT) mice at the age of 4
weeks were switched to high-fat diet and were monitored for 90 (males,
panel A) or 120 (females, panel B) days. Ten males and 12 females of
each genotype (transgenic and nontransgenic) were analyzed. The mean
body weights ± SE are plotted. Also included on the
graphs are the weight gain curves for 12 nontransgenic
males and females of the same age but fed normal mouse chow (NT,
control).
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We also examined the weight of various fat pads. As can be seen in Fig. 6
, the gonadal and subcutaneous fat pad weights of both
the transgenic males and females were significantly decreased relative
to their nontransgenic littermates. Again, both the gonadal and
subcutaneous adipocytes isolated from the transgenic mice were smaller
in size than those from the nontransgenic mice (Table 1
), suggesting
that there was decreased lipid accumulation resulting from increased
ß1AR responsiveness.

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Figure 6. Fat Pad Weights of Mice on a High-Fat Diet
Six mice from each group in Fig. 5 were killed, and the gonadal (GF),
subcutaneous (ScF) and brown fat (BF) pads were weighed. The fat pad
weight as a percentage of total body weight was calculated for each
animal; presented is the average value ± SE. *,
P < 0.01; **, P < 0.025; ***,
P < 0.005; +, P < 0.1.
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The lipolytic activity of adipocytes from the transgenic animals on the
high-fat diet was also elevated relative to the controls (Fig. 7
). As was seen in mice fed normal chow, nontransgenic
females had higher lipolytic activity in their gonadal fat pads than
did nontransgenic males.

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Figure 7. Lipolysis Assays with Purified Adipocytes from Mice
on a High-Fat Diet
Mature adipocytes from five transgenic and nontransgenic males after 80
days on the diet and from four transgenic and nontransgenic females
after 120 days on diet were purified and pooled. Lipolysis was induced
by 0.1 µM and 100 µM isoproterenol (panel
A) or dobutamine (panel B), and glycerol release assays were performed.
The experiment was repeated with the same number of animals, and the
averaged data and deviations are presented.
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Histological analysis of the WAT and BAT of the ß1AR
transgenic mice was also carried out. The subcutaneous and gonadal WAT
from control mice exhibited typical unilocular adipocytes (Fig. 8
, A and B, respectively). The unilocular adipocytes in
the transgenic WAT depots appeared smaller in size (Fig. 8
, C and D),
confirming the data presented in Table 1
. In addition to the unilocular
adipocytes, the gonadal adipose tissue from both males (Fig. 8C
) and
females (not shown) contained readily visible adipocytes with
multilocular lipid droplets. Moreover, the subcutaneous fat of both
sexes had many large loci of these multilocular adipocytes (Fig. 8D
for
males; not shown for females). There were no apparent differences in
the interscapular BAT morphology between the transgenic (Fig. 8E
) and
nontransgenic mice (not shown).

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Figure 8. Morphology of the Cells in Different Adipose Depots
Hematoxylin and eosin-stained adipose tissue from subcutaneous (A and
C), gonadal (B and D), and interscapular BAT (E). Sections are from
age-matched nontransgenic (A and B) and transgenic (C and D) males fed
normal mouse chow. Magnification, 80x.
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Because the multilocular cells had the appearance of brown adipocytes,
we tested whether there was UCP expression in the various WAT depots.
Detectable levels of UCP RNA were found in the subcutaneous adipose
tissue of transgenic, but not nontransgenic, males and females (Fig. 9
and not shown). UCP RNA was also found at very low
levels in the gonadal fat of transgenic, but not nontransgenic, males
(Fig. 9
). In contrast, both transgenic and nontransgenic periovarian
WAT had UCP RNA, although there was 2- to 3 times more in the
transgenic tissue (not shown). There was no effect on UCP expression in
the interscapular BAT of either males (Fig. 9
) or females (not shown).
The relative levels of UCP expression paralleled the appearance of
multilocular cells in the WAT depots of the mice, indicating that the
ß1AR transgene induced the appearance of brown adipocytes
in WAT depots.
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DISCUSSION
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Activation of all ß-ARs results in the generation of cAMP, via
coupling of G proteins to adenylyl cyclase. This increase in cAMP
leads, in turn, to the activation of protein kinase A and, ultimately,
the stimulation of lipolysis in white adipocytes and thermogenesis in
brown adipocytes (1). The ßAR subtypes were originally classified by
their pharmacological properties. More recently, these receptor
subtypes have been cloned, and analysis of their expression has
indicated that the ß3-subtype is the most abundant in
rodent adipose tissue (5). It has been suggested that the various ßAR
subtypes play different roles in adipocyte metabolism and that the
ß3AR is primarily responsible for the regulation of
lipolysis in adipose tissue.
That 77% of the maximally stimulated adenylyl cyclase activity in
mouse adipose tissue is due to activation of the ß3AR is
consistent with this suggestion (5). However, mice that lack
ß3ARs due to targeted mutagenesis of the gene encoding
this receptor are only mildly obese relative to wild type mice (6). Our
results show that increased expression of a functional
ß1AR in the presence of ß3ARs results in
mice with increased adipose cell lipolytic activity. This increased
activity caused less lipid storage in adipose tissue, especially in
response to a high-fat diet. Thus, our data support the notion that it
may be the overall amount of ßAR in adipose tissue that determines
its lipolytic rate. Indeed, in the ß3AR-knockout mice,
there was a 2-fold increase in the level of ß1AR
transcripts, and activation of these receptors may have prevented
excessive adiposity.
The adipose tissue of the ß1AR-transgenic mice was more
lipolytic both in vivo and in vitro, indicating
that ligand is not limiting in the animal. There was an even more
striking decrease in the EC50 of the lipolytic response
in vitro to both isoproterenol and dobutamine by the
transgenic adipocytes. That the sensitivity of the response was
affected more than the maximal response itself is consistent with the
notion that there are spare receptors; that is, there was a saturation
of the response to ligand even when the additional receptors were not
fully occupied. However, in the transgenic mice, activation of
lipolysis would occur at lower ligand concentrations than in
nontransgenic mice and thus, the adipocytes of the former would be more
lipolytic. In support of this, unlike what was observed with transgenic
mice on a high-fat diet, we found that the ß1AR transgene
did not protect mice from monosodium glutamate-induced obesity (V.
Sololeva, unpublished); because monosodium glutamate is thought to
decrease adrenergic signaling (17), the additional receptors provided
by the ß1AR transgene most likely could not be activated
and, therefore, no effects on lipid accumulation were seen.
Female mice were less affected by the expression of the
ß1AR transgene than were male mice. This is in contrast
to what was observed with the ß3AR knockout mice, where
females had, on average, higher fat stores than males (6). We also
observed that adipocytes from nontransgenic females were more
responsive to ß-stimulated lipolysis in vitro than were
those isolated from nontransgenic males. These results indicate that
adipose tissue from female mice are naturally more responsive to
ß-adrenergic stimulation, perhaps due to higher levels of
ß3AR. Hence, addition of more ß1ARs could
have a smaller effect than it does in males because the response to
ligand may be already saturated, as discussed above. Conversely, if the
ß3AR receptors were more active in female mice than in
males, their absence would have a greater effect, as was seen in the
knockout mice.
Human adipose tissue differs from that of mice in that the
ß3AR is present at much lower levels while the levels of
ß1AR and ß2AR are higher (1, 23). Moreover,
ß3AR levels in guinea pig adipose tissue are also low,
and ß1-agonists are effective inducers of lipolysis in
this species (24). However, it has been shown that
ß3-specific agonists can stimulate lipolysis in human
adipocytes isolated from the omental depot (25). In addition, mutations
in the gene for this receptor were found to be associated with a lower
resting metabolic rate, greater weight gain, and an earlier onset of
non-insulin-dependent diabetes mellitus (26, 27, 28). In some cases the
correlation between the phenotype and the presence of the mutated gene
was of borderline statistical significance, and there was little or no
evidence that the mutation was present more frequently in obese than
lean individuals. That the ß1AR can also have an effect
on the total lipolytic activity of adipose tissue, as shown here,
indicates that there may be compensatory increases in the activity of
this receptor (or the ß2AR) in humans bearing the
ß3AR mutation. This could explain, at least in part, why
there is not a stronger correlation between increased adiposity and the
mutant ß3AR gene in humans.
The increased ß1AR activity also caused the appearance of
brown adipocytes in WAT depots. It is not known whether brown and white
adipocytes develop from the same or different stem cells. Brown and
white adipocytes are very similar at the level of gene expression.
Although there are quantitative differences in the levels of a number
of genes that are expressed in WAT and BAT, only UCP has been found to
be uniquely expressed in the latter (reviewed in Ref.29). It has been
suggested that there is a pool of intraconvertible cells that can
differentiate to either cell type in vivo after cold
induction, although it is not known what determines the ultimate
differentiation fate (11, 30). In the mice studied here, the
ß1AR transgene was under the control of the aP2 promoter,
which only functions in adipocytes and not in stem cells (reviewed in
Ref.29). The increase in brown adipocytes in WAT depots could be due
to a catecholamine-triggered proliferation of brown adipocytes or to
interconversion between the two cell types, perhaps at an early stage
in adipocyte differentiation. Further experiments are needed to address
this issue; however, the ß1AR transgenic mice should
provide a useful model for the study of this phenomenon, given the
larger pool of brown fat cells in their WAT depots.
There are a number of reasons why the ß1AR transgenic
mice have decreased adipose stores. The increased lipolytic activity of
their adipocytes in general, coupled with the greater number of brown
adipocytes, may lead to increased energy expenditure through heat
production by BAT (18). Alternatively, increased energy expenditure in
other organs, such as liver or skeletal muscle, may occur. Whether
expression of the ß1AR transgene in brown or white
adipocytes is most important for preventing obesity induced by a high
fat diet can be tested by creating transgenic mice that overexpress the
ß1AR only in BAT, using the UCP-regulatory region
(19).
Because only one line of transgenic mice was studied, it is formally
possible that the in vivo phenotypic effects described
herein were due to the transgene insertion. However, the in
vitro studies showing that there was more ß1AR
activity in the adipose tissue indicate that the decreased lipid stores
in vivo are the direct result of increased receptor
expression. It is also unlikely that the phenotypic changes were the
result of the transgene insertion because all of the studies were
carried out in mice heterozygous for the transgene; any insertion would
have to cause a dominant mutation that only affected adipose
tissue.
In conclusion, we have developed transgenic mice that were used to
study ß-adrenergic activation of lipolysis in adipocytes and the
differentiation of the two types of adipose tissue, BAT and WAT. This
genetic approach allowed us to study the role of the ß1AR
specifically in adipose tissue, without treating animals with
pharmacological agents that may have pleiotropic effects on many
tissues. We showed that it is the total ßAR activity of adipose
tissue, rather than the particular subtype, that may determine its
overall lipolytic state. These and similarly genetically engineered
mice should be useful in the study of pharmacological treatment of
obesity.
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MATERIALS AND METHODS
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Transgenic Mice
A 2.4-kb fragment containing the human ß1AR cDNA
(31) was ligated downstream of the 5.4-kb aP2 enhancer/promoter region
(12) in the pBluescript SK vector (Stratagene, Inc., La Jolla, CA); the
SV40 small tumor antigen splice site and polyadenylylation signal
sequences were added 3' of the ß1AR cDNA. The DNA used
for injections was liberated from the plasmid as a 8.5-kb
HindIII/NotI fragment.
DNA was injected as previously described (12). Swiss Webster mice
(males and females) were purchased from the National Institute of
Health Frederick Cancer Research Facility (Frederick, MD). Transgenic
mice were identified by PCR and Southern blot analysis, as previously
described (14) (not shown).
Transgenic and nontransgenic littermates of the same sex were housed
together, four animals per cage, with food and water ad
libitum. Except where noted, animals were fed standard mouse chow
(Purina 5008, Ralston-Purina, St. Louis, MO). For the high-fat diet
experiments, groups of transgenic and nontransgenic littermates were
fed powdered chow containing40% fat (3.2 kcal/g) (BioServe
Biotechnologies, Laurel, MD) for either 90 days (males) or 120 days
(females).
RNA Analysis
RNAs from different tissues and fat pads were purified by
guanidine thiocyanate extraction and CsCI gradient centrifugation (32).
Northern blot analysis (33) was carried out, and the following probes
were used for hybridizations: rat UCP cDNA (34), mouse ß-actin cDNA
(P. Denberg, unpublished), mouse ß3AR cDNA (2), mouse aP2
cDNA (35), and SV40 donor/acceptor splice site (12). Hybridization to
mouse ß-actin and aP2 probes were done as controls for RNA loading
and integrity.
Body and Tissue Weights
Body weights were performed on a regular basis (usually every 10
days) in all experiments. Three- or 4-month-old mice were used to
measure fat pad weight. After the mice were killed, three fat pads were
analyzed: gonadal (GF), subcutaneous inguinal (ScF), and subcutaneous
intrascapular brown (BF) fat. For the GF and ScF measurements, the
bilateral fat pads from each animal were combined and weighed.
Adipocyte Purification
Adipocytes were isolated from gonadal fat pads using collagenase
digestion according to Rodbell (36) with minor modifications (37).
Briefly, the gonadal adipose tissues from several animals (six to eight
mice on the normal diet and three mice on the high-fat diet) were
pooled and minced in Krebs-Ringer bicarbonate buffer (pH 7.5) with 3%
BSA, 2.5 mM glucose, and 1 mg/ml collagenase type II
(C-6885, Sigma Chemical Co., St. Louis, MO). The tissues were incubated
for 3040 min at 37 C in a 5% CO2 incubator. After
complete collagenase digestion, the adipocytes were separated from the
stromal-vascular fraction and undigested tissue pieces by filtration
through a 250-µm mesh tissue sieve (E-C Apparatus Corp., St.
Petersburg, FL) and centrifugation at 400 x g for 1
min. The fat cake was washed three times with the same buffer without
collagenase. The purified adipocytes were used for adenylyl cyclase and
lipolysis assays.
Adipocyte Size
Adipocytes from whole tissue were fixed in osmium tetroxide, and
the size of the cells was determined by light microscopy using a
reticule. The number of cells was calculated for each 8-µm increment,
and the average size was determined as the arithmetical mean of the
population. The cell size in nanometers was converted to the nanogram
amount of lipid per cell according to the formula developed by Hirsch
and Gallian (38).
Adenylyl Cyclase Assays
For analysis of adenylyl cyclase activity, membranes were
obtained from purified peritoneal adipocytes in cell lysis buffer [2
mMTris-HCI, pH 7.5, 2.5 mM MgCI2,
0.1 mM EGTA, 1 mM dithiothreitol and protease
inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, aprotinin, and pepstatin A)] at +4 C. After Dounce
homogenization (five strokes with a type B pestle), sucrose was added
to 0.25 M. The homogenate was centrifuged at 1100 x
g for 3 min. The supernatant was saved and the pellet was
rehomogenized in buffer and spun down a second time. The supernatants
were combined and spun at 39,000 x g for 10 min. The
pellet was washed in the same buffer with 0.25 M sucrose
and repelleted. The crude membrane pellet was resuspended in buffer
containing 20 mM HEPES, pH 7.4, 1 mM
MgCI2, 1 mM dithiothreitol, and 0.3
mM phenylmethylsulfonyl fluoride and frozen in liquid
nitrogen. The crude membranes were assayed for adenylyl cyclase
activity in the presence of 200 µM ATP and varying
concentrations of the ßAR agonist isoproterenol (Sigma) (Ref. 39, as
modified by Ref.40). Protein concentrations were determined using the
Bio-Rad protein assay (Bio-Rad, Inc., Richmond, CA).
Lipolysis Assays
An aliquot of the purified adipocytes was fixed in 2%
osmium tetroxide and counted (38). Aliquots of isolated gonadal
adipocytes were incubated in a total volume of 1 ml Krebs-Ringer
bicarbonate buffer at 37 C, 5% CO2, for 1 h in the
presence of adenosine deaminase type VIII (1 µg/ml) (Sigma).
Different concentrations of either isoproterenol or the
ß1AR-selective agonist, dobutamine (Research Biochemicals
International, Natick, MA) were added to the reactions, as described in
the figures. The release of glycerol was measured in aliquots of the
infranatant according to the manufacturers directions (Triglyceride
kit 320-UV, Sigma).
Statistics
All statistical analyses were performed using the one-tailed
Students t test.
Experimental Animals
All animal studies were conducted in accord with the principles
and procedures outlined in the "Guidelines for Care and Use of
Experimental Animals."
 |
ACKNOWLEDGMENTS
|
---|
We thank P. Wright, M. Talluri, and B. van den Hoogen for
excellent technical assistance; S. Fried for her advice on the
adipocyte analysis; M. Birnbaum for helpful discussions; and J.
Himms-Hagen for critical reading of the manuscript. The UCP cDNA was a
kind gift from D. Ricquier, and P. Denberg provided the ß-actin
cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Susan R. Ross, Room 526 CRB, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6142.
Supported by grants from the American Heart Association (to S.R.R),
NIMH (to M.M.R.), Tobacco Research Council (to M.M.R.) and NIDDK (to
B.M.S.).
Received for publication May 22, 1996.
Revision received October 3, 1996.
Accepted for publication October 7, 1996.
 |
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