Disruption of the RIIβsubunit of PKA reverses the obesity syndrome of agouti lethal yellow mice
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Communicated by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, November 14, 2007 (received for review September 2, 2007)
Abstract
Agouti lethal yellow (Ay) mice express agouti ectopically because of a genetic rearrangement at the agouti locus. The agouti peptide is a potent antagonist of the melanocortin 4 receptor (MC4R) expressed in neurons, and this leads to hyperphagia, hypoactivity, and increased fat mass. The MC4R signals through Gs and is thought to stimulate the production of cAMP and activation of downstream cAMP effector molecules such as PKA. Disruption of the RIIβregulatory subunit gene of PKA results in release of the active catalytic subunit and an increase in basal PKA activity in cells where RIIβis highly expressed. Because RIIβis expressed in neurons including those in the hypothalamic nuclei where MC4R is prominent we tested the possibility that the RIIβknockout might rescue the body weight phenotypes of the Ay mice. Disruption of the RIIβPKA regulatory subunit gene in mice leads to a 50% reduction in white adipose tissue and resistance to diet-induced obesity and hyperglycemia. The RIIβmutation rescued the elevated body weight, hyperphagia, and obesity of Ay mice. Partial rescue of the Ay phenotypes was even observed on an RIIβheterozygote background. These results suggest that the RIIβgene mutation alters adiposity and locomotor activity by modifying PKA signaling pathways downstream of the agouti antagonism of MC4R in the hypothalamus.
The rapid increase in obesity in the human population and the associated increased risk for ailments such as diabetes and cardiovascular disease has driven an intense effort to understand the physiological pathways that regulate body weight. Both induced and spontaneous mutations in the mouse genome have been instrumental in identifying the genes and cell types that play a role in energy balance in mammals. We have previously determined that mice lacking the RIIβregulatory subunit of PKA exhibit a 10% reduction in body weight and a 50% decrease in white adipose tissue (WAT) and are resistant to diet-induced obesity and hyperglycemia (1, 2). RIIβknockout (KO) mice have a 2-fold increase in their nocturnal locomotor activity and an increase in resting metabolic rate as measured by oxygen consumption (1, 3, 4). These studies have established that signaling through the RIIβ–PKA pathway can have dramatic effects on both body weight and energy expenditure in mice. The RIIβsubunit of PKA is highly expressed in WAT, brown adipose tissue (BAT), and brain (1, 5), but significant expression is also seen in several other tissues including thyroid, testis, and ovary (6–8). The contribution of each of these tissues to the overall RIIβKO phenotype has not yet been resolved.
The arcuate nucleus region (ARC) of the hypothalamus serves as a center to integrate signals that control feeding and energy expenditure. Two anatomically distinct populations of leptin-responsive neurons exist in the ARC; those that express neuropeptide Y (NPY) and agouti-related protein (AgRP) and those that express proopiomelanocortin (POMC) (9, 10). The NPY/AgRP and POMC neurons project from the ARC to the paraventricular hypothalamus (PVH) and other regions that express the Gs-coupled melanocortin receptors MC3R and MC4R. Activation of the MC4R byα-melanocyte-stimulating hormone (α-MSH) released from POMC neurons is thought to stimulate the cAMP pathway and lead to a decrease in food intake and an increase in energy expenditure. AgRP acts to antagonize the action ofα-MSH on both MC3R and MC4R. A knockout of the MC4R gene causes obesity (11), and disruption of NPY (12, 13) or AgRP (14) production leads to more subtle changes in food intake and body weight.
Agouti protein is normally expressed in the skin, but a chromosomal deletion in agouti lethal yellow (Ay) mice places the agouti gene next to a constitutively active promoter causing agouti to be expressed almost ubiquitously, including the hypothalamus (15–18). Mice carrying just a single Ay allele are obese, hypoactive, hyperphagic, hyperglycemic, and hyperinsulinemic (19, 20). Injection of a viral vector expressing agouti only in the PVH also caused mice to become hyperphagic and gain weight (21). These studies support the hypothesis that antagonism of melanocortin receptors in the PVH is at least partially responsible for the obesity syndrome in Ay mice (11, 22–24). Because agouti is a specific antagonist of MC4R but not MC3R, the MC4R is thought to be the key player in body weight regulation (22).
The intracellular signaling pathways through which melanocortin receptors exert their effects in the hypothalamus are not well understood. It is clear that cAMP levels rise dramatically after melanocortin receptor (MCR) stimulation and that PKA is activated; however, other signaling pathways including the MAP kinase pathway and the inositol phospholipid/Ca2+ pathways have been implicated in MCR signaling (25–30). RIIβis expressed throughout the hypothalamus in many of the same regions that also contain MCRs including the ARC, PVH, dorsal medial hypothalamus (DMH), and ventral medial hypothalamus (VMH) (5, 31–33). Each of these regions has distinct effects on food intake and energy expenditure. Because RIIβKO mice exhibit a phenotype that is consistent with increased anorexigenic tone downstream of MCR, we hypothesized that deletion of RIIβmight alleviate the obesity syndrome of Ay mice. We found that deletion of RIIβreduces both food intake and adiposity in Ay mice. The deletion of RIIβnot only rescues the hypoactivity normally seen in Ay mice but increases nocturnal activity to levels that surpass WT controls and approach the hyperactivity phenotype of the RIIβKO mice. These data suggest that the RIIβgene mutation is modifying the signaling pathway downstream of the agouti antagonism of melanocortin receptors in the hypothalamus.
Results
Deletion of RIIβDecreases the Body Weight and Linear Growth of Ay Mice.
The PKA holoenzyme is composed of two catalytic subunits and two regulatory subunits that are either type I (RIα,β) or type II (RIIα,β). Previous studies in our laboratory have determined that in RIIβKO mice there is an increase in basal PKA activity as well as a compensatory increase in the type I PKA holoenzyme that is half-maximally activated by lower cAMP levels than the type II holoenzyme (1, 5, 34, 35). The mating strategy depicted in Fig. 1 A was used to derive Ay/RIIβdouble mutants (Ay/RIIβKO) and control animals. Previous studies have shown that Ay mice begin to significantly differ in their body weight relative to controls by 6–8 wk of age (24, 36, 37). We found that female Ay/RIIβKO mice were visibly smaller than Ay (Fig. 1 B), and they maintained a body weight similar to WT at least through 19 wk of age (Fig. 1 C). A similar result was found in males, but the rescue was not as complete (Fig. 1 D).
Generation of Ay/RIIβKO mice and effects on body weight. (A) Mating scheme to generate Ay mice lacking RIIβ(Ay/RIIβKO) and littermate controls. (B) Female Ay/RIIβKO mouse and littermate controls at 20 wk of age. Ay/RIIβKO mice are visibly smaller than Ay controls but still retain the yellow color of Ay mice. (C) Growth curves of females from 6 to 19 wk of age. **, P < 0.001 vs. all groups. (D) Growth curves of males from 6 to 19 wk of age. **, P < 0.001 vs. WT; *, P < 0.01 vs. WT and Ay; #, P < 0.05 vs. WT.
Ay mice also have increased linear growth that correlates with elevated circulating IGF-1 levels (38). We compared linear growth at the time that mice were killed and verified that female Ay mice were significantly longer than WT controls. We also determined that linear growth was normalized in Ay/RIIβKO mice (Table 1). The rescue of both body weight and linear growth in Ay/RIIβKO mice is consistent with our hypothesis that the RIIβKO phenotype is due to increased activation of PKA downstream of the melanocortin receptors. Significant reductions in body weight were also found in male Ay/RIIβKO mice compared with Ay controls (Fig. 1 D). However, the extent of the rescue was not as complete as in females, and male Ay/RIIβKO mice still weighed significantly more than WT mice at all ages examined. Linear length was not significantly different among the genotypes of males analyzed at 19–20 wk of age.
The RIIβKO background rescues the Ay body weight phenotypes in female mice
Deletion of RIIβIncreases Locomotor Activity and Decreases Adiposity, Food Intake, and Serum Leptin in Ay Mice.
The Ay mice exhibited decreased levels of locomotor activity as previously published (36). However, deletion of RIIβraised the locomotor activity of Ay mice above that of WT controls (Fig. 2), and Ay/RIIβKO mice were ≈1.5 times as active as controls. In both females and males the locomotor activity of Ay mice was approximately half that of WT. In contrast, the RIIβKO mice are twice as active as WT, as we have previously published (3, 4), and it appears that this phenotype of hyperactivity persisted in the Ay/RIIβKO double mutants.
Locomotor activity. The number of ambulations per hour measured over 3 days for females (A) and males (B). Each ambulation is equivalent to 8 cm of distance traveled. Black bars on the x axis indicate lights-off. (C) The total distance traveled for females in meters over three consecutive 12-h dark cycles and two 12-h light cycles. **, P < 0.001, P < 0.05 (WT vs. Ay), and P < 0.01 (WT vs. RIIβKO). (D) The total distance traveled for males as described in C. **, P < 0.001; #, P < 0.05 and P < 0.001 (WT vs. RIIβKO).
Deletion of RIIβresulted in a 34% reduction in adiposity in RIIβKO mice as compared with controls, consistent with our previous findings (1). Deletion of RIIβalso reduced the adiposity of Ay mice. Female Ay/RIIβKO mice had a 56% reduction in adiposity compared with Ay (Fig. 3 A). The weights of three major WAT pads were significantly reduced in Ay/RIIβKO mice (Table 1). Liver weights were also significantly reduced, and visual inspection of the liver revealed that the fatty liver typical of Ay mice was rescued to a more normal size and color in Ay/RIIβKO mice. Male Ay/RIIβKO mice were also significantly leaner than Ay controls (Fig. 3 B), with a 17% reduction in total adiposity. These reductions were more modest than those seen in females of similar age. However, in younger, 13- to 14-wk-old males we observed a 40% reduction in adiposity of Ay/RIIβKO mice compared with Ay controls (Fig. 3 C).
Adiposity, feeding, and leptin. (A) Female Ay/RIIβKO mice are significantly leaner than Ay controls. **, P < 0.001; *, P < 0.01, P < 0.001 (WT vs. Ay), and P < 0.01 (WT vs. RIIβKO, Student's t test). (B) Male Ay/RIIβKO mice are significantly leaner than Ay mice but significantly more obese than WT mice. (C) Decreased adiposity in 13.5-wk-old male Ay/RIIβKO mice compared with Ay/RIIβHET. Shown is WAT pad weight as a percentage of body weight at 13.5 wk of age. *, P < 0.003 (Student's t test). (D) Average food intake at 18 wk of age in females. Ay mice consume more food than WT controls (P < 0.01), but food intake is decreased in Ay/RIIβKO mice. #, P < 0.05. (E) Food intake in male Ay/RIIβKO mice. Plotted is the average daily food intake over 7 consecutive days measured in grams per mouse per day. #, P < 0.05. (F) Serum leptin levels are decreased in female Ay/RIIβKO mice. **, P < 0.001 (WT vs. Ay and Ay vs. Ay/RIIβKO).
Ay mice were hyperphagic as expected, but food intake was significantly decreased in female Ay/RIIβKO mice (Fig. 3 D). A similar reduction in food intake was found in male Ay/RIIβKO mice (Fig. 3 E). Feeding in Ay/RIIβKO mice was similar to RIIβKO mice, and, consistent with previous reports (1, 4), the RIIβKO mice tended to eat slightly more than controls.
Leptin is an adipose tissue-specific hormone that is secreted in proportion to body fat and is one of the key satiety signals (39). The hypothalamus normally responds to increased leptin levels by stimulating neuronal pathways that reduce food intake. In Ay mice and other mouse models of obesity there are very high levels of leptin, as shown in Fig. 3 F, but the animals are leptin-insensitive, which contributes to their hyperphagia (24, 40). Because decreased serum leptin levels in RIIβKO mice and other strains have been a good indicator of adiposity (1, 2, 41), we measured serum leptin levels in females and found that they were ≈80% lower in Ay/RIIβKO compared with Ay mice (Fig. 3 F and Table 1). This decrease in leptin is consistent with the decreased adiposity of the Ay/RIIβKO mice.
RIIβHaploinsufficiency Can Attenuate the Obesity Syndrome of Ay Mice.
To test whether a heterozygous deficiency in RIIβwould be sufficient to partially rescue the Ay phenotype we first characterized the phenotype of the RIIβheterozygotes in more detail. RIIβheterozygous mice have a lean body weight phenotype that is intermediate between WT and KO. Female heterozygotes had a 25% reduction in WAT pad weight (Fig. 4 A) and a 60% reduction in leptin levels (WT, 4.5 ± 2 ng/ml, n = 4; RIIβheterozygotes, 1.8 ± 0.4 ng/ml, n = 6; RIIβKO, 1.1 ± 0.2 ng/ml, n = 6). There was no increase in nocturnal locomotor activity (Fig. 4 B). Male RIIβheterozygotes were 14% leaner than WT controls. Both male and female RIIβheterozygous mice also tended to be slightly hyperphagic, similar to RIIβKO mice (Fig. 4 C).
Partial rescue of Ay phenotypes on RIIβheterozygous background. (A) Female adiposity. (B) Female locomotor activity. (C) Food intake. (D) Ay/RIIβHET mice have decreased body weight. **, P < 0.001 (paired Student's t test). (E) Decreased white fat pad weight. (F) Decreased food intake. ⋀, P = 0.02. All data shown are for female mice.
The Ay mice were crossed onto an RIIβheterozygous background, and parameters associated with the obesity of the Ay mice were measured. Ay/RIIβheterozygote females had decreased body weight (Fig. 4 D) but did not show the complete rescue of body weight seen for the Ay/RIIβKO mice (Fig. 1 C). WAT pad weight (Fig. 4 E) and food intake (Fig. 4 F) were partially rescued on the RIIβheterozygous background compared with Ay (see Fig. 3 A and D for comparison with the KO rescue). Leptin levels were also decreased (data not shown), consistent with the change in adiposity. Activity levels of Ay/RIIβheterozygotes were increased compared with Ay but were still less than WT. Adiposity and body weight were also significantly decreased in male Ay/RIIβ+/- mice compared with Ay mice (data not shown). These data suggest that a 50% reduction in RIIβis enough to partially alleviate some of the obesity syndrome of Ay mice but does not elicit the hyperactivityphenotype seen with RIIβKO and Ay/RIIβKO mice.
RIIβKO Mice Respond Normally to Stimulation of Melanocortin Receptors with MTII.
Cell culture models have established that the melanocortin receptor agonist melanotan II (MTII) increases intracellular cAMP levels in cells transfected with either MC3R or MC4R. In vivo administration of MTII causes an acute decrease in food intake in animals that have been fasted and refed, and this response is thought to be mediated by activation of the MC4R on neurons in the PVH (22, 42). The rescue of hyperphagia in the Ay mice on an RIIβKO background could be due to (i) an increased sensitivity to MCR ligands if neurons have compensated for the loss of RIIβby forming more type I kinase, which activates at a lower threshold of cAMP (34, 43), or (ii) an increased constitutive PKA activity because a major regulatory subunit, RIIβ, is absent, and therefore a fraction of the C subunit is free in the cell. To distinguish between these possibilities, we tested for increased sensitivity to MTII in RIIβKO mice. Mice were fasted overnight and then refed after i.p. injection of either saline or MTII. At a dose of 10 mg/kg MTII, both WT and RIIβKO mice responded with a significant decrease in food intake during the first 4 h (Fig. 5). A low dose of 1 mg/kg MTII was tested to see whether the KO mice would respond to a dose that does not reduce feeding in WT mice, and the results showed that both WT and RIIβKO mice had a similar small response that was not significant (data not shown). These results indicate that the MCR pathway continues to function in the absence of RIIβ–PKA and that there is not a substantial change in sensitivity to agonist. We favor the second possibility, that the constitutive tone of PKA activity is elevated in MCR-expressing neurons and that this decreases feeding and increases energy expenditure. Indeed, RIIβKO mice are eating substantially less than would be expected for animals with a leptin level that is 1.1 ng/ml compared with the WT average of 4.5 ng/ml.
RIIβKO mice have normal feeding response to the MC3/4R agonist MTII. Female WT and RIIβKO mice responded similarly to an injection of 10 mg/kg MTII given i.p. in saline. Mice were fasted for 20 h before injection, and food was made available 15 min after injection.
Discussion
Although a great deal is known about the hormones, neuropeptides, and hypothalamic cell types that participate in body weight regulation, much less is understood about the interplay of intracellular signaling systems that underlie the regulation of feeding, energy expenditure, and fat storage. The MCR signaling system is a key regulator of body weight. Profound obesity develops in both mice and humans when MC4R is inactivated (11, 44), when the gene encoding the MCR ligandαMSH is deleted (45), or when the MC3/4R antagonist AgRP (23) or the MC4R antagonist agouti are overexpressed in the hypothalamus (19). Conversely, overexpression ofαMSH leads to leanness (46), and AgRP KO mice remain relatively lean as they age compared with WT mice (14). Downstream signaling pathways from the melanocortin receptors have been studied in cell culture and nonhypothalamic tissue, and the results support the prediction for a Gs-coupled receptor: stimulation of cAMP production, activation of PKA, and changes in phosphorylation and gene expression. However, other signaling pathways have also been shown to be activated, resulting in activation of the inositol phospholipid pathway and release of Ca2+ (26), and activation of MAPK (27, 30). In addition, cAMP can activate Rap-1 guanine nucleotide exchange factors (Epacs) and cyclic nucleotide-gated ion channels, and any of these non-PKA signaling pathways could play a role in melanocortin receptor signaling.
Our initial reports on the lean and hyperactive phenotypes of RIIβKO mice were not able to conclude which specific tissues were responsible for the phenotypes. A role for the induced expression of UCP1 in BAT was recently ruled out when we demonstrated that the lean and hyperactive phenotypes of RIIβKO mice were not rescued in a UCP-1 KO background (3) and that reexpression of RIIβin BAT does not reverse the phenotypes (M.A.S. and G.S.M., unpublished data). A cross between the RIIβKO and ob/ob mice demonstrated that the RIIβlean and hyperactive phenotype partially overcame the loss of leptin and suggested that the phenotypes of RIIβKO mice arise from specific changes in leptin-responsive neuronal pathways (4). The results reported here show that the RIIβKO background reverses the hyperphagia of Ay mice, stimulates locomotor activity to levels above WT, and reduces adiposity significantly in both males and females. We found that even the heterozygous RIIβbackground was sufficient to partially reduce hyperphagia and body weight of the Ay mice. The rescue of the Ay phenotype suggests that the regulation of feeding and adiposity by the melanocortin receptor signaling pathway depends on PKA in vivo and that the phenotypes of the RIIβKO mouse are likely due to increased PKA activity in the MC4R-expressing neurons of the hypothalamus that are the targets for agouti antagonism. However, it is also possible that the PKA mutation is affecting another pathway in the complex neuronal circuitry regulating energy homeostasis to effectively rescue the agouti phenotype. The neuronal basis of the RIIβbody weight phenotype was recently confirmed by preliminary results in our laboratory showing that reexpression of RIIβonly in neurons using a Cre recombinase-dependent approach reverses both the lean phenotype and the hyperlocomotor activity (M.A.S. and G.S.M., unpublished data).
Several transgenic and mutant mouse lines have been crossed to Ay mice to examine their ability to modify the Ay phenotypes. Overexpression ofαMSH decreased body weight in female Ay mice (46), whereas deletion of NPY had no effect in Ay mice (47). Crosses of Ay with attractin (Atrn)- and mahogunin (Mgrn1)-deficient mice have had more dramatic rescue effects, similar to the Ay/RIIβmice. Ay/Atrn and Ay/Mgrn1 double mutants have reduced body weight and adiposity and also exhibit increased locomotor activity compared with Ay mice (36, 37, 48), suggesting that attractin (a transmembrane protein that binds agouti) and mahogunin (an E3 ubiquitin ligase) act to block the action of agouti on MC4R-expressing neurons.
The rescue of hyperphagia and obesity in Ay mice by deletion of RIIβsuggests that in the absence of the regulatory subunit RIIβthere is more unregulated PKA catalytic subunit in MCR-expressing neurons in the hypothalamus (Fig. 6). We have previously reported a compensatory increase in the type I PKA holoenzyme in RIIβKO tissues, and the type I PKA was found to be more sensitive to cAMP activation than the type II holoenzyme (1, 5, 34, 35). This compensatory increase in type I PKA occurs robustly in BAT and WAT, but a weaker compensation also occurs in brain. The net effect of having unregulated C subunit as well as a more sensitive type I PKA would be an overall increase in the basal activity of PKA signaling in hypothalamic neurons that normally respond to the anorexigenic peptideα-MSH. We predict that this could be responsible for the lean phenotype and increased energy expenditure in RIIβKO mice. Ay mice ectopically express the MC4R antagonist agouti in hypothalamic nuclei, as shown in Fig. 6, and this is expected to prevent the MC4R-dependent stimulation of cAMP production and subsequent PKA activation. We propose that the RIIβmutation acts downstream of MC4R and provides a direct stimulation of PKA activity even in the presence of agouti, thereby causing a rescue of agouti-dependent phenotypes.
Model of the interactions of PKA–RIIβwith agouti. In the absence of the predominant neuronal regulatory subunit RIIβ, it is proposed that there is unregulated PKA activity and an increase in type I kinase in key hypothalamic neurons that are also expressing MC4R. The result is an overall increase in PKA signaling downstream of the melanocortin receptors resulting in decreased feeding and increased energy expenditure in Ay/RIIβKO mice. Immunohistochemistry detection of RIIβexpression in the hypothalamic areas of the PVH and surrounding structures is shown in green with DNA counterstained in blue (To-Pro-3).
Our experiments do not directly address the specific brain regions that are most important for the observed interaction between the RIIβKO and Ay. Although agouti is an antagonist for both the type 1 and type 4 MCR, the MC4R is most closely associated with the regulation of body weight, feeding, energy expenditure, and body length, which are the parameters affected by ectopic agouti expression in Ay mice. The MC4R KO mouse and to a lesser extent the heterozygotes display a phenotype similar to that seen for Ay. One area of high expression of MC4R is the paraventricular nucleus (PVH) of the hypothalamus. Lesions of the PVH lead to hyperphagia (49, 50), and stereotaxic injection of MC4R ligands in this region regulate both feeding and energy expenditure (51, 52). The RIIβprotein is clearly expressed in the PVH, as shown by immunohistochemistry in Fig. 6, and we have also recently shown that a Sim1-Cre recombinase transgenic (gift from Bradford Lowell, Harvard University, Boston) will activate RIIβexpression in the PVH specifically using the RIIβneolox animals described in Methods [see supporting information (SI) Fig. 7]. Ectopic agouti expression is likely to be affecting this neuronal pathway by antagonizing the response of the MC4R toαMSH, as depicted in Fig. 6, and this would lead to the Ay body weight phenotypes. In a recent report Balthasar et al. (53) developed an MC4R null mouse line in which the gene could be reactivated by Cre recombinase. Using Sim1-Cre to reactivate the MC4R in the PVH they showed that this completely reversed the hyperphagia and body length increase associated with the MC4R KO and partially rescued the obesity. Remarkably, the reexpression of MC4R in the PVH did not rescue the diminished energy expenditure of the MC4R KO, suggesting that POMC neuronal projections to other regions of the brain are involved in the energy expenditure phenotype. It is likely that the reversal of agouti-induced hyperphagia and body length increase seen in Ay mice by inactivation of RIIβis due to increased PKA function in the PVH. The elevation of locomotor activity we see in RIIβKO and Ay/RIIβKO mice may depend on other sites of RIIβexpression in the hypothalamus or elsewhere. Cell-type specific reexpression of RIIβwill be helpful to resolve this question in the future.
Methods
Generation of Ay/RIIβKO Mice.
Homozygous RIIβneolox mice contain a loxed neomycin resistance cassette in the 5′untranslated region of Prkar2b, are phenotypically identical to RIIβnull mice (1, 5), show no expression of RIIβ, and were used in the above crosses to Ay mice (M.A.S., unpublished data). Homozygous RIIβneolox mice are referred to as RIIβKO mice in this study. Ay mice on a C57BL/6J background (strain B6.Cg-Ay/J) were purchased from The Jackson Laboratory. Mice for all experiments were littermates generated from mating Ay/RIIβHET and RIIβHET mice (Fig. 1 A). Mice were maintained on a rodent chow diet (Picolab 20, 4.5% fat). The Ay allele was identified by coat color at the time of weaning (48). All procedures were approved by the Institutional Care and Use Committee of the School of Medicine of the University of Washington.
Body Weight and Adiposity.
Mice were killed at 20–23 wk of age, and adiposity was determined by weighing the three major WAT pads (inguinal, reproductive, and retroperitoneal). In addition, we weighed interscapular BAT located on the dorsal side of the mouse near the neck region. Body length was measured as the distance from the nose to the anus.
Locomotor Activity and Food Intake.
Locomotor activity was measured at 16 wk in a plastic activity chamber (47×25×21 cm) equipped with photobeams. Daily food intake was measured in home cages every 24 h for 7 consecutive days and averaged.
Leptin Measurements.
Mice were killed with CO2 gas. Whole blood was collected between 1200 and 1500 hours by cardiac puncture. The serum was collected and assayed for leptin concentration by ELISA.
MTII Treatment.
Mice were individually housed for 2 wk before injection. Mice were fasted for 20 h and then given an i.p. injection of either saline or MTII (Bachem) at a dose of 1–10 mg/kg of body weight. Food was made available 15 min after injection, and food intake was monitored at 2, 4, 8, and 24 h after refeeding.
Data Analysis and Statistics.
Values shown in all figures and in the table are the means ± SEM for each genotype. Statistical analyses were conducted with Prism 4 (GraphPad). Comparisons among the four genotypes (WT, Ay, RIIβKO, and Ay/RIIβKO) were done with one-way ANOVA and Newman–Keuls multiple-comparison test for post hoc analysis unless noted otherwise. Growth curves were analyzed with a one-way ANOVA with repeated measures and Newman–Keuls multiple-comparison test, or a two-tailed, paired Student t test when Ay and Ay/RIIβHET mice were compared. Comparisons of Ay and Ay/RIIβHET mice were analyzed with a two-tailed, unpaired Student t test.
Acknowledgments
We thank the Clinical Nutrition Research Unit Laboratory Core at the University of Washington for leptin assays, Michelle Matias for help with fat pad analysis, and Sara Shum for assistance with mouse breeding. This work was supported by National Institutes of Health Grants 5 T32 NS 07332 (to T.A.C.) and GM32875 (to G.S.M.).
Footnotes
- *To whom correspondence should be addressed at: Department of Pharmacology, University of Washington School of Medicine, Box 357750, 1959 Northeast Pacific Street, Seattle, WA 98195 . E-mail: mcknight{at}u.washington.edu
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Author contributions: T.A.C., M.A.S., and G.S.M. designed research; T.A.C., M.A.S., and L.Y. performed research; M.A.S. contributed new reagents/analytic tools; T.A.C., M.A.S., and L.Y. analyzed data; and T.A.C. and G.S.M. wrote the paper.
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The authors declare no conflict of interest.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0710607105/DC1.
- © 2008 by The National Academy of Sciences of the USA
