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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ratio of {alpha}- 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adrenergic receptors (AR) are known to participate in the regulation of adipose tissue development and metabolism. There are two major classes of ARs, {alpha} 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 {alpha}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 {alpha}2/ß ARs in different adipose tissue depots affects the lipolytic rate, with depots containing higher levels of {alpha}2ARs more prone to lipogenesis. Thus, the ratio of functional {alpha}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 ß123 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 {alpha}- 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo) but not in other tissues of this mouse strain (Fig. 1BGo). 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. 9Go). 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. 1Go and 9Go). 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 16–18 h. The multiple bands seen after hybridization to the ß3AR probe are due to alternative splicing, variable transcript initiation, and alternative polyadenylylation (41, 42).

 
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 2Go 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.

 
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. 3AGo, 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. 3BGo). 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. 3AGo).



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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.

 
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. 4Go. 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. 4BGo). Moreover, the average size of the adipocytes in the transgenic mice was significantly smaller than that of the nontransgenic mice (Table 1Go).



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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 13–14 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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Table 1. Size of the Adipocytes Isolated from the Different Fat Depots of the ß1AR Male Transgenic Mice

 
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. 5Go, 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).

 
We also examined the weight of various fat pads. As can be seen in Fig. 6Go, 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 1Go), 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. 5Go 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.

 
The lipolytic activity of adipocytes from the transgenic animals on the high-fat diet was also elevated relative to the controls (Fig. 7Go). 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.

 
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. 8Go, A and B, respectively). The unilocular adipocytes in the transgenic WAT depots appeared smaller in size (Fig. 8Go, C and D), confirming the data presented in Table 1Go. In addition to the unilocular adipocytes, the gonadal adipose tissue from both males (Fig. 8CGo) 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. 8DGo for males; not shown for females). There were no apparent differences in the interscapular BAT morphology between the transgenic (Fig. 8EGo) 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.

 
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. 9Go and not shown). UCP RNA was also found at very low levels in the gonadal fat of transgenic, but not nontransgenic, males (Fig. 9Go). 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. 9Go) 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 30–40 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 manufacturer’s directions (Triglyceride kit 320-UV, Sigma).

Statistics
All statistical analyses were performed using the one-tailed Student’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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