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Molecular Endocrinology, doi:10.1210/me.2004-0194
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Molecular Endocrinology 18 (9): 2302-2311
Copyright © 2004 by The Endocrine Society

The Role of Uncoupling Protein 1 in the Metabolism and Adiposity of RIIß-Protein Kinase A-Deficient Mice

Michael A. Nolan, Maria A. Sikorski and G. Stanley McKnight

Department of Pharmacology, University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: G. Stanley McKnight, Department of Pharmacology, Box 357750, University of Washington, Seattle, Washington 98195-7750. E-mail: mcknight{at}u.washington.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice lacking the RIIß regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RIIß–/– mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RIIß null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RIIß and UCP1 (RIIß–/–/Ucp1–/–) were created, and the key parameters of metabolism and body composition were studied. We discovered that RIIß–/– mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RIIß–/– mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RIIß null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RIIß mutant mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MOUSE MODELS HAVE dramatically impacted the direction and progress of diabetes and obesity research (1, 2). Disruption of the RIIß gene resulted in one of the first examples of a healthy mouse with low body mass index, resistance to diet-induced obesity, and increased basal metabolic rate (BMR) (3, 4, 5, 6). These phenotypes are thought to result from a change in activity or localization of protein kinase A (PKA) activity in either brown adipose tissue (BAT), white adipose tissue (WAT), or brain. These three tissues are the major sites of RIIß expression in the mouse, although lower levels are seen in thyroid gland, testicular Sertoli cells, and ovarian granulosa cells (7, 8). BAT, WAT, and the brain are also key players in the coordination of body weight and adiposity through regulation of energy storage, energy expenditure, and feeding behavior. Examination of RIIß–/– WAT and BAT demonstrates that the loss of RIIß results in a compensatory increase in RI{alpha} and a switch from type II to type I holoenzyme. The type I holoenzyme dissociates at a lower threshold of cAMP compared with type II, and this leads to release of free C subunit, an increase in basal PKA activity, and subsequent degradation and down-regulation of PKA by proteolysis (4, 6, 9).

In RIIß–/– BAT, UCP1 protein content was increased, and the observed elevation in basal oxygen consumption suggested that overexpression of UCP1 was functioning to promote energy expenditure through thermogenesis, leading to a lean phenotype (4). UCP1 uncouples oxidative phosphorylation from ATP synthesis by allowing protons to cross the inner mitochondrial membrane independent of the ATP synthase (10). Whereas other genetic factors influence an animal’s overall cold sensitivity (11), UCP1 is primarily responsible for adaptive thermogenesis in response to sympathetic stimulation of BAT (12, 13, 14). Norepinephrine release stimulates ß3-adrenergic receptors to increase cAMP levels and PKA activity (15). This activation induces cytoplasmic (lipolysis) and nuclear (PGC-1{alpha} and Ucp1 transcription) events that increase thermogenesis. Ucp1–/– mice are not obese (13), but increased UCP1 protein is associated with leanness in studies in addition to the RIIß–/–. For example, transgenic expression of UCP1 under the adipocyte-specifc promoter aP2 leads to elevated UCP1 in WAT pads and leanness (16). Endogenous alterations in BAT UCP1 are also thought to be the cause of increased energy expenditure and leanness in both Eif4ebp1–/– (17) and Cidea–/– (18) mice.

Here we report that the induction of UCP1 protein in RIIß knockout mice is not due to an increase in mRNA but is a posttranslational event that correlates with an increase in BAT mitochondrial DNA content. We also find that the RIIß–/– mice have a 2-fold elevated nocturnal locomotor activity that indicates a significant increase in physical activity-dependent energy expenditure in addition to the increased basal oxygen consumption previously reported. Elevated nocturnal activity has now been observed in several mutant mouse lines that have a lean phenotype including mice deficient in acyl coenzyme A:diacylglycerol transferase (Dgat), melanin-concentrating hormone (MCH), MCH-1 receptor (MCH1R), and attractin (mahogany) (19, 20, 21, 22).

We bred double knockout (DKO) mice deficient in both RIIß and UCP1, and they no longer have an elevated basal metabolic rate but retain the nocturnal hyperlocomotor behavior and lean phenotype characteristic of RIIß–/– mice. We suggest that the nocturnal locomotor activity and leanness of RIIß–/– mice may stem from PKA changes in the brain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thermoregulation of UCP-1 in RIIß–/– BAT
Induction and suppression of the sympathetic nervous system (SNS) activity in BAT by exposure to different ambient temperatures has been widely used to study UCP1 gene and protein expression in rodents (12, 14, 23, 24, 25). To examine the regulation of UCP1 in RIIß–/– BAT, mice were exposed to either a thermoneutral (30 C) or cold (4 C) environment for 1 wk. UCP1 protein was elevated 4-fold in the RIIß–/– BAT compared with wild type (WT) at 30 C (Fig. 1AGo). Cold exposure increased UCP1 protein in both genotypes, concurrent with a 6-fold increase in UCP1 mRNA (Fig. 1BGo). Within each temperature group, however, there was no difference in UCP1 mRNA level between WT and RIIß–/– BAT (Fig. 1BGo). This result was surprising because we had previously shown that the RIIß mice had increased basal PKA activity in BAT, and UCP1 is known to be a PKA-responsive gene (4, 6, 26, 27). The induced protein levels in the absence of an increase in mRNA could be due to either an increase in the synthesis or stability of UCP1 protein in the RIIß–/– BAT. One stabilizing influence might be the level of mitochondria because UCP1 is known to reside in the inner mitochondrial membrane. We measured mitochondrial DNA content per cell and found that it was 2-fold higher in RIIß–/– BAT compared with WT controls when the animals were housed at thermoneutrality and remained elevated in mice challenged at 4 C (Fig. 1CGo). This suggests that an increase in the number of mitochondria per brown adipocyte may be responsible for the increase in UCP1 protein and the increase in basal oxygen consumption.



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  		Fig. 1. Thermoregulation of UCP1 in RIIß–/– BAT

BAT from male mice housed at either 30 C or 4 C for 1 wk were analyzed for UCP1 protein (A), UCP1 mRNA (B), and mitochondrial DNA (C). Northern blot analysis of PGC-1{alpha} (D) and UCP1 (E) mRNA from BAT before (room temperature, RT) or after (4 C 3 Hr) a 3-h cold exposure. Data are represented as means ± SEM, n = 3–4 per genotype per temperature. (*, significant difference from WT 30 C; X, significant difference from WT RT; P < 0.05).

 
Induction of PGC-1{alpha} Transcription Is Unaffected in RIIß–/– BAT
Transcription of UCP1 is robustly induced by synergism of PGC-1{alpha} and nuclear receptors. This coactivator is rapidly induced in BAT after a cold challenge, enhances expression of the UCP1 gene, and is also involved in induction of mitochondria and the differentiation and proliferation of BAT (26, 28, 29). Despite the induced levels of mitochondria and UCP1 protein in RIIß–/– BAT, the levels of peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA were identical with that of WT in the long-term thermoneutral and cold exposure experiments (data not shown). At room temperature, PGC-1{alpha} mRNA expression was also unchanged in RIIß–/– BAT (Fig. 1DGo). After a 3-h cold exposure, both WT and RIIß–/– BAT induce PGC-1{alpha} mRNA 20-fold. The levels of UCP1 mRNA from the same animals are also shown to demonstrate the normal induction of UCP in RIIß–/– BAT (Fig. 1EGo). These findings support the conclusion that increased UCP1 protein in RIIß–/– BAT results from a posttranscriptional event.

RIIß–/– Mice Have Increased Locomotor Activity
Nocturnal hyperlocomotor activity has been reported in mouse models of genetic leanness stemming from various disruptions of the energy balance: Dgat–/–, MCH–/–, MCH1R–/–, and Atrn–/– mice (19, 20, 21, 22). To examine this phenotype in RIIß–/– mice, animals were individually housed in a beam break apparatus (San Diego Instruments, San Diego, CA) for three dark phases of the light cycle to measure locomotor activity (Fig. 2Go, A and B). All mice demonstrated a slight decrease in total activity over the course of the first 24-h period, attributable to habituation to the measurement chamber; however, RIIß–/– mice were remarkably hyperactive compared with WT mice in each of the successive dark phases, displaying at least a 2-fold increase in total distance traveled (Fig. 2Go, C and D). Female daytime locomotor activity measured during lights-on phases did not differ between genotypes; however, male RIIß–/– mice remained hyperactive compared with WTs during the day. The increased nocturnal locomotor activity was not a result of the reduced adiposity in RIIß–/– mice because a 24-h fast of WT mice did not induce hyperlocomotor activity even though this duration of fasting causes a drastic reduction in adiposity (data not shown). Fasting also did not cause a significant change in the activity of the RIIß–/– mice.



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  		Fig. 2. RIIß–/– Mice Exhibit Nocturnal Hyperactivity

Continuous recording of locomotor activity displayed in 1-h increments over three dark cycles (denoted by black bars over graph) for male (A) and female (B) mice. All mice were from 12–17 wk old. Cumulative distance traveled in the three 12-h lights-out (nights 1–3) and two lights-on (days 1–2) cycles for male (C) and female (D) mice. Data are represented as means ± SEM, n = 12–18 mice per genotype.

 
Breeding of RIIß–/–/UCP1–/– Mice, BAT Histology, and UCP3 Compensation
To determine whether increased UCP1-dependent BAT activity is a direct cause of the lean phenotype of the RIIß–/– mice, we bred DKO mice lacking both RIIß and Ucp1. The breeding strategy for generation of WT (RIIß+/+/Ucp1+/+); RIIß knockout (RKO) (RIIß–/–/ Ucp1+/+); Ucp1 knockout (UKO) (RIIß+/+/ Ucp1–/–), and DKO (RIIß–/–/Ucp1–/–) mice is shown in Fig. 3AGo. As expected, DKO mice exhibited no gross abnormalities and were born in the expected Mendelian ratio. Maintenance of a mixed C57BL6:129Sv/J background was intentional due to the cold sensitivity of Ucp1–/– mice of either genetic background (11). Histological examination of BAT from the four genotypes of mice housed at room temperature revealed that DKO BAT more closely resembled UKO BAT in lipid droplet size, demonstrating that the RIIß mutation is affecting BAT activity upstream of UCP1 (Fig. 3BGo).



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  		Fig. 3. BAT Histology and UCP3 Expression in [RIIß–/–/Ucp1–/–] Mice

A, Breeding strategy to generate WT [RIIß+/+/Ucp1+/+], RKO [RIIß–/–/Ucp1+/+], UKO [RIIß+/+/Ucp1–/–] and DKO [RIIß–/–/Ucp1–/–] mice on a mixed C57BL6/129SV/J background. Only male mice were examined from these crosses. B, Histological sections (x40) from intrascapular BAT stained with hematoxylin/eosin (36 ). Scale bar, 50 µm. Analysis of UCP3 protein from BAT (C), WAT (D), and skeletal muscle (E). Images are representative samples of three male mice from each genotype. Fold increases are relative to the WT after densitometric quantification and averaging band intensities.

 
Although neither UCP2 nor UCP3 has been shown to functionally replace UCP1, their expression in BAT, WAT, and skeletal muscle has been tied to the metabolic state of an individual. For example, overexpression of human UCP3 in mouse skeletal muscle results in a lean phenotype (30). Also, UCP2 expression is increased in Ucp1–/– BAT (13). To examine the combined effects of the RIIß and Ucp1 deletions on the expression of other uncoupling protein isoforms, we determined the relative levels of UCP2 (mRNA) and UCP3 (protein) in the four genotypes of mice in this study. RKO, UKO, and DKO BAT all exhibited a 2- to 3-fold increase in UCP3 protein over WT BAT (Fig. 3CGo). As expected, UCP2 mRNA was elevated 2-fold in UKO BAT, a phenotype that did not change in the DKO (data not shown), suggesting that the compensation for loss of UCP1 in BAT is not enhanced by the genetic removal of RIIß.

WAT UCP3 protein content, however, did demonstrate a synergistic effect of the two mutations. RKO WAT had a slight increase in UCP3 protein compared with WT and UKO levels (Fig. 3DGo), but DKO WAT had a 10-fold increase in UCP3 protein compared with the other genotypes (Fig. 3DGo). In contrast, WAT UCP2 mRNA levels are unchanged in all groups (data not shown). Furthermore, skeletal muscle expression of UCP3 protein is unchanged among all four genotypes (Fig. 3EGo), as we would expect because neither UCP1 nor RIIß is expressed in this tissue. It is unclear what the mechanism or significance is of the increase in UCP3 in WAT in the DKO mice.

Increased Basal Resting Oxygen Consumption (VO2) in RKO Mice Requires UCP1
RKO mice have an elevated BMR as measured by VO2 (4). Mice placed in an Oxymax chamber (Columbus Instruments, Columbus, OH) were monitored for VO2 for 2.5 h after a 90-min acclimation period (Fig. 4AGo). The first 30 min of recording was averaged to calculate the basal VO2 of each mouse. RKO mice had a higher basal rate of oxygen consumption than all other genotypes (Fig. 4BGo). The restoration of normal oxygen consumption in DKO mice indicated that UCP1 is required for this measure of increased energy expenditure in RKO mice and suggests that the elevation of UCP3 in DKO WAT does not measurably increase VO2. After the BMR measurement window, mice were injected with the ß3-adrenergic receptor agonist, CL-316,243, and placed back in the Oxymax chamber. This pharmacological treatment results in a BAT-dependent elevation in oxygen consumption. Mice lacking UCP1 do not increase VO2 after this treatment (13) (Fig. 4BGo). RKO mice did not increase VO2 in response to ß-adrenergic stimulation above their already higher basal value, suggesting that they are already in an induced state, whereas WT mice showed a 30% increase in oxygen consumption after CL-316,243 injection. The results demonstrate that the increase in VO2 in RKO mice is UCP1 dependent because it is returned to normal in the DKO mice, and that DKO BAT is no longer responsive to ß-receptor activation as expected because the mice lack UCP1.



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  		Fig. 4. Increased VO2 in RIIß–/– Mice Is UCP1 Dependent

A, Oxymax traces (Columbus Instruments) recorded from male mice (12–17 wk old) after a 90-min acclimation period. A 1 mg/kg injection of CL-316,243 was given to mice 45 min after the acclimation (Inj. and arrow on graph). B, Oxygen consumption for 30 min windows before (Basal) and 30 min after (CL-induced) CL-316,243 injection (indicated on the trace with black lines). Data are presented as means ± SEM (n = 6–8 per genotype), (*, significant elevation over WT basal; P < 0.05).

 
RIIß–/– Hyperactivity Is Independent of UCP1
The nocturnal hyperactivity shown in Fig. 2Go is also a likely contributor to the increased energy expenditure and might lead to a lean phenotype. To determine whether this change in energy expenditure is UCP1 dependent, locomotor activity of DKO mice was measured. DKO mice were hyperactive compared with UKO controls, exhibiting the same level of activity as RKO mice in the dark phase of the light cycle (Fig. 5Go). These findings suggest that UCP1-dependent thermogenesis and locomotor activity are two independent mechanisms of up-regulated energy expenditure in RIIß–/– mice.



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  		Fig. 5. RIIß–/– Mice Are Hyperactive Independent of UCP1

Total distance traveled in a 24-h interval, separated into the subjective night (dark bar) and day (light bar) phases of the light cycle. All mice in this experiment were males (12–17 wk old). Data are presented as means ± SEM (n = 4–8 per genotype), (*, significance; P < 0.05).

 
Disruption of UCP1 Does Not Rescue the Lean Phenotype of RIIß–/– Mice
Leanness in the RKO mouse is characterized by a decrease in WAT mass as a percent of body weight (3, 4). Upon dissection, body weight, BAT mass, and WAT mass (epididymal, retroperitoneal, and inguinal pads weighed individually) were recorded (Table 1Go). The RKO mice weighed significantly (18%) less than their WT littermate controls and the DKO mice were also 18% lighter than the corresponding littermate UKO mice. All animals that had a disruption of the UCP1 gene had significantly higher BAT mass compared with WT animals. In this study, the BAT from RKO mice was reduced in mass compared with WT, consistent with the increased metabolic activity and lowered triglyceride content of BAT due to the RIIß knockout (Fig. 3Go). The lean phenotype of the RKO mice is clearly evident in both Fig. 6Go and Table 1Go. These mice had a 50% reduction in percent adiposity compared with either WT or UKO mice. The DKO mutant missing both RIIß and UCP1 remained lean, although total adiposity for this group was only reduced by 32% compared with the control UKO group. As expected, serum leptin levels directly correlated with the percent adiposity of individual mice of all genotypes (data not shown). The results indicate that the lean phenotype of RKO mice is not due to induced UCP1 protein in BAT and suggests that the loss of RIIß in other tissues may be contributing to both the nocturnal hyperactivity and the lean phenotype.


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  		Table 1. Comparison of BAT and WAT Pad Weights upon Dissection

 


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  		Fig. 6. Percent Adiposity Remains Low in [RIIß–/–/Ucp1–/–] Mice

Percent adiposity of male mice (12–17 wk old) on a standard chow diet. Individual mice are plotted by genotype along with means (–) ± SEM (n = 6–16 per genotype) (*, significance; P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The RKO mouse has a disruption in energy homeostasis that leads to an abnormally low level of triglyceride storage in WAT. The reduced adiposity of RKO mice is reflected by low circulating leptin levels, but the mice do not respond with sufficient hyperphagy to replenish WAT stores. As reported previously, there is no significant increase in food consumption in RKO mice as measured in kilocalories per animal (4). Our previous studies had demonstrated that the loss of RIIß in both WAT and BAT led to a compensatory switch from an RIIß containing holoenzme to an RI{alpha} holoenzyme with an increased sensitivity to cAMP. This is accompanied by increased expression of UCP1 protein in BAT and an increase in basal oxygen consumption.

Considerable precedent exists to support the association between an increase in UCP1-dependent energy expenditure and leanness in mouse models. The overexpression of UCP1 in WAT and BAT with a transgene driven by the fat-specific aP2 promoter resulted in lower adiposity (16) as did the disruption of the Cidea protein, a mitochondrial component that was shown to suppress Ucp1 activity (18). Overexpression of UCP3 in muscle caused an increase in energy metabolism and a decrease in adiposity in transgenic mice (30). There is also evidence supporting a role of BAT in the regulation of adiposity in mice because a toxic transgene expressing diphtheria toxin A chain behind the Ucp1 promoter caused destruction of a large percentage of BAT adipocytes and led to increased susceptibility to diet-induced obesity (31). Targeted disruption of all three ß-receptors also led to obesity in mice especially when fed a high-fat diet due to a loss of diet-induced thermogenesis (32).

BAT is innervated by the SNS, and activation of the SNS occurs in response to many factors including cold, stress, and high leptin. Sympathetic stimulation of BAT causes an increase in cAMP, activation of PKA and other signaling molecules, induction of genes including those coding for PGC1{alpha} and UCP1, enhanced mitochondriogenesis, as well as a stimulation of lipolysis and release of fatty acids (10). In BAT, these changes lead to activation of the UCP1-dependent uncoupling of oxidative phosphorylation and the generation of heat. The increase in UCP1 protein and the increase in basal oxygen consumption in RKO mice suggested that the mutation in RIIß had bypassed the ß-receptors and directly activated the BAT thermogenic system, leading to increased energy expenditure and a lean phenotype.

An examination of Ucp1 gene expression in RIIß-deficient BAT revealed that despite the apparent increase in basal PKA activity and sensitivity to cAMP, there was no induction of UCP1 mRNA and the increased UCP1 protein depended only on posttranscriptional events. Another PKA inducible gene, PGC1{alpha}, was also unaffected by the mutation in RIIß, indicating that the switch from a type II to type I kinase had not changed the gene expression profile of cAMP-responsive genes. Nevertheless, the RKO BAT has properties associated with increased sympathetic activation that include an increase in the number of mitochondria per cell and an increase in basal oxygen consumption. One hypothesis to explain the dissociation between these aspects of BAT activation would be that some responses (mitochondriogenesis, UCP1 protein stabilization) are more sensitive to the modest increase in PKA activity than the gene expression response.

To determine the role of UCP1 in the metabolic phenotype of RIIß–/– mice, we produced DKO animals missing both proteins. These DKO mice revealed that the elevated basal oxygen consumption of RKO mice depended on UCP1 expression and in the absence of UCP1, the DKO BAT no longer responded to the ß3-specific agonist CL-316,243 with induced oxygen consumption. In the DKO mice, the BAT also reverted to a weight and histological appearance resembling inactivated BAT with large triglyceride droplets. The leanness of RKO mice persisted in the DKO mice, although there was a slight increase overall in adiposity of DKO compared with RKO that may reflect a minor contribution of UCP1 to the phenotype. In addition to demonstrating that UCP1 is not required for the lean phenotype of RKO mice, these results also make it unlikely that BAT activation is a major part of the phenotype although other influences of the RIIß-deficient BAT on overall energy metabolism cannot be ruled out without producing a BAT-specific knockout.

Although RIIß knockout mice behaved in a similar way in our previous open field studies conducted during the light phase (33), we discovered that both male and female RIIß knockout mice exhibit a 2-fold increase in nocturnal locomotor activity. This exercise-induced increase in energy expenditure may be a major factor in the overall regulation of energy balance in these mice. Nocturnal hyperactivity is not simply the result of low adiposity because a 24-h fast of WT mice did not increase their nocturnal activity substantially and fasting also did not cause a significant change in the activity of RIIß knockout mice (Sikorski, M. A., and G. S. McKnight, unpublished data). The hyperactivity is probably not directly causing the increase in basal oxygen consumption because oxygen consumption was measured in a small chamber that limits locomotion and done during the morning hours when mice of all four genotypes were relatively inactive. Nevertheless, it is possible that the increase in physical activity during the dark phase has effectively endurance trained skeletal muscle, altering muscle fiber type from fast-twitch (type II) to slow-twitch (type I) and increased oxygen consumption by muscle. Arguing against this conclusion is the fact that the resting oxygen consumption of DKO mice remains the same as WT despite the nocturnal hyperactivity of DKO mice.

Hyperlocomotor activity has been observed in several other genetic mutants that maintain a low adiposity, including mice lacking MCH (20), and the MCH1R (21). The MCH1R is most highly expressed in the striatum and nucleus accumbens, brain regions that also show the highest levels of RIIß expression. Our previous work with RKO mice has shown that the greatest disruption of kinase activity in the central nervous system occurs in the striatum (33). Loss of RIIß leads to an increase in free catalytic subunit, and although some of it is successfully reincorporated into stable holoenzyme with other R subunits in the brain, a substantial fraction is left in a constitutively active form that is then rapidly down-regulated by protein degradation. Because a major coupling mechanism of MCH1R is through Gi to inhibit adenylate cyclase and cAMP production, it is intriguing to suggest that a loss of the MHC1R (or its agonist) could lead to an elevated activation of PKA in MCH1R-expressing neurons. The RIIß gene is also expressed in other brain regions including the hypothalamus where several key orexigenic (neuropeptide Y, agouti-related protein) and anorexigenic ({alpha}-MSH) act on Gi- and Gs-coupled receptors (respectively) to affect cAMP generation and PKA activation (34). An increase in unregulated PKA activity in the hypothalamus would be expected to be anorexigenic based on the properties of these peptide/receptor systems.

This study demonstrates that UCP1 protein is induced in RIIß knockout BAT by a posttranscriptional mechanism but that the increase in UCP1 and the coordinate increase in basal oxygen consumption is not the cause of the lean phenotype of these mice. Genetic ablation of Ucp1 does not normalize the adiposity of RIIß null mice and they also retain the nocturnal hyperlocomotor phenotype that we have shown in this study to be associated with the RIIß mutation. Although these results do not rule out BAT as a potential contributor to the lean phenotype, we favor the idea that loss of brain RIIß protein is influencing nocturnal energy expenditure and perhaps modifying feeding behavior to maintain a lean phenotype and resistance to diet-induced obesity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
The generation of RIIß–/– and Ucp1–/– mice has been previously described (4, 6, 13, 35). Adult mice on a mixed C57BL/6:129Sv/J background were group housed and maintained on a 12-h light, 12-h dark cycle unless otherwise noted. All animals were age and sex matched and had access to standard mouse chow (LabDiet 5053, PMI Nutrition International, Brentwood, MO) and water ad libitum. For short-term cold exposure (3 h, 4 C) and long-term (1 wk) exposure to thermoneutrality (30 C) and cold (4 C), mice were individually housed. All procedures were approved by the Institutional Animal Care and Use Committee of the School of Medicine of the University of Washington, in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals.

Western Blotting
Intrascapular BAT, epididymal WAT, and skeletal muscle were homogenized in lysis buffer [250 mM sucrose, 20 mM Tris-Cl (pH 7.6), 0.1 mM EDTA, 0.5 mM EGTA, 10 mM dithiothreitol, 1% Triton X-100, 0.5% deoxycholic acid] supplemented with protease and phosphatase inhibitors (1 µg/ml leupeptin, 3 µg/ml aprotinin, 40 µg/ml soybean trypsin inhibitor, 0.5 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1 µM microcystin-LR, 0.2 µM NaF, 0.2 µM orthovanadate), sonicated, and cleared by centrifugation (10,000 x g, 15 min). After the protein concentration of the soluble infranatant (BAT and WAT) or supernatant (skeletal muscle) was determined by the Bradford method (Bio-Rad, Hercules, CA), samples were diluted into 1x sample buffer [62.5 mM Tris-Cl (pH 6.8), 2% (wt/vol) sodium dodecyl sulfate (SDS), 5% glycerol, 0.05% (wt/vol) bromophenol blue], boiled 5 min, then brought to 5% (vol/vol) ß-mercaptoethanol. Thirty micrograms of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH), and stained with Ponceau-S. Membranes were blocked [5% (wt/vol) BSA in PBS (10 mM sodium phosphate, 150 mM NaCl at pH 7.2) for 30 min] and probed for UCP1 [1:1000 (vol/vol) (Calbiochem, San Diego, CA)] or UCP3 [1:1000 (vol/vol), Affinity Bio Reagents (Golden, CO)] at 4 C overnight. After three washes in PBS-T [0.1% (vol/vol) Tween 20 in PBS], membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody [1:10,000 (vol/vol) in PBS-T] containing 5% (wt/vol) nonfat dry milk. After three washes in PBS-T, bound antibodies were detected using ECL (Amersham Biosciences Corp., Piscataway, NJ) and exposure to HyperFilm ECL (Amersham). Densitometric quantification was performed using the NIH Image program (version 1.63).

Solution Hybridization
The steady-state amount of UCP1 mRNA was determined by solution hybridization as previously described (6). Briefly, total nucleic acid was isolated from BAT by proteinase K digestion and phenol/chloroform extraction. Samples were hybridized overnight with approximately 5000 cpm of [32P]CTP-labeled antisense RNA probe at 70 C under paraffin oil. Free probe was then digested with ribonuclease (RNase) A and T1 for 1 h at 37 C. Samples were precipitated with 10% trichloroacetic acid and collected on Whatman (Brentford, UK) GF/C glass microfiber filters to trap hybridized probe. The amount of RNase-resistant probe was measured by liquid scintillation counting. The results were converted to molecules of mRNA per cell based on the specific activity of the probe and Hoechst stain (Sigma, St. Louis, MO) fluorimetric measurement of DNA concentration from the phenol/chloroform extracted sample.

BAT Mitochondrial DNA Content
The relative amount of mitochondrial DNA was determined by scintillation counting of dot-blot hybridization. DNA was extracted from BAT by proteinase K digestion followed by phenol/chloroform extraction. One microgram of DNA was denatured in 15 µl 2 M NaCl, 0.1 M NaOH, and 5 µl were spotted in duplicate onto a Nylon membrane (Roche Molecular Biochemicals, Indianapolis, IN). Hybridization of a [32P]deoxy-ATP-labeled probe of the mitochondrial DNA-encoded 16S ribosomal RNA gene (l-rRNA, courtesy of R. Palmiter, University of Washington) to BAT DNA was confirmed by autoradiography and quantified by excision of the spotted DNA and liquid scintillation counting. The specificity of the probe was confirmed by Southern blotting of EcoRI-digested BAT DNA, which gave the expected single band.

Northern Blotting
Total RNA was extracted from intrascapular BAT pads using Trizol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s protocol after precipitation in ethanol and resuspension in RNase-free H2O. Equal amounts of RNA (5–10 µg) were fractionated by electrophoresis through 1.2% (wt/vol) agarose in 40 mM 3-(N-morpholino) propane-sulfonic acid, 10 mM sodium acetate, 1 mM EDTA, 2% (vol/vol) formaldehyde (37% solution), transferred to a nylon membrane (Roche), and fixed by ultraviolet illumination. 28S and 18S ribosomal RNA was visualized by methylene blue [0.03% (wt/vol) in 0.3 M ammonium acetate] staining to determine equal loading and transfer of RNA to the membrane. Membranes were prehybridized for 1 h at 68 C in Church & Gilbert Buffer [250 mM sodium phosphate, 1 mM EDTA, 1% (wt/vol) BSA, 7% (wt/vol) SDS], and probed for UCP1 ([1100 bp XhoI fragment from cDNA], and PGC-1{alpha} (800-bp EcoRI fragment from cDNA). Probes were generated by random priming (Roche), purified over a Sephadex G-50 column (NICK column, Amersham), and heat denatured before hybridizing to filters at 65 C overnight in Church & Gilbert buffer. After hybridization, membranes were washed twice in 2x SSC [0.3 M NaCl, 30 mM sodium citrate, 0.5% (wt/vol) SDS, 0.1% (wt/vol) sodium pyrophosphate] for 15 min at room temperature, then up to 45 C. The membranes were then washed in 0.5x SET [0.5% (wt/vol) SDS, 5 mM Tris, 2.5 mM EDTA, 0.1% sodium pyrophosphate] up to 45 C. Membranes were air dried and exposed to x-ray film (X-Omat, Eastman Kodak) at –80 C for 8–16 h. Densitometric quantitation was performed using NIH Image (version 1.63), and relative mRNA levels were determined.

Histology
After fixation in formaldehyde, tissues were serially dehydrated in ethanol and processed in paraffin. Embedded specimens were sectioned on a microtome at 8 µm and stained with hematoxylin and eosin. Images were taken at x40 magnification on a Zeiss Axioscop2 (Carl Zeiss Ltd., Oberkochen, Germany). Scale bar and image processing were performed with AxioVision 3.1 software (Carl Zeiss Ltd.).

Oxygen Consumption Measurements
Oxygen consumption (VO2) was determined for mice in an Oxymax chamber (Columbus Instruments) at room temperature. The chamber was 4 x 8 in., allowing limited locomotion and the entire apparatus was housed in an isolated room away from other animals and stimuli. Air flow to the cage was 500 ml/min. VO2 was recorded for a total of 4 h (between 0800 and 1300 h). Basal VO2 was determined as the average of measurements for the 30 min window after a 90-min acclimation period. After 2 h of recording, the chamber was opened, mice were injected ip with 1 mg/kg CL-316,243 diluted in PBS (Wyeth/Ayerst Pharmaceuticals), and returned to the cage to continue recording. After a 30-min recovery period, CL-induced VO2 was taken as the average of the next 30 min.

Locomotor Activity Measurements
Animals were housed individually in cages in a beam-break locomotor activity chamber (San Diego Instruments, San Diego, CA) at 1600 h for up to three consecutive nights. Ambulations are scored as interruption of consecutive beams in 30-min intervals and converted to meters based on the distance between the beams. Nocturnal activity is taken as the total distance traveled in the dark phases (1900–0700 h).

Percent Adiposity Determination
Immediately after cervical dislocation, mice were weighed and dissected to remove the epididymal, retro-peritoneal, and inguinal WAT pads, which were each weighed individually. The sum of these three WAT pads divided by the body weight of the animal was used as a measure of percent adiposity.

Statistical Analysis
Data are presented as the mean ± SEM, and comparisons among genotypes or treatment groups were analyzed by single factor ANOVA and post hoc t tests. P < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
The authors thank Leslie P. Kozak (Pennington Biomedical Research Institute, Baton Rouge, LA) for providing Ucp1–/– mice and comments on the manuscript; David Cummings (University of Washington) for contributions to experimental design, and Thomas Su (University of Washington) for technical support of this work.


   FOOTNOTES
 
This work was supported by GM32875 (to G.S.M.). M.A.N. is a recipient of training grant support from National Institutes of Health (T32 GM07270).

Abbreviations: BAT, Brown adipose tissue; BMR, basal metabolic rate; DKO, double knockout; MCH, melanin-concentrating hormone; MCH1R, MCH-1 receptor; PGC-1{alpha}, peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha}; PKA, protein kinase A; RKO, RIIß knockout; RNase, ribonuclease; SDS, sodium dodecyl sulfate; SNS, sympathetic nervous system; UCP, uncoupling protein; UKO, UCP1 knockout; VO2, oxygen consumption; WAT, white adipose tissue; WT, wild-type.

Received for publication May 10, 2004. Accepted for publication June 3, 2004.


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