Genetic Dissection of the Functions of the Melanocortin-3 Receptor, a Seven-transmembrane G-protein-coupled Receptor, Suggests Roles for Central and Peripheral Receptors in Energy Homeostasis*

Background: Conditional gene targeting methods were used to investigate the role of melanocortin-3 receptors (MC3Rs). Results: MC3Rs expressed in the brain are not sufficient to defend against diet-induced obesity but can improve metabolic homeostasis. Conclusion: The role of MC3Rs in energy homeostasis involves central and peripheral actions. Significance: This is the first evidence suggesting a role for central and peripheral MC3Rs in energy homeostasis. The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a “lox-stop-lox” (LoxTB) 5′ of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3rTB/TB mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.


MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5 of the translation initiation codon of the mouse
Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r TB/TB mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent path-ways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.
Obesity of Mc3r Ϫ/Ϫ mice is independent of hyperphagia and is thought to result from altered metabolism. However, whether MC3Rs regulate appetite remains unclear. Several groups have reported that inhibition of food intake by nonselective melanocortin agonists or activation of melanocortin neurons using serotonergic drugs does not require functional MC3Rs (6, 19 -22). However, hyperphagia induced by melanocortin receptor antagonists may involve MC3Rs (23,24). In other situations, activation of MC3R has been reported to either stimulate (25,26) or inhibit (24) feeding behavior. Analyzing feeding behavior of Mc3r Ϫ/Ϫ mice has also yielded inconsistent results, with reports of hypophagia (6), normal food intake (3), or hyperphagia during the lights-on period (4). There is also evidence suggesting that MC3R in the periphery may regulate food intake (27,28). A consensus on the role of MC3Rs in appetite regulation has not been established.
MC3Rs regulate feeding-associated behaviors, modulating the expression of food anticipatory activity (FAA) (8,10,29). FAA involves a timing mechanism that triggers increased food seeking behaviors anticipating nutrient availability. Although FAA exhibits characteristics suggesting a circadian mechanism, the location and molecular mechanisms of the putative "food-entrainable oscillators" governing the expression of this behavior remain enigmatic and controversial (29,30).
To further investigate the functions of MC3Rs expressed in the brain and periphery in the control energy homeostasis, we inserted a lox-stop-lox (LoxTB) sequence to block Mc3r transcription. Homozygous carriers of the LoxTB MC3R allele (Mc3r TB/TB ) display an obese phenotype similar to that observed in Mc3r Ϫ/Ϫ mice (13,16). LoxTB MC3R mice can be used in conjunction with transgenic strains expressing Cre recombinase for cell type-specific reactivation to map functions associated with receptors expressed in discrete regions (31). Transgenic mice expressing Cre controlled by the rat nestin promoter and enhancer (Nes-Cre) were initially used to rescue expression in the nervous system (32). Surprisingly, the obese phenotype is only partially rescued in Nes-Cre;Mc3r TB/TB mice, suggesting that functions of peripheral MC3Rs impact on adiposity. The VMH, and in particular the dorsomedial VMH, exhibits dense Mc3r expression (3). VMH neurons impact on adiposity by regulating ingestive behaviors and the autonomic nervous system (33,34) and modulate the expression of FAA (30). We therefore investigated the impact of restoring MC3R signaling in the VMH on energy homeostasis using steroidogenic factor-1 (SF-1) Cre transgenic mice (35). Analysis of SF1-Cre;Mc3r TB/TB mice indicates that the action of VMH MC3R results in a partial rescue of the body composition phenotype, attenuating the change in fat mass while not affecting lean mass, and markedly improves metabolic homeostasis. However, actions of MC3R in neurons residing outside the VMH are required for the expression of food anticipatory activity.

EXPERIMENTAL PROCEDURES
Gene Targeting and Animal Husbandry-A targeting vector was generated using recombineering within SW106 cells (36 Assessment of Movement, Food Intake, and Energy Expenditure-Assessment of food intake, movement in the x axis, energy expenditure (24 h, resting and non-resting), and the respiratory exchange ratio (RER, an indicator of whole body substrate preference) involved a 16-chamber comprehensive laboratory animal monitoring system housed as described previously (5,10). FAA was assessed using running wheels as described previously (10). The impact of Mc3r TB genotype on DIO was studied used a purified high fat diet (HFD, 60% kJ/fat).
In Situ Hybridization Histochemistry-Fresh frozen brains were collected and processed for in situ hybridization as described previously (37). Coronal sections (20 m) were cut on a cryostat and thaw-mounted onto Superfrost Plus slides (VWR Scientific, West Chester, PA). Hypothalamic sections were collected in a 1:6 series from the diagonal band of Broca (bregma 0.50 mm) caudally through the mammillary bodies (bregma Ϫ5.00 mm). Antisense 33 P-labeled rat MC3R riboprobe (corresponding to bases 808 -1204; GenBank accession number NM_008561.3) (0.2 pmol/ml) was denatured, dissolved in hybridization buffer along with tRNA (1.7 mg/ml), and applied to slides. Controls used to establish the specificity of the MC3R riboprobe included slides incubated with an equivalent concentration of radiolabeled sense MC3R riboprobe or radiolabeled antisense probe in the presence of excess (1000ϫ) unlabeled antisense probe. Slides were covered with glass coverslips, placed in a humid chamber, and incubated overnight at 55°C. The following day, slides were treated with RNase A and washed under conditions of increasing stringency. Slides were dipped in 100% ethanol, air-dried, and then dipped in NTB-2 liquid emulsion (Eastman Kodak Co.). Slides were developed 16 days later and covered with glass coverslips.
Gene Expression Analysis-RNA isolated from liver tissue using TRIzol was converted to biotin-labeled cDNA using the Ambion Illumina total prep RNA amplification kit. Pooled samples were hybridized on 24 arrays using three MouseRef-8 v2 Expression BeadChips for the analysis of 18,138 genes based on RefSeq release 22 and supplemented with MEEBO and RIKEN FANTOM2 content. Each probe is represented with an average of 30-fold redundancy (5-fold minimum). The resultant array data (gene level data) were preprocessed by GenomeStudio software version 1.0.6 (Illumina Inc., San Diego, CA) to give quantile-normalized gene level data. Each record in the subject-level data file represented a single gene with columns providing the expression intensity for each of 24 subjects and the p value for each gene on the array. The expression of candidate genes of interest was assessed using a 7900HT fast real-time PCR system and total RNA samples from individual mice as described previously (9,10). To test whether removal of the LoxTB sequence restores expression and function, we removed the LoxTB sequence globally using EIIa-Cre mice (38).  In chow-fed conditions, the increase in FM associated with loss of MC3Rs was attenuated in Nes-Cre;Mc3r TB/TB mice (Fig.  2, A-C, n ϭ 5/group). The effects of the Nes-Cre and Mc3r TB genotypes on body composition was assessed using two-way analysis of variance. FFM was significantly reduced by the Nes-Cre transgene (p Ͻ 0.05), with no effect of Mc3r TB genotype (p ϭ 0. 30). An effect of the Nes-Cre transgene on body composition has been reported (39) and may have masked the impact of the Mc3r TB genotype on FFM. Mc3r TB genotype had a signif-icant impact on FM (p Ͻ 0.05) and adiposity (FM as percentage of total weight, p Ͻ 0.005). There was no significant effect of the Nes-Cre transgene on either parameter. There was a trend (p ϭ 0.06) for an effect of Mc3r TB genotype on body weight that was not observed in Nes-Cre;Mc3r TB/TB mice.

RESULTS
In mice fed HFD for 6 weeks, the Nes-Cre transgene had no impact on the obese phenotype of Mc3r TB/TB mice (Fig. 2, D-F). Analysis of the impact of Nes-Cre and Mc3r TB genotype on body weight and composition using two-way analysis of variance suggested a significant effect of Mc3r TB genotype on FM (p Ͻ 0.05) and adiposity (p Ͻ 0.01). There was a also a trend for an impact of Mc3r TB genotype on body weight (p ϭ 0.06). However, the Nes-Cre genotype had no effect on any of these parameters, irrespective of the Mc3r TB genotype.
The SF1-Cre Transgene Modifies the Impact of Mc3r TB Genotype on Whole Body Substrate Metabolism-We next assessed the metabolic phenotype of Mc3r TB/TB and SF1-Cre;Mc3r TB/TB mice. Food intake, activity, and energy expenditure were measured simultaneously in WT (n ϭ 16), Mc3r TB/TB (n ϭ 8), and SF1-Cre;Mc3r TB/TB mice (n ϭ 8). Note that the "WT group" consisted of eight SF1-Cre transgenic and eight normal littermates; because the SF1-Cre genotype had no significant effect on any of the parameter, these data were pooled for statistical analysis. Body weight and composition for the mice used are shown in Fig. 4A. Food intake was not affected by genotype (Fig.  4B). Presentation and interpretation of energy expenditure data have been recently discussed (40,41). Energy expenditure data are thus presented per mouse and normalized for either FFM or total body weight (Fig. 4, C-J). Energy expenditure expressed in kJ/h per mouse was lower in Mc3r TB/TB mice in the mid-dark period (Fig. 4C), although this effect was subtle and did not translate into significant differences in 24-h energy expenditure (Fig. 4D). Energy expenditure expressed per kg of fat-free mass was also not significantly different between genotype. However, when expressed per kg of body weight, there was a significant reduction in Mc3r TB/TB mice, but not in SF1-Cre;Mc3r TB/TB mice (Fig. 4, G and H). Further evidence for a modifier effect of the SF1-Cre transgene on the energy expenditure phenotype of Mc3r TB/TB mice came from regression analysis of energy expenditure data as a function of body weight (5) and from analyzing the respiratory exchange ratio (RER). Resting energy expenditure and total energy expenditure correlated with body weight in Mc3r TB/TB and in SF1-Cre;Mc3r TB/TB mice; the regression line for Mc3r TB/TB mice was lower when compared with SF1-Cre;Mc3r TB/TB mice (Fig. 4, I and J). The RER is an indicator of energy derived from oxidizing glucose or fatty acids (42) and was lower in Mc3r TB/TB mice when compared with WT and SF1-Cre;Mc3r TB/TB mice (Fig. 4, K and L). This observation is surprising given that our previously published results from studies analyzing the RER of melanocortin receptor knock-out mice (4,10,43). The differences in RER were not due to altered food intake (Fig. 4B), whereas the mice were also in a positive energy balance (weight gain in grams: for WT, 0.5 Ϯ 0.1 g/day; for Mc3r TB/TB mice, 0.4 Ϯ 0.1 g/day; and for SF1-Cre; Mc3r TB/TB mice, 0.4 Ϯ 0.1 g/day).
A hallmark of the Mc3r-deficient phenotype is reduced physical activity (3,4,6,10,44). Activity measured using either cross-beam breaks or running wheels was reduced in Mc3r TB/TB mice during the dark period (Fig. 5, A-D). The physical activity phenotype associated with Mc3r deficiency was only partially rescued in SF1-Cre;Mc3r TB/TB mice (Fig. 5, A-C), indicating that actions of MC3R expressed in SF1(ϩve) neurons are not sufficient to completely restore locomotor behav-ior to normal levels. Changes in physical activity could impact on energy expenditure. Non-resting energy expenditure, the portion of energy expenditure attributable to movement and feeding, is reduced in Mc3r Ϫ/Ϫ mice (5), and in this study, it was reduced by 30% in Mc3r TB/TB mice but was normal in SF1-Cre; Mc3r TB/TB mice (Fig. 5E). Resting energy expenditure was not significantly affected by genotype (Fig. 5E).
Improved Metabolic Profile of SF1-Cre;Mc3r TB/TB Mice-Obesity and insulin resistance are associated with fatty liver disease and altered expression of lipogenic enzymes (45). To assess the impact of Mc3r deficiency and obesity on liver metabolism, we performed a microarray analysis of gene expression. This analysis indicated that changes due to MC3R signaling and obesity were attenuated in SF1-Cre;Mc3r TB/TB mice (Fig. 6, A and B). Using a 1.5-fold up-or down-regulation as a threshold, 219 genes met the criteria in Mc3r TB/TB mice, whereas 117 met the criteria in SF1-Cre;Mc3r TB/TB mice. Use of a more stringent criteria (2-fold change) resulted in a more stark difference, with 63 genes meeting the criteria in Mc3r TB/TB mice and only 23 meeting the criteria in SF1-Cre; Mc3r TB/TB mice. Using pathway analysis indicated an enrichment for genes involved in the metabolism of xenobiotics by cytochrome P450 (19 genes, p Ͻ 0.0001), arachidonic acid metabolism (12 genes, p Ͻ 0.0001), linoleic acid metabolism (eight genes, p Ͻ 0.0001), glutathione metabolism (eight genes, p Ͻ 0.001), peroxisome proliferator-activated receptor signaling (eight genes, p Ͻ 0.005), and biosynthesis of unsaturated fatty acids/fatty acid metabolism (six genes, p Ͻ 0.001). We assessed changes in expression of selected genes using quantitative RT-PCR (Fig. 6C). Analysis of serum lipids indicated significantly lower levels of total triglycerides TG in Mc3r TB/TB mice. However, a similar effect was observed in SF1-Cre; Mc3r TB/TB mice (Fig. 6D). Although the pattern of gene expres- sion and RER suggests altered fat oxidation, ␤-hydroxybutyrate levels were not affected by genotype (Fig. 6D).
Obesity is associated with a mild state of inflammation that contributes to the insulin-resistant phenotype (46). Inspection of the microarray results suggested that induction of inflammatory processes altered in Mc3r TB/TB mice was attenuated in SF1-Cre;Mc3r TB/TB mice. We therefore measured secreted factors linked to inflammatory processes. Significant increases in the serum levels of monocyte chemoattractant protein-1 (Mcp1) and resistin, but not in Il-6, were observed in Mc3r TB/TB mice (Fig. 6E). This aspect of the metabolic phenotype was improved in SF1-Cre;Mc3r TB/TB mice (Fig. 6E).
FAA Is Not Rescued in SF1-Cre;Mc3r TB/TB Mice-FAA was assessed using the restricted feeding (RF) protocol previously employed by our laboratory (10). Based on the recommendations of our Institutional Animal Care and Use Committee, the amount of food provided, although initially reduced to induce the behavior, was then modified to "clamp" weight loss at 10 -15% (10). Weight loss was therefore not significantly different between groups (WT, 3 WT mice subjected to RF, where food is provided during the lights-on period to mice housed in a light-dark cycle, displayed FAA (Fig. 7). A reduction in total activity was observed, consistent with an effect of reduced caloric intake (Fig. 7, A and B). This reduction was most severe particularly during the dark period (Fig. 7, C and D) as activity in the lights-on period was increased by RF (Fig. 7, E and F). FAA, which is the amount of activity during the 2-h period prior to food presentation, was also increased relative to activity at the same time during ad libitum feeding (Fig. 7, G-I).
As predicted based on our earlier observation using Mc3r Ϫ/Ϫ mice, the reduction of activity in the dark period, stimulation of activity in the lights-on period, and expression of FAA during RF were attenuated in Mc3r TB/TB mice (Fig. 7, A-I). Indeed, activity of Mc3r TB/TB mice was actually moderately increased during RF when compared with baseline values (Fig. 7, A and B). In contrast, the increase in activity during the lights-on period, and more specifically during the 2-h period preceding food presentation, during RF was attenuated in Mc3r TB/TB mice (Fig.  7, E-I). This behavioral deficit in the response to RF was not significantly modified in SF1-Cre;Mc3r TB/TB mice (Fig. 7, A-I). Mc3r Ϫ/Ϫ mice subject to RF exhibit an altered rhythmicity in the expression of clock genes in the cortex (10). Altered expression of clock genes in the cortex was observed in both Mc3r TB/TB and SF1-Cre;Mc3r TB/TB mice subject to RF (Fig. 8) (n ϭ 5-6/group).

DISCUSSION
These experiments had three objectives. The first was to generate and validate the LoxTB MC3R mouse strain as a model for the genetic dissection of MC3R function. This strategy using Cre-mediated recombination to reactivate transcription was successfully used for the conditional targeting of the mouse Mc4r gene (31,47). Homozygous carriers of the LoxTB MC3R allele (Mc3r TB/TB ) mice displayed loss of Mc3r expression and altered body composition and behavioral characteristics previously reported to be associated with loss of MC3Rs by our laboratory (4, 5, 10) and others (3,6,7,48). We determined that removing the lox-stop-lox sequence globally using EIIa-Cre rescued the obese phenotype. Collectively, these results indicated the successful inhibition of Mc3r transcription through insertion of the LoxTB sequence and indicated that removing this sequence using Cre-mediated recombination restores functionality. Although we had inserted an IRES-acGFP sequence downstream of the MC3R open reading frame, the level of expression was insufficient for detecting GFP immunoreactivity. This model will be an important genetic tool for the dissection of neural circuits involved in the expression of feeding-related behaviors and may also be useful for investigating the functions of this receptor in non-neural cell types.
Mc3r TB/TB mice exhibited an obese phenotype associated with hyperinsulinemia, hyperleptinemia, and evidence of a mild inflammatory condition associated with obesity (46). The obese phenotype appears to be metabolic as food intake is normal. Analysis of liver gene expression indicated the effects of loss of MC3R signaling and obesity on fatty acid metabolism and suggested altered immune function. Of note, Mc3r TB/TB mice exhibited a marked increase in the expression of proteins belonging to the cell death-inducing DFF45-like effector (CIDE) family that localize to lipid droplets and the endoplasmic reticulum; up-regulation correlates with obesity, hepatic steatosis, and insulin resistance (49,50).
A Role for Peripheral MC3Rs in Protecting against Obesity?-Melanocortin neurons form a critical node in the nutrient sensing networks governing the expression of ingestive behaviors and maintaining metabolic homeostasis in response to internal signals of metabolic status (1,8). These actions are thought to be mediated primarily by the two melanocortin receptors that are expressed in the central nervous system (MC3R and MC4R). Loss of MC4R function causes hyperphagia, increased lean mass, and obesity; restoring MC4R expression in the nervous system using the same approach described for these experiments was sufficient to rescue this phenotype (31). These results imply that the functions of MC4R expressed outside the nervous system have a minimal impact on the obese phenotype.
Loss of MC3R function is also associated with obesity, although this phenotype is more dependent on dietary fat content. In chow-fed conditions, loss of MC3R function has a subtle impact on body weight, increasing adiposity by reducing lean mass and increasing fat mass. When exposed to high fat diets, MC3R-deficient mice display accelerated weight gain. Our initial hypothesis was that the impact of MC3Rs on energy homeostasis and protection from obesity involved actions in the central nervous system. However, the results of the current study are surprising and important in suggesting that the impact of the interaction between Mc3r genotype and diet on obesity may result from actions outside the central nervous system. Although neural MC3R can compensate for loss of peripheral actions in chow-fed conditions, neural MC3R had no impact on obesity in situations of increased dietary fat consumption.
How the actions of peripheral MC3Rs impact on obesity is not clear. However, MC3Rs are expressed in the periphery. For example, MC3Rs expressed in the renal cortex and medulla regulate natriuresis and salt-sensitive hypertension (51). MC3Rs expressed by the immune system regulate inflammation (52). Whether and how MC3Rs expressed in these organ systems impact on increased propensity for diet-induced obesity is not clear. One possibility is that the regulation of inflammation by MC3Rs impacts on obesity. Obesity is associated with a mild inflammatory condition (46); whether inhibiting inflammation prevents obesity is less clear, although cases where inhibiting the inflammatory responses protects against DIO have been reported (53,54). Another possibility is evidence for a role for peripheral melanocortin receptors in regulating some food intake (19,27,28). Intraperitoneal injections of high doses of the melanocortin analog melanotan-II in Mc4r Ϫ/Ϫ mice reduces food intake, whereas central administration has the opposite effect (28). Moreover, melanotan-II administered peripherally poorly penetrates the central nervous system, suggesting that the site (or sites) of action may be peripheral (27,55). Peripheral administration of melanocortin analogs also improves glucose homeostasis in Mc4r Ϫ/Ϫ mice, suggesting actions at receptors that may reside in peripheral tissues (19). The relevance of these data to our findings is, however, not clear. Future studies involving a more thorough investigation of the impact of Cre transgenes expressed in neural and non-neural tissues on the obesity syndrome observed in Mc3r TB/TB mice are clearly warranted and could yield important information on the roles of melanocortins in periphery that impact on obesity and diabetes.
VMH MC3Rs Regulate Systems Involved in Metabolic Homeostasis but Not in the Expression of Anticipatory Behaviors-Another objective was to investigate the functions of MC3R expressed in the VMH. Neurons residing in the VMH regulate appetite and protect against obesity (34,56). As restoring expression in the nervous system had no impact on the accelerated DIO phenotype, we considered it unlikely that VMH MC3R signaling would alter the accelerated DIO phenotype associated with loss of MC3R. Our experiments therefore focused on studies using mice maintained on a low fat diet, investigating the role of VMH MC3R in the body composition phenotype and the expression of feeding-related behaviors. The analysis of body composition and metabolic phenotype of SF1-Cre;Mc3r TB/TB mice produced two important observations. First, VMH MC3R signaling had a modest impact on fat mass and had no effect on the lean mass phenotype. This result suggests that altered body composition observed with loss of MC3R involves SF1(Ϫve) neurons outside the VMH. Indeed, based on our observations of Nes-Cre;Mc3r TB/TB mice, the actions of receptors expressed in the periphery may be important. Resolving this issue will require further analysis using other neuron-selective Cre transgenics that are free of the body composition phenotype observed in Nes-Cre mice. Second, the analysis of whole body energy expenditure using indirect calorimetry indicates that VMH MC3R signaling is involved in regulating metabolism. Mc3r TB/TB mice exhibited a reduction in non-resting energy expenditure, which we have observed previously in Mc3r Ϫ/Ϫ mice (5 , p Ͻ 0.05). Note that for A, C, E, and G, the x axis is the same; activity during ad libitum feeding (AL) is in the blue shaded region. All mice were subject to 19 days of restricted feeding (RF). After 5 days of RF, the amount of food provided was increased incrementally to protect mice from excessive weight loss. A and B, restricted feeding results in reduced 24-h activity in WT mice, but not in was not due to reduced food intake; food intake was not affected by genotype, and the mice all gained weight while housed in metabolic chambers. Possible factors impacting on substrate preference include impaired insulin action, indicated by hyperinsulinemia in Mc3r TB/TB mice but not in SF1-Cre;Mc3r TB/TB mice. The liver expression profile also suggests altered fatty acid metabolism in Mc3r TB/TB mice, with SF1-Cre;Mc3r TB/TB mice exhibiting a significant attenuation of this response.
Previous studies reported that the RER in Mc3r Ϫ/Ϫ mice is increased (3,4). However, in these studies, the Mc3r TB genotype appeared to have an inhibitory effect. The reasons for this discrepancy are not clear. As discussed above, differences in food intake are not a factor. One difference between this and the reported studies is genetic background. The current study used mice on a mixed FVB;B6 background when compared with previous experiments using Mc3r Ϫ/Ϫ mice backcrossed more than seven generations onto the B6 background. The analysis of triglycerides and ␤-hydroxybutyrate, although displaying changes due to Mc3r TB genotype, also yielded results that were not anticipated and differed markedly from that previously reported. Again, this could be due to the effects of genetic background and/or from the state of the mice when the samples were collected (fasted versus fed). Another consideration when using genetic tools is the issue of compensation. Reactivation of MC3Rs in this model involves spatial and temporal variability in the expression of Cre recombinase. The possibility that developmental compensation has altered the timing and pattern of MC3R expression and has in some way affected the phenotype of the mice being studied cannot be excluded. Future studies may involve the use of transgenics where Cre expression can be manipulated temporally.
Finally, results from the analysis of FAA in SF1-Cre; Mc3r TB/TB mice suggest that VMH MC3R signaling is not sufficient, in of itself, to regulate the expression of this complex behavior. The attenuated expression of FAA in Mc3r Ϫ/Ϫ mice was associated with abnormal clock activity in the cortex, indicated by the analysis of clock gene expression (10). We observed that the expression of the clock gene is also altered in the cortex Mc3r TB/TB mice and in SF1-Cre;Mc3r TB/TB mice. The role of the known clock in the expression of rhythms anticipating food intake is uncertain (57). The significance of this result in explaining the FAA phenotype is thus unclear. However, it does suggest abnormal rhythmicity in the activity of clocks expressed in the cortex and suggests that this phenotype is not rescued by VMH MC3R signaling. These results, although negative, nevertheless are an important first step in mapping the MC3R-dependent neural pathways involved in the regulation of anticipatory activity.
In summary, we report the development a new genetic model for investigating of the functions of MC3Rs. We have used this model to analyze functions of MC3R expressed in SF1(ϩve) neurons in the VMH. Our data suggest that the actions of MC3R in these neurons significantly impact on metabolic homeostasis but are not sufficient to restore body composition to normal or for regulating expression of complex behaviors associated with food anticipation. This strain will be important for future experiments mapping the MC3R signaling pathways in the CNS regulating the expression of food anticipatory behavior and body weight homeostasis.