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(The FASEB Journal. 2005;19:1482-1491.)
© 2005 FASEB

Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin

Yubin Zhang*, Gail E Kilroy*, Tara M. Henagan*, Vera Prpic-Uhing*, William G. Richards{dagger}, Anthony W. Bannon{dagger}, Randall L. Mynatt* and Thomas W. Gettys*,1

* Division of Experimental Obesity, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA; and
{dagger} Amgen Inc., Thousand Oaks, California, USA

1Correspondence: 6400 Perkins Rd., Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA. E-mail: gettystw{at}pbrc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse lines with targeted disruption of the cocaine amphetamine-related transcript (CART), melanocortin receptor 3 (MCR3), or melanocortin receptor 4 (MCR4) were used to assess the role of each component in mediating the anorectic and metabolic effects of leptin, and in regulating the partitioning of nutrient energy between fat and protein deposition. Leptin was administered over a 3 day period using either intraperitoneal or intracerebroventricular routes of injection. The absence of MCR4 blocked leptin’s ability to increase UCP1 mRNA in both brown and white adipose tissue, but not its ability to reduce food consumption. In contrast, deletion of MCR3 compromised leptin’s ability to reduce food consumption, but not its ability to reduce fat deposition or increase UCP1 expression in adipose tissue. Leptin-dependent effects on food consumption and adipocyte gene expression were unaffected by the absence of CART. Repeated measures of body composition over time indicate that the absence of either MCR3 or MCR4, but not CART, increased lipid deposition and produced comparable degrees of adiposity in both lines. Moreover, modest increases in fat content of the diet (4 to 11%) accentuated fat deposition and produced a rapid and comparable 10–12% increase in % body fat in both genotypes. The results indicate that nutrient partitioning, as well as the anorectic and metabolic responses to leptin, are dependent on integrated but separable inputs from the melanocortin 3 and 4 receptor subtypes.— Zhang, Y., Kilroy, G. E., Henagan, T. M., Prpic-Uhing, V. Richards, W. G., Bannon, A. W., Mynatt, R. L., Gettys, T. W. Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin.


Key Words: leptin • melanocortin receptors • uncoupling proteins • adipose tissue


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CONSTITUTIVE ECTOPIC EXPRESSION of Agouti protein in lethal yellow (Ay) mice disturbs the balance between energy intake and expenditure, and produces a profound increase in body weight (1 , 2) . The subsequent discovery of agouti related protein (AGRP) and recognition of its functional overlap with agouti (3) suggested that antagonism of melanocortin signaling played an important role in energy balance (1 , 2) . Support for the concept that leptin used melanocortin as an effector system to regulate energy balance was provided by observations that leptin regulated AGRP expression (4) and transgenic overexpression of AGRP led to obesity in mice (5) . Our understanding of the neural circuits that coordinate energy balance and responses to leptin was further advanced by identification of leptin-responsive neurons in the hypothalamus (6 7 8 9 10 11 12) , where two populations of neurons within the arcuate nucleus mediate leptin’s effects on food intake and SNS activation (4 , 11 12 13 14) . Leptin increases CART and {alpha}-MSH, a post-translational anorexigenic product of POMC (12 , 15 16 17 18) , and simultaneously decreases the orexigenic peptides, NPY and AGRP (4 , 6 , 12) . Thus, through reciprocal regulation and integration of these orexigenic and anorexigenic signals, leptin produces coordinated changes in food intake and energy metabolism.

NPY acts through distinct receptors to regulate food intake and SNS activity (6 , 10) , while {alpha}-MSH and AGRP compete for common receptors on target neurons within each system (3 , 19 20 21) . Currently, it is not known how CART peptides exert their effects. {alpha}-MSH is an agonist for both the melanocortin-4 (MCR4) and melanocortin-3 (MCR3) receptors (22) , while AGRP functions as an antagonist at both receptor subtypes and can increase food intake by virtue of its ability to antagonize the anorectic effects of {alpha}-MSH (3 , 20) . Short loop feedback systems between NPY/AGRP and POMC nuclei involving MCR3 and gamma amino butyric acid (GABA) have also been proposed (23) . Collectively, these studies illustrate that melanocortin signaling serves the dual purpose of integrating cross talk between leptin-responsive nuclei and translating leptin-dependent signals to downstream effector systems that regulate energy balance.

A combination of pharmacological and genetic approaches has been used to assess the respective roles of CART peptides and melanocortin receptor subtypes in mediating biological responses to leptin. Responses to central administration of melanocortin receptor agonists (24 25 26 27 28) and/or antagonists (26 , 29 30 31) support a primary role for MCR4, but not MCR3, in both the anorectic and metabolic effects of leptin. The phenotype of MCR4 null mice is consistent with this prediction in that MCR4 null mice are hyperphagic (32 33 34) , hypometabolic (35) , and obese. However, the hyperphagia that develops in MCR4 null mice 6–8 wk after weaning may be secondary to development of resistance to peripheral leptin (34 , 35) , given the effective anorectic response to centrally administered leptin in these mice (34) . Responsiveness to exogenous leptin has not been reported in CART- or MCR3 null mice, but both lines show evidence of modest disturbances in energy balance (36 37 38) . Using CART-, MCR3-, and MCR4 null mouse lines on an outbred, obesity-resistant background, we show that the MCR4 is not required for leptin to reduce food intake but is required for leptin to affect changes in gene expression in adipose tissue. The absence of MCR3 compromised leptin’s ability to reduce food intake but not its ability to affect changes in adipocyte gene expression or fat deposition. However, the absence of either MCR3 or MCR4 produced significant and comparable increases in adiposity that were further exacerbated by a modest increase in dietary fat content.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ethylenediamine tetraacetic acid (EDTA), sodium cholate, Triton X-100, bovine serum albumin (BSA), guanidinium thiocyanate, TES, sucrose, and other common chemicals were from Sigma Chemicals (St. Louis, MO, USA). Trizol LS Reagent was from Life Technologies (Gaithersburg, MD, USA). Taq DNA polymerase, MMLV reverse transcriptase were from Promega (Madison, WI, USA). Hot start Taq polymerase (Ex-Taq) used for real time PCR was from Takara Bio Inc. (Shiga, Japan). 2-Mercaptoethanol was acquired from J. T. Baker, Inc. (Phillipsburg, NJ, USA).

Experimental Animals
Breeding pairs of mice with targeted deletion of the cocaine amphetamine-related transcript (CART–/–), melanocortin receptor 3 (MCR3–/–), or melanocortin receptor 4 (MCR4–/–) were obtained from Charles River Labs (Wilmington, MA, USA) and the respective genotypes confirmed by PCR. The mouse lines were originally developed on an outbred Black Swiss/129 background (BSw;129) by Amgen (Thousand Oaks, CA, USA) and archived with Charles River for maintenance. Breeding pairs of wild-type BSw;129 mice (+/+) were also obtained for use as controls. Breeding colonies of each line were established and used to produce the mice of each genotype used in the present studies. All studies were reviewed and approved by the Pennington Biomedical Research Center IACUC committee.

Experiment 1
Twelve to 14 male mice of all four genotypes were obtained at weaning and studied at ~5.5 wk of age after a 2 wk adaptation period. The mice were weaned onto Purina Rodent Diet (#5001) and housed in a controlled environment at 22°C on a 12 h light/dark cycle with free access to food and water. Mice of each genotype were randomly assigned to receive intraperitoneal injections of vehicle (PBS) or leptin (20 µg/g bw/d) using the 3 day protocol we described previously (39 , 40) . In brief, 100 µL injections were given 2 h after the start of the light cycle each day and all mice were killed 1 h after the last injection on day 3. Animals were weighed before injection on day 1 and food consumption was measured each day during the 3 day protocol. Mice were weighed at sacrifice on day 3, and the interscapular brown adipose tissue (BAT), epididymal white adipose tissue (WAT), inguinal WAT, and retroperitoneal WAT depots were carefully dissected and weighed. Total RNA was isolated from BAT and inguinal WAT for assessment of UCP1 mRNA induction.

Experiment 2
Twelve to fourteen male mice of each genotype were obtained at weaning and studied two wk later at ~5.5 wk of age. Mice of each genotype were randomly assigned to receive intracerebroventricular (ICV) injections of vehicle (artificial cerebrospinal fluid, aCSF) or leptin (2 µg/mouse/d) using the 3 day protocol we described previously (39 , 40) . In brief, on the day before starting the experiment, mice were anesthetized by inhalation of isoflurane and a guarded, blank 27-gauge 0.5 inch needle was used to create a guide injection site 0.7 mm posterior to bregma and 1.0 mm lateral to midline at a depth of 4.0 mm (41) . In the experiments proper, a 10.0 µL Hamilton 1700 series gastight syringe (Hamilton, Reno, NV) and the guide injection site were used to give daily ICV injections of either artificial cerebrospinal fluid (aCSF) or murine leptin in a volume of 2–5 µL. Thereafter, mice were monitored to insure full recovery. Injections were given 2 h after the start of the light cycle and mice were killed 1 h after the last injection on day 3. Animals were weighed before injection on day 1 and food consumption was measured each day during the 3 day protocol. Mice were weighed at sacrifice on day 3, and the BAT, epididymal WAT, inguinal WAT, and retroperitoneal WAT depots were carefully dissected and weighed. Total RNA was isolated from BAT and inguinal WAT for assessment of UCP1 mRNA induction.

Experiment 3
Ten male MCR4–/– mice received daily IP injections of vehicle (PBS) or the selective ß3-adrenoceptor agonist, CL 316,243 (1 µg/d/g bw) for 3 days to mimic sympathetic stimulation of adipose tissue. The mice were killed 2 h after the last injection on day 3 and tissues were harvested and processed as before for measurement of UCP1 mRNA.

Experiment 4
Male MCR3–/– null (n=10), MCR4–/– null (n=9), and wild-type (n=5) mice were weaned onto Purina Rodent Diet (# 5001, 4% fat) at 3 wk of age and housed in a controlled environment at 22°C on a 12 h light/dark cycle with free access to food and water. At 10 wk of age, all mice were switched to the Purina Mouse Diet (#5015, 11% fat), and maintained with ad lib access to this diet through 14 wk of age. Body weights were obtained for each animal at 3 wk of age and weekly thereafter. At 6 wk of age, fat mass (FM), lean mass and fluid mass were determined in triplicate for each animal by nuclear magnetic resonance (NMR) using a Bruker Mice Minispec NMR analyzer (Bruker Optics, Inc., Billerica, MA). Lean mass was added to liquid mass to produce the variable fat free mass (FFM) that was used for analysis. Repeated measures of body composition were obtained for each animal at 6, 8, 10, 12, and 14 wk of age.

Preparation of RNA
In Exps 1–3, interscapular BAT and inguinal WAT pads were carefully dissected and homogenized with Trizol LS Reagent using an Ultraturax Tissuemizer (Tekmar, Cincinnati, OH, USA) according to manufacturer’s specifications. Total RNA was isolated and purified as described (42) .

Real-time PCR
Real time PCR was used to measure UCP1 mRNA expression in RNA samples from BAT and inguinal WAT as described (40) . In brief, conventional PCR was used to amplify UCP1 cDNA from a BAT cDNA library. The UCP1 cDNA fragment was purified, sequenced, quantitated, and used to prepare standards by serial 10-fold dilutions of the purified cDNA. The slope of the resulting standard curve relating log mass of the UCP1 cDNA standards to cycle threshold was –3.2 and dilutions of unknown cDNA samples produced changes in cycle threshold which paralleled the standard curve. In the assay proper, duplicate dilutions of each standard and unknown sample were amplified and the mass of mRNA in unknown samples was estimated from the standard curve by inverse calibration (40 , 43) . Cyclophilin standard curves were used to determine the mass of this housekeeping gene in each sample and after correcting RNA to reflect cyclophilin differences, UCP1 mRNA levels were expressed as fmol/µg total RNA.

Methods of analysis
The estimated concentration of UCP1 mRNA in each sample was obtained by inverse calibration from standard curves as described in Materials and Methods. Group means for UCP1 mRNA, total food consumption, total weight of WAT depots, and body weight changes were analyzed using a two-way ANOVA with mouse line and leptin treatment as main effects. The same design was used to analyze measures of body composition (fat mass, fat free mass, fat mass/unit body weight, fat free mass/unit body weight) at each time point. The line x treatment interaction was tested using residual variance (animal within line x treatment) as the error term, and post hoc testing of group means within each experiment and genotype was made using the Bonferroni correction using the pooled error term to calculate standard errors. Protection against type I errors was set at 5% ({alpha}=0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leptin-induced effects on food consumption, adipose tissue mass, and body weight
Cumulative food consumption over the 3 day period was similar among vehicle-treated WT, MCR3–/–, and CART–/– mice, and all were ~30% lower than control MCR4–/– mice during the same period (Table 1 ). Both routes of leptin administration produced significant 20–30% reductions in food intake among WT, MCR4–/–, and CART–/– mice, while neither route of administration reduced cumulative food consumption in MCR3–/– mice (Table 1) . To further explore this outcome, average daily food consumption was examined to see if MCR3–/– mice were uniformly unresponsive to leptin on each of the three days of the experiment. Regardless of route of injection, leptin did not decrease food intake on the first two days of either experiment in this genotype. In fact, a slight increase in food consumption was produced by IP leptin on day 1 followed by a modest nonsignificant reduction on day 2 and a significant reduction (P<0.05) on day 3 (IP vehicle, 4.14±0.33 g; IP leptin, 3.35±0.24 g). When leptin was given ICV, it produced a significant increase (P<0.05) in food consumption on day 1 (ICV vehicle, 3.44±0.27 g; ICV leptin, 4.28±0.36 g; n=6), had no effect on day 2, and produced a significant decrease (P<0.05) on day 3. This biphasic response to leptin in MCR3–/–mice was unique among the genotypes and illustrated why cumulative food intake over the 3 day period was unaffected by leptin.


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  		Table 1. The effect of leptin and route of administration on body weight, adipose tissue and food intake in wild-type, MCR4–/–, MCR3–/–, and CART–/– micea,b

The combined mass of epididymal, retroperitoneal, and inguinal WAT depots was used as a surrogate measure of carcass fat and used to evaluate leptin effects on fat deposition among the groups. At this age (6 wk), fat mass was similar among vehicle-treated WT, MCR3–/–, and CART–/– mice, and in all cases nearly twofold lower than fat mass in MCR4–/– mice (Table 1) . Both routes of leptin injection reduced fat deposition in WT and MCR3–/– mice, although leptin appeared more effective in WT than MCR3–/– (Table 1) . The small reductions in fat mass of CART–/– mice after leptin treatment did not reach significance. Despite an effective anorectic response, neither IP nor ICV leptin reduced fat mass in MCR4–/– mice (Table 1) .

Body weights of WT and MCR3–/– mice were indistinguishable (~21 g) on day 1 of the study, and lower (P<0.05) than CART–/– (~23 g) and MCR4–/– (~26 g) mice of the same age (Table 1) . Analysis of differences between ending and starting body weights showed that leptin, regardless of route of administration, reduced body weight in WT mice (Table 1) . Leptin also reduced body weight in MCR3–/– and CART–/– mice, although surprisingly, the reduction produced by ICV leptin did not reach statistical significance in MCR3–/– mice (Table 1) . In contrast, body weights, like fat mass, were unaffected by either route of leptin administration in MCR4–/– mice (Table 1) . Collectively, these results show that all four genotypes were responsive to leptin, although the nature of the response differed among the groups.

Induction of UCP1 mRNA in brown and white adipose tissue
Based on our previous demonstration that norepinephrine is required for leptin-dependent effects on adipocyte gene expression (14) , the induction of UCP1 mRNA in BAT and inguinal WAT was used as a surrogate measure of leptin-dependent SNS activation to examine the role of MCR3, MCR4, and CART in this response. Both routes of leptin administration produced significant (P<0.05) increases in UCP1 mRNA in BAT from WT, MCR3–/–, and CART–/– mice, with no apparent difference in efficacy among the genotypes (Fig. 1 , Fig. 2 ). In contrast, regardless of route of administration, leptin had no effect on UCP1 mRNA expression in MCR4–/– mice. Despite this impaired response in MCR4 mice, basal UCP1 mRNA levels did not differ among the four groups (Figs. 1 , 2) .



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  		Figure 1. Leptin-dependent induction of UCP1 mRNA in BAT from wild-type, MCR3–/–, MCR4–/–, and CART–/– mice given IP injections of vehicle or leptin (20 µg/g bw/d) for 3 days as described in Materials and Methods.



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  		Figure 2. Leptin-dependent induction of UCP1 mRNA in BAT from wild-type, MCR3–/–, MCR4–/–, and CART–/– mice given ICV injections of vehicle or leptin (2 µg/mouse/d) for 3 days as described in Materials and Methods.

UCP1 mRNA levels were 50-fold lower in inguinal WAT than BAT, and in contrast to BAT, were not increased by IP leptin in any of the four genotypes. Examination of values contributing to each mean indicated a broad range of UCP1 mRNA concentrations in WT and MCR3–/– mice receiving IP leptin, with few intermediate values. The range of UCP1 mRNA levels in inguinal WAT from MCR4–/– and CART–/– was even more pronounced, but intermediate values were evident in these two groups. Although there is a suggestion of subgroups of responsive and nonresponsive mice in the WT and MCR3–/– mice, the experiment was insufficiently powered to test this possibility.

A different outcome was observed when leptin was administered ICV in that it significantly (P<0.05) increased UCP1 mRNA in inguinal WAT from WT, MCR3–/–, and CART–/– mice (Fig. 3 ). However, ICV leptin was unable to increase UCP1 mRNA in inguinal WAT from MCR4–/– mice (Fig. 3) . Notwithstanding the lack of response to IP leptin in inguinal WAT from all genotypes, the pattern of responses to ICV leptin in this tissue mirrors the pattern observed in BAT, indicating that the MCR4 is required for leptin to increase UCP1 mRNA expression in both BAT and WAT.



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  		Figure 3. Leptin-dependent induction of UCP1 mRNA in inguinal WAT from wild-type, MCR3–/–, MCR4–/–, and CART–/– mice given ICV injections of vehicle or leptin (2 µg/mouse/d) for 3 days as described in Materials and Methods.

Increased fat deposition in several mouse models of obesity is associated with development of resistance to ß-adrenergic modulation of gene expression in adipose tissue (40 , 44 45 46 47) . To rule out this possibility for the failure of leptin to affect changes in UCP1 mRNA in MCR4–/– mice, MCR4–/– mice were injected with the selective ß3-adrenoceptor agonist, CL316,243 for 3 days to selectively target adipose tissue and mimic sympathetic stimulation. CL316,243 produced a robust increase in UCP1 mRNA in both BAT (vehicle, 3.06±1.44 fmol UCP1 mRNA/µg RNA; CL-316,243, 14.45±4.28 fmol UCP1 mRNA/µg RNA) and inguinal WAT (vehicle, 0.05±0.01 fmol UCP1 mRNA/µg RNA; CL-316,243, 0.33±0.18 fmol UCP1 mRNA/µg RNA), indicating that adipose tissue from MCR4–/– is fully responsive to ß3-adrenoceptor stimulation. These results support the conclusion that the failure of leptin to increase UCP1 mRNA in BAT and WAT of MCR4–/– mice is due to a failure of leptin to increase sympathetic outflow rather than an inability of adipose tissue of these mice to respond to sympathetic stimulation. Collectively, the results support an essential role for the MCR4 in mediating leptin’s effects on adipocyte gene expression but not food intake. The results also support an essential role for the MCR3 in modulating leptin’s anorectic effects, but not its effects on adipocyte gene expression.

Nutrient partitioning in MCR3–/– and MCR4–/– null mice on BSw;129 genetic background
Based on our findings of altered leptin signaling in MCR3–/– and MCR4–/– mice, growth and nutrient partitioning were examined in MCR3–/– and MCR4–/– mice. Body weights of WT and MCR3–/– mice were indistinguishable at 3 wk of age and weaning them onto a low fat (4%) rodent chow produced growth curves that were nearly superimposable through 10 wk of age (Fig. 4 A). MCR4–/– mice were already 20–25% heavier than WT and MCR3–/– mice at weaning (P<0.05) and this difference was maintained through 10 wk of age (P<0.05). Body composition was not determined at the 3 wk time point, but by 6 wk of age, fat mass (FM) was 40% higher in MCR3–/– compared with WT mice (P<0.05), whereas fat free mass (FFM) did not differ between these groups (Fig. 4B, C ). At this time point, FM in MCR4–/– mice was 34% (P<0.05) and 87% (P<0.05) higher than MCR3–/– and WT mice, respectively (Fig. 4B ). FFM in MCR4–/– mice was also 15–20% greater (P<00.05) than the other two groups (Fig. 4C ), indicating that the difference in body weight between MCR4–/– and the other groups was not solely due to larger fat stores. Group differences in carcass composition were further magnified by 10 wk of age such that FM was 69% higher in MCR3–/– than WT mice (P<0.05) while the difference between MCR4–/– and MCR3–/– had grown to 54% (P<0.05, Fig. 4B ). At the 10 wk time point, FM in MCR4–/– mice was 2.6-fold higher than WT mice (P<0.05, Fig. 4B ). In contrast, the relative rankings of FFM among the groups changed very little between 6 and 10 wk, although the difference between WT and MCR3–/– did reach significance at 10 wk (P<0.05, Fig. 4C ).



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  		Figure 4. Change in body weight (A), fat mass (B), and fat free mass (C) over time in wild-type, MCR3–/–, and MCR4–/– null mice weaned onto low fat (4%) rodent chow at 3 wk of age and switched to higher fat (11%) breeder chow at 10 wk of age. Body weights were determined at weekly intervals and body composition was determined using a small animal Bruker Minispec NMR at 6, 8, 10, 12, and 14 wk of age.

Fat content of the diet was increased from 4% to 11% at 10 wk by shifting the mice from rodent chow to a breeder diet. The increased caloric density resulted in an immediate change in trajectory of the growth curves for MCR3–/– and MCR4–/– mice, but was without effect in WT mice (Fig. 4A ). After 4 wk on the higher fat diet, MCR3–/– mice were 20% heavier than the WT mice (P<0.05, Fig. 4A ). The higher fat diet also magnified the body size difference between MCR4–/– and WT mice (P<0.05), but the change in diet had only a modest effect on the relative difference between body weight of MCR4–/– and MCR3–/– mice between 10 (MCR4–/– 29% heavier) and 14 wk (MCR4–/– 38% heavier, see Fig. 4A ). Fat deposition was an important component of the increased body weight in MCR3–/– and MCR4–/– mice during this period (Fig. 4B ), but in contrast to MCR3–/– mice, MCR4–/– continued to increase FFM between 10–14 wk. Thus, the increase in body weight of MCR3–/– mice during this period was almost entirely due to an increase in FM whereas the growth of MCR4–/– mice included both FM and FFM (Fig. 4B, C ). The increased caloric density at 10 wk had little effect on the growth trajectory of WT mice (Fig. 4A ) or the composition of the gained weight during this period (Fig. 4B, C ).

To examine relative body composition and group differences in nutrient partitioning over time, FM and FFM were expressed as a function of body weight (Fig. 5 A, B). These variables provide body weight adjusted measures of fatness and leanness, and show that WT mice maintained essentially constant body composition from 3 to 10 wk of age while increasing body weight from 12 to 28 g. Percentage body fat varied between 12–13% while lean body mass varied between 74 to 75% during this period (Fig. 5A, B ). The increase in caloric density of the diet at 10 wk resulted in a small increase in body fat (13 to15%) and a commensurate decrease in lean body mass (75 to72%) among WT mice. Percent body fat was similar between MCR3–/– (16±2%) and MCR4–/– (18±2%) mice at 6 wk of age and significantly greater than WT mice (P<0.05). And in contrast to WT mice, percentage body fat increased to comparable extents in MCR3–/– (16±2% to 21±3%) and MCR4–/– mice (18±2% to 25±4%) between 6 and 10 wk (Fig. 5A ). The switch to higher fat diets at 10 wk produced a significant increase in fat deposition such that at 12 and 14 wk of age, percentage body fat in both MCR3–/– and MCR4–/– was near 35% (Fig. 5A ). Thus despite large differences in body weight at 12 and 14 wk (Fig. 5A ), the degree of adiposity was comparable between MCR3–/– and MCR4–/– mice at these times (Fig. 5A ). The increase in % FM was paralleled by a comparable decrease in % FFM between these two groups (Fig. 5B ). The preservation of longitudinal growth in MCR4–/– mice, but not MCR3–/– mice, resulted in significant differences in body size. Despite this difference in body size, the absence of either receptor subtype produced comparable degrees of adiposity. In addition, these data show that nutrient partitioning toward fat deposition is comparably enhanced by increased caloric density in MCR3–/– or MCR4–/– mice.



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  		Figure 5. The change in ratio of fat mass to body weight (A) or fat free mass to body weight (B) over time in wild-type, MCR3–/–, and MCR4–/– null mice weaned onto low fat (4%) rodent chow at 3 wk of age and switched to higher fat (11%) breeder chow at 10 wk of age. Repeated measures of body composition were determined in each animal using a small animal NMR, and group means for each ratio were calculated from the respective ratios of individual mice in each group at each time point.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings from the present study are that targeted deletion of MCR3 or MCR4, but not CART, results in 1) receptor-specific alterations in the behavioral and metabolic responses to leptin, 2) disordered nutrient partitioning that produces comparable increases in % fat mass and decreases in % fat free mass at substantially different growth rates, and 3) disordered nutrient partitioning that is further exacerbated by modest increases in dietary fat content. In the present studies, mouse lines with targeted deletion of MCR3, MCR4, and CART were produced on an outbred Black Swiss;129 background. The obesity-resistant, leptin responsive phenotype of Bsw;129 mice provided a sensitive readout of how the absence of MCR3, MCR4, or CART affected leptin signaling and nutrient partitioning. Therefore, the metabolic phenotype of the background strain was particularly well suited to assessing how the absence of melanocortin receptors affected energy balance and nutrient partitioning. The original studies with targeted deletion of MCR3 (36 , 37) , MCR4 (32) , or CART (38) used a leptin-resistant, obesity-prone, mixed background for production of each line. In each case, the C57BL/6J;129 chimeras expressing the disrupted allele were backcrossed with C57BL/6J to produce mice that were homozygous for the targeted allele. Although not congenic on the C57BL/6J background, it seems likely that the obesigenic characteristics of the background strain created a higher threshold for detecting changes in energy balance associated with each gene deletion. For instance, on the mixed C57BL/6J background, the phenotype of MCR3–/– mice (36 , 37) was subtle with respect to body weight and obesity, whereas in the current study, MCR3–/– mice were 20% heavier with 2.4-fold greater adiposity than wild-type mice at comparable ages. The mixed C57BL/6J background also muted differences between control and MCR4–/– mice at early ages (32 , 48) , although the development of postweaning hyperphagia produced a rapid divergence from controls, regardless of the background strain (32 , 34 , 35 , 48) . In the present studies, MCR4–/– mice were clearly heavier and fatter than wild-type mice at weaning, with the difference in adiposity increasing to 4-fold in the 10 wk after weaning. Therefore, with deletion of MCR3 or MCR4 receptors, alterations in energy balance and nutrient partitioning were readily detected when the alleles were targeted on the Bsw;129 background.

A second particularly interesting observation regarding the phenotypes of MCR3–/– and MCR4–/– mice used here was that nutrient partitioning was similarly disordered, despite significant differences in food intake and body weight between these strains. This conclusion became evident when fat mass and fat free mass were expressed per unit of body weight, providing repeated measures of relative adiposity and leanness over time. When examined in this way, it became evident that wild-type mice maintained a stable body composition from weaning to 14 wk, despite a nearly threefold increase in body weight. During the same period, a comparable increase in body weight of MCR3–/– mice was accompanied by a doubling of % body fat from 16 to 33% and a decrease in % lean mass from 70 to 57% (see Fig. 5A, B ). However, despite the fact that MCR4–/– mice were 25 to 38% heavier than MCR3–/– mice at weaning and 14 wk, respectively, the changes in % body fat (18 to 35%) and % lean mass (70 to 54%) were indistinguishable from MCR3–/– mice. Therefore, it seems likely that the higher food intake of MCR4–/– mice supported the continuation of longitudinal growth that is reflected in their higher body weights. However, the larger and heavier MCR4–/– mice are no more obese than 25% smaller MCR3–/– mice that typically consume ~30% less energy. Collectively, these data support the conclusion that the absence of MCR3 or MCR4 produce a similar disordering of nutrient partitioning that occurs at significantly different rates of energy intake. This conclusion is supported by previous studies showing that MCR4–/– mice retain an increased propensity to deposit fat even when they are pair-fed to wild-type mice (35) .

The phenotypes of mice with targeted deletions of MCR3, MCR4, or CART indicate that energy balance is disturbed in each model (32 , 35 36 37 38) , and infer that maintenance of energy balance requires the collective integration of input from each system. In the present experiments we have used the absence of these key components to examine how the respective pathways affect leptin’s ability to simultaneously regulate energy intake and energy utilization. Identifying the signaling networks used to coordinate control of the respective components of energy balance has been the focus of many studies (4 , 6 7 8 9 10 11 12 13) . Although melanocortin receptors are the conduits that relay signals conveyed by AGRP and POMC-derived peptides, the role of each receptor subtype in translating the simultaneous effects of leptin on ingestive behavior and energy metabolism has not been clearly established. The role of CART peptides in the process also is not clear. Thus, a primary goal of the current study was to evaluate how the absence of MCR3, MCR4, and CART affect representative components of the leptin response, and determine whether the signals conveyed through each system can serve interchangeable or redundant roles.

Our findings support the conclusion that leptin uses MCR3 and MCR4 to provide unique and separable inputs into the maintenance of energy balance. In the case of food intake, we observed that leptin’s ability to reduce ingestive behavior was not compromised by the absence of MCR4, but was by the absence of MCR3. Marsh (34) used a similar experimental protocol with MCR4–/– mice on a C57BL/6J;129 background and showed that the absence of MCR4 compromised the ability of peripherally, but not centrally injected leptin, to reduce food intake. Anorectic responses of MCR4–/– mice in the present study were comparable using both routes of leptin injection, so this subtle difference from Marsh et al. (34) is likely due to the characteristic development of resistance to peripheral leptin in C57BL/6J mice (40 , 46 , 49 50 51) . Thus, both the present and earlier (34) studies support the conclusion that leptin does not require the MCR4 to reduce food intake, and illustrate how characteristics of the background strain can confound inferences of receptor function based on phenotype. For instance, given the present findings it is tempting to speculate that the hyperphagia and obesity that develop in MCR4–/– mice on a C57BL/6J background (32 , 34 , 35) are exacerbated by the development of leptin resistance. Support for this idea was provided by Ste. Marie et al. (35) , who showed that pair-feeding MCR4–/– mice to WT mice prevented the rapid postweaning increase in weight seen in non-pair-fed MCR4–/– mice. However, fat deposition in pair-fed MCR4–/– mice was still higher than wild-type controls (35) , demonstrating that increased fat deposition in MCR4–/– mice is enhanced by but not dependent on their hyperphagia. Given that the mice in the present study were responsive to peripheral leptin but still hyperphagic, it seems more likely that mechanisms independent of leptin resistance are increasing food consumption.

Studies in rats using pharmacological approaches show that simultaneous activation or inhibition of MCR3 and MCR4 reciprocally regulate food intake (19 , 24 , 26 , 29 , 31) . Additional studies (30 , 52) support an essential role for the MCR4 in leptin’s anorectic effects. It is unclear why results from pharmacological vs. genetic approaches differ regarding the role of MCR4 in leptin signaling, but several possibilities merit consideration. First, the pharmacological studies were conducted in rats while the current and previous genetic studies (34 , 35) were all in mice. It also seems likely that the nature of pharmacological approaches (i.e., selectivity of available ligands, sites of actual drug delivery, biological half-life), and the acute nature of the measured responses could be contributing to different outcomes. In the present studies, the receptor subtypes were never present to provide signaling input before or during the course of the 3 day leptin injection protocol. Thus, the more chronic nature of the measured responses and the total absence of input from deleted receptors strongly supports the conclusion that the MCR4 is not required for leptin to reduce food intake in mice. A similar conclusion is supported from studies with CART–/– mice, where deletion of this gene did not alter leptin’s ability to reduce food intake.

Our finding that the absence of MCR3 compromised leptin’s ability to reduce food consumption was somewhat of a surprise based on the lack of hyperphagia observed in MCR3–/– mice (36 , 37) , and the reported failure of ICV injection of MCR3 agonists to reduce food intake (28) . Collectively, these studies provide little indication that the absence of MCR3 would compromise leptin’s anorectic effects. However, recent studies suggest that the MCR3 may function as an autoreceptor that regulates crosstalk between NPY and POMC neurons (12 , 23) . GABA-regulated inhibitory postsynaptic currents (IPSC) are increased by MCR3 agonists and play key roles in regulating the resting potentials of arcuate neurons, and therefore their ability to be hyperpolarized (NPY/AGRP) or depolarized (POMC) by leptin (12 , 23) . The MCR3 may play an important role in setting leptin response thresholds in these neurons. Thus, the chronic absence of this integrative loop in MCR3–/– mice may disturb the coordinated functioning of NPY and POMC neurons, and explain why leptin actually increased food consumption on day 1 in these mice, followed by a delayed decrease by day 3. This pattern of response was unique to MCR3–/– mice and suggests that temporal organization of the anorectic response was altered by absence of the MCR3.

Based on our previous work (14 , 39 , 53) , leptin-induced up-regulation of UCP1 mRNA in BAT and WAT was used as a measure of its ability to increase SNS outflow and regulate adipocyte gene expression. The absence of MCR3 did not compromise induction of UCP1 by leptin in either BAT (IP and ICV) or WAT (ICV only), although the induction with IP leptin was not significant in inguinal WAT. The reasons are unclear, but UCP1 mRNA also was not increased in inguinal WAT among any of the groups receiving IP leptin (see Fig. 3 ). The observation that both routes of leptin administration were equally effective in increasing UCP1 mRNA in BAT from WT and MCR3–/– mice argues against the involvement of leptin resistance. In fact, the proposed role of MCR3 in regulating resting membrane potential in POMC neurons (12 , 23) predicts that its absence would decrease GABA-mediated IPSCs, thereby lowering membrane potential and making POMC neurons more responsive to depolarization by leptin. Current experiments were not designed to evaluate changes in leptin sensitivity among the strains, but it will be interesting to determine in future studies whether absence of MCR3 modifies the threshold for activation of the SNS by leptin. Like MCR3, the absence of CART did not compromise the ability of leptin to increase UCP1 in either BAT or WAT.

The present experiments show that absence of MCR4 compromised the ability of leptin to increase UCP1 expression in BAT and WAT, irrespective of route of administration. Using a selective ß3-adrenoceptor agonist as a sympathomimetic, we further show that MCR4–/– mice respond to ß3-adrenoceptor activation with robust increases in UCP1 mRNA in both tissues. This additional control argues that the lack of UCP1 induction by leptin in MCR4–/– mice is not due to an inability of their adipose tissue to respond to sympathetic stimulation, and is most likely due to an inability of leptin to increase SNS outflow. Coupled with the finding that both routes of leptin reduced food intake (Table 1) , leptin resistance is not a viable explanation. Evidence that leptin increases SNS activity is compelling (14 , 54 55 56 57) , and recent work indicates that CART peptides and {alpha}-MSH mediate this effect by activating preganglionic neurons within the autonomic nervous system (10 , 58) . Ste. Marie et al. (35) used MCR4–/– mice and found that IP leptin was unable to increase UCP1 in BAT. However, their previous work showed that MCR4–/– mice were resistant to the effects of IP leptin but responded to ICV leptin with a reduction in food intake (34) . Therefore, the unique characteristics of MCR4–/– mice on the C57BL/6J background make it difficult to distinguish between leptin resistance and a requirement for MCR4 to increase UCP1. Satoh et al. (52) used a pharmacological approach in rats and found that ICV injection of a MCR3/MCR4 antagonist blocked leptin’s ability to increase UCP1 mRNA in BAT. Rahmouni et al. (59) used direct multifiber recording techniques to measure firing rates in sympathetic nerves innervating the kidney and found that leptin was unable to increase renal sympathetic nerve activity in MCR4–/– mice. Our findings are consistent with this study and support the conclusion that the MCR4 is necessary for SNS activation by leptin. However, Haynes et al. (58) simultaneously measured firing rates in sympathetic nerves innervating BAT and the kidney, and found that ICV injection of an MCR3/MCR4 antagonist had no effect on leptin-dependent firing rates in BAT, but blocked leptin-dependent increases in renal firing rates. This finding suggests the interesting possibility that leptin utilizes different or redundant signaling systems (i.e., CART or melanocortin receptor subtypes) to regulate sympathetic outflow to different anatomical regions. While this is possible, the current studies establish that the absence of MCR4, but not MCR3 or CART, blocks leptin’s ability to induce adipocyte gene expression in fat depots (BAT and WAT) from separate anatomical locations.

In conclusion, the present studies make a strong case that the anorectic and metabolic effects of leptin are dependent on integrated but separable inputs from the melanocortin 3 and 4 receptor subtypes, but not CART. This conclusion is consistent with the more severe disturbance of energy balance noted in double MCR3/MCR4 knockouts (37) , and the present studies showing that the absence of MCR3 or MCR4 produce mice with comparably disturbed nutrient partitioning. The collective phenotypic and mechanistic evidence supports the view that MCR3 and MCR4 play complementary, but nonredundant roles in regulating nutrient partitioning and translating behavioral and metabolic responses to leptin.


   ACKNOWLEDGMENTS
 
The authors acknowledge the excellent technical assistance of Mandy Shirah and the editorial assistance of Jenny Monceaux. This work was supported by a Research Grant from the American Diabetes Association (T.W.G.) and U.S. Public Health Service Grants DK 053872 (T.W.G.), DK 064156 (T.W.G.), and DK60747 (R.L.M.).

Received for publication February 18, 2005. Accepted for publication April 27, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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