Double Leptin and Melanocortin-4 Receptor Gene Mutations Have an Additive Effect on Fat Mass and Are Associated with Reduced Effects of Lept
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《内分泌学杂志》
Neuropeptides Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana 70808
Abstract
Melanocortin-4 receptors (MC4Rs) are involved in the regulation of food intake, sympathetic nervous activity, and adrenal and thyroid function by leptin. The role of MC4Rs in regulating energy balance by leptin was investigated using double heterozygote or homozygous leptin (Lepob) and Mc4r gene mutant mice. Double heterozygous or homozygous mutants were generated by crossing MC4R knockout (Mc4r–/–) mice, backcrossed onto C57BL/6J, with B6.V-Lepob mice. Energy expenditure was measured using indirect calorimetry. The effect of leptin on food intake, weight loss, insulin, and corticosterone was compared for Lepob/LepobMc4r–/– mice and Lepob/Lepob mice. Double heterozygous and homozygous mutants exhibited an additive effect on fat mass. The 2-fold increase in body weight associated with severe obesity of Lepob/Lepob mice was associated with a significantly higher 24 h total and resting energy expenditure. The effect of obesity on energy expenditure was attenuated by 50% in Lepob/Lepob Mc4r+/– and Lepob/Lepob Mc4r–/– mice. Loss of MC4Rs did not affect basal food intake of Lepob/Lepob mice but was associated with partial leptin resistance in terms of food intake and weight loss. Leptin suppression of insulin and corticosterone in Lepob/Lepob mice were not significantly affected by Mc4r genotype. These results suggest a complex interaction between the Lep and Mc4r genes in energy homeostasis and suggest that MC4Rs retain significant anti-obesity function in the obese leptin-deficient state. Increased adiposity with double mutations may involve a reduction in energy expenditure. MC4Rs might have a modest role in the regulation of energy balance by exogenously administered leptin, primarily effecting food intake.
Introduction
LEPTIN, AN ADIPOCYTOKINE produced predominantly by adipose tissue, has been strongly implicated in the regulation of energy homeostasis (1). The obese (ob) mutation in the leptin gene (Lep), described in 1950 (2), is recessive and results in extreme obesity, hypercorticosteronemia, and type II diabetes (2, 3). The Lep gene encodes a 167 amino acid secreted protein that normalizes appetite, reduces body weight, and restores insulin sensitivity of leptin-deficient mice when administered peripherally or intracerebroventricularly (4, 5, 6, 7). The B6.V-Lepob strain carries a CT mutation, converting codon 105 from arginine to a stop codon, resulting in a truncated inactive protein (1). Serum leptin levels fall rapidly with caloric restriction, providing a signal of negative energy balance (3). Lepob/Lepob mice exhibit many of the behavioral and neuroendocrine abnormalities associated with fasting, including reduced gonadal and thyroid function, and increased adrenal activity (3). Most, if not all, of the regulation of food intake and metabolism by leptin are observed when recombinant protein is applied into the third ventricle (6).
In the arcuate nucleus of the hypothalamus (ARC), neurons expressing proopiomelanocortin (POMC) or that coexpress neuropeptide Y (NPY) and agouti-related peptide (AgRP) are involved in the regulation of energy balance by leptin (8). POMC and AgRP/NPY neurons project from the ARC to hypothalamic and extrahypothalamic sites within the brain involved in the regulation of neuroendocrine function, autonomic nervous activity, and ingestive behavior (9). The two groups of neurons have opposing effects on feeding and metabolism. Leptin deficiency is associated with a modest reduction in Pomc expression and an increase in the expression of AgRP and Npy mRNA (10, 11, 12, 13). Mice heterozygous or homozygous for a null Pomc gene and humans with Pomc mutations are obese and hyperphagic (14). Conditional knockout of the long form of the leptin receptor in POMC neurons also results in a modest increase in body weight (15). Conversely, double Lepob/Lepob Npy knockout mice exhibit an attenuated obese phenotype associated with reduced food intake compared with Lepob/Lepob mice (16).
Melanocyte-stimulating hormones (MSHs), derived from the posttranslational processing of POMC, regulate appetite and energy expenditure primarily through the melanocortin-4 receptor (MC4R) (17, 18). The stimulation of renal sympathetic nervous activity by -MSH and by leptin is impaired in heterozygous and abolished in homozygous Mc4r mutant mice (19). MC4Rs might also be involved in the stimulation of brown adipose tissue (BAT) thermogenesis by leptin (20). Mice and humans bearing null mutations of the Mc4r gene or overexpressing agouti, an antagonist of the MC4R, exhibit an obese phenotype correlating with the degree of functional impairment (21, 22). The obese phenotype of Mc4r mutant mice comprises hyperinsulinemia, hyperleptinemia associated with delayed peripheral and hypothalamic leptin resistance, accelerated longitudinal growth, as well as obesity and hyperphagia that is sensitive to dietary fat content (18, 21, 23).
Mice with impaired MC4R action attributable to gene deletion or expression of agouti protein, an antagonist of the melanocortin-1 receptors and MC4Rs, rapidly develop resistance to centrally and peripherally administered leptin (6, 18, 21, 24). Boston et al. (25) reported that leptin sensitivity is restored in lethal yellow (Ay/a) mice when backcrossed onto the B6-V.Lepob strain, with a partial leptin resistance still observed in males. In the same study, overexpression of agouti in adrenalectomized Lepob/Lepob mice had an additive effect on obesity. These results suggest that MC4R regulation of energy balance involves leptin-dependent and -independent functions and that residual MC4R activity functions to prevent additional weight gain of Lepob/Lepob mice. The interpretation of these experiments are confounded, however, by recent observations of MC4R-independent stimulation of adipogenesis by agouti (26, 27). Moreover, intracerebroventricular administration of the potent nonselective melanocortin receptor agonist melanotan-II to Ay/a mice still reduces food intake, mediated through MC4Rs (18), suggest significant retention of MC4R function (28). To further investigate the contribution of MC4Rs to the regulation of energy balance by leptin, we crossed the null Mc4r gene onto the B6-V.Lepob strain. Overall, our results are consistent with MC4Rs being involved in both leptin and leptin-independent pathways in the regulation of energy intake and expenditure.
Materials and Methods
Animal husbandry
The Pennington Biomedical Research Center Institutional Animal Care and Use Committee reviewed and approved these experiments. Mc4r–/– mice backcrossed 12+ generations onto the B6 background were genotyped as described previously (29). Lepob/+ Mc4r+/– heterozygotes were generated by crossing B6.V-Lepob/Lep+ mice (The Jackson Laboratory, Bar Harbor, ME) with Mc4r–/– mice. Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice were then produced by mating Lepob/Lep+ Mc4r+/– mice and genotyped as described previously (29). For the leptin-treatment experiments, male and female Lepob/Lep+ Mc4r–/– mice were crossed to produce Lepob/Lepob Mc4r–/– mice; age-matched Lepob/Lepob controls were purchased from The Jackson Laboratory. Mice were housed on a 12-h light, 12-h dark cycle (lights on, 0600–1800 h), and food intake was measured in mice housed in wire-mesh caging fed low-fat diets (low-fat diet 12450B, 10% kJ/fat, Research Diets, New Brunswick, NJ) at 1000 h and adjusted for spillage. Fat mass and fat-free mass were measured using nuclear magnetic resonance (NMR) (Bruker Mice Minispec NMR Analyzer; Bruker Optics, Billerica, MA) (30). Unless otherwise stated, all data presented are from studies using female mice.
Indirect calorimetry
Indirect calorimetry was performed using a 16 chamber Oxymax system (Columbus Instruments, Columbus, OH), as described previously (29). Chambers were housed in a temperature-controlled incubator set at 28 C. Mice were acclimated 3–7 d to housing in metabolic chambers. Data are presented as total 24-h energy expenditure (in kilojoules per day) or resting energy expenditure (kilojoules per day, mean of the lowest 10% of value assumed to be represent periods with minimal activity). Energy expenditure data were also adjusted for fat-free mass measured by NMR. The respiratory exchange ratio (RER), also known as the respiratory quotient, is the ratio of CO2 produced to O2 consumed. RER is inversely related to fat oxidation and positively related to carbohydrate utilization. That is, an increased RER value is an indicator that the animal is using carbohydrates as the preferred fuel substrate.
Blood chemistries
Blood samples were taken between 1000 and 1200 h from mice that had been fasted for 24 h (experiment 1) or 4 h (experiment 3). Insulin (CrystalChem, Downer’s Grove, IL) and corticosterone (Diagnostic Systems Laboratories, Webster, TX) levels were measured using commercially available assays.
Leptin treatment
Three experiments examining the response of Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/+ mice were performed. In experiment 1, 6-month-old Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice were continuously infused with leptin (10 μg/d; R & D Systems, Minneapolis, MN) using osmotic minipumps (Alzet; Durect, Cupertino, CA). Mice were anesthetized using isoflurane, with pumps soaked in sterile PBS for 4 h, and then implanted beneath the skin on the dorsal surface
In experiment 2, Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/+ mice were treated with leptin, supplied by Dr. A. Parlow (National Hormone and Pituitary Program), administered by ip injection to minimize the stress of surgical implantation of osmotic pumps. At approximately 16 wk of age, mice were treated with two ip injections of leptin per day (0900 and 1700 h) at a dose of 1 mg/kg·d (i.e. 0.5 mg/kg per injection, twice daily) for 5 d, after a 2-d lead-in period whereby 0.9% saline was injected ip at the same times. NMR analysis was performed before and after leptin treatment; food intake and body weight were measured daily throughout the experiment. The response of Lepob/Lepob Mc4r–/– mice to leptin injections was compared with that of female age-matched Lepob/Lepob mice (The Jackson Laboratory).
For the third experiment investigating neuroendocrine hormones, mice from experiment 2 were allowed to recover for 2 weeks, such that body weight and daily food intake had returned to baseline. The mice were then treated with either leptin ip as described for experiment 2 or 0.9% saline, for 2 d. On the final day, food was removed at 0700 h, and animals were given a final dose of leptin or saline at 0900 h and then killed at 1100 h.
Statistics
All data are presented as mean ± SE. Sigmastat software (SPSS, Chicago, IL) was used for statistical analysis. Normality of data distribution was analyzed by Kolmogorov-Smirnov test. Analysis of two or multiple groups of data used either Student’s t test or ANOVA, respectively, for normally distributed data. Food intake data were analyzed using two-way ANOVA (genotype and treatment) with repeated measures (treated and untreated). Least significant difference or Games-Howell post hoc tests were performed after ANOVA analysis for data exhibiting homogeneous or nonhomogeneous variance, respectively. Data were separated into different gender groups before analysis. Pearson correlations were performed for normally distributed data. Statistical significance was assumed for P values < 0.05.
Results
Body weight and food intake of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice
Fat mass was significantly increased in double heterozygotes compared with single heterozygotes and wild-type (WT) mice (genotype effects on fat mass and percentage body fat, P < 0.001; on fat-free mass, not significant) (Fig. 1, A and C). Between 4 and 10 wk of age, double heterozygotes gained 3-fold more fat mass compared with WT littermates and 2- to 2.5-fold more compared with single heterozygotes (Fig. 1, B and D). A 20% increase in fat mass, with no change of fat-free mass, was also observed for female Lepob/LepobMc4r–/– mice compared with Lepob/Lepob controls between 4 and 5 months of age (Fig. 1, E and F) but was not associated with a significant increase in body weight (Fig. 1G). Basal food intake of Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/– mice was not significantly different from Lepob/Lepob littermates (food intake: Lepob/Lepob, 80 ± 9 kJ/d, n = 7; Lepob/Lepob Mc4r+/–, 78 ± 6 kJ/d, n = 12; Lepob/Lepob Mc4r–/–, 75 ± 7 kJ/d, n = 9).
Energy expenditure and physical activity of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice
Increased fat mass of Lepob/Lepob Mc4r–/– mice in the absence of hyperphagia might indicate reduced fatty acid oxidation and/or energy expenditure. RER was, however, not significantly affected by Mc4r genotype (representative RER data from one of three experiments: Lepob/Lepob, 0.97 ± 0.01; Lepob/Lepob Mc4r+/–, 0.96 ± 0.005; Lepob/Lepob Mc4r–/–, 0.96 ± 0.01; n = 4–6 per group).
The almost 2.0-fold gain in body weight of Lepob/Lepob and Mc4r–/– mice compared with WT mice was associated with a significant increase in total and resting energy expenditure, as reported previously (17, 23, 29, 31) (Fig. 2, A and D). Analysis of the net increase in total or resting energy expenditure, per animal or adjusted for fat-free mass, indicated that the increased energy expenditure associated with severe obesity was attenuated by 50% in Lepob/Lepob Mc4+/– and Lepob/LepobMc4r–/– mice compared with Lepob/Lepob mice (7–8 kJ/d compared with 15 kJ/d) (Table 1). The differences of 24-h energy expenditure of Lepob/LepobMc4r–/– or Lepob/LepobMc4r+/– mice as separate groups were still not significantly different. However, pooling data from Lepob/Lepob mice that were either heterozygous or homozygous for the null Mc4r allele, both of which would be expected to exhibit a phenotype based on studies demonstrating gene-dosage effects of melanocortin mutants on obesity (19, 21, 32) indicated that energy expenditure of Lepob/LepobMc4r(+/– or –/–) mice was significantly (P < 0.05) lower compared with Lepob/Lepob mice.
The interpretation of energy expenditure from lean and obese subjects is difficult because of the heterogeneity in metabolism of tissues and organs (33, 34, 35, 36). Although body weight or estimates of surface area (body weight to the power 0.75) are often used to normalize data, this is not accurate, because neither accounts for variation in body composition. Although not ideal, fat-free mass is the best predictor of resting expenditure in humans (35). For experiments comparing a combination of Lep and Mc4r mutant genes, a similar pattern of changes in energy expenditure was observed whether energy expenditure was expressed per animal (Figs. 2, A and D, 3A) or adjusted for fat-free mass (Fig. 2, B and C, and Table 1). When compared between Lepob/Lepob mice of different Mc4r genotypes, adjustment for body mass also yielded similar results (Fig. 2, E and F, and data not shown). The differences of energy expenditure between Lepob/Lepob Mc4r–/– and Lepob/Lepob mice thus appear to be due to changes in resting or basal metabolic rate and are not due to behavioral differences in spontaneous locomotory movements (Fig. 3, B and C).
Lepob/Lepob mice lacking MC4R are partially resistant to the effects of bi-daily leptin injections on food intake and weight loss
There was no significant difference in food intake of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice treated with leptin by osmotic pump. In experiment 2, leptin was delivered by bi-daily ip injections (two injections of 0.5 mg/kg) for 5 d to Lepob/Lepob Mc4r–/– mice (n = 6) and Lepob/Lepob mice (n = 7). Injections of approximately 25 μg are likely to produce significantly elevated spikes in circulating leptin levels and should thus be considered as a pharmacological, rather than physiological, method of treatment. Basal food intake of Lepob/Lepob Mc4r–/– mice was not significantly different from Lepob/Lepob controls (Fig. 4, B and C). However, the inhibition of food intake by bi-daily leptin injections was significantly impaired in Lepob/Lepob Mc4r–/– mice compared with Lepob/Lepob controls (Fig. 4), with Lepob/Lepob Mc4r–/– mice consuming approximately 50% more food during 5 d of leptin treatment than Lepob/Lepob mice (142 ± 12 vs. 92 ± 7 kJ). Analysis by two-way ANOVA with repeated measures indicated a significant effect of leptin treatment on food intake (P < 0.001) that was dependent on Mc4r genotype (genotype-treatment interaction, P < 0.05).
Leptin treatment, irrespective of the method of delivery or genotype, resulted in a significant reduction of body weight (Fig. 5, A, C, and D). Weight loss associated with leptin injections was more rapid when compared with leptin infusion (12% in 5 d of injections compared with 9–10% in 14 d for leptin infusion). This might be due to differences in the dose used (10 vs. 45–50 μg/d) or the age of mice (6–7 months for mice implanted with osmotic pump, 3 months for mice given injections). Weight loss of Lepob/Lepob Mc4r–/– during leptin treatment was 60% of that observed for Lepob/Lepob mice; however, the difference was statistically significant for experiment 2 only (Fig. 5, A and C). In experiment 1, weight loss correlated with cumulative food intake over the 14 d of leptin treatment (Fig. 5B). When data from all three groups of experiment 1 was pooled (n = 18), approximately 80% of the difference in weight loss could be explained by differences in food intake (R2 = 0.781; P < 0.001). Similar significant correlations were also observed within each genotype (data not shown). When the data from both groups used for experiment 2 was pooled, differences in cumulative food intake over the 5 d of injection accounted for approximately 70% of the variation in weight loss (R2 = 0.682; P < 0.01).
In experiment 2, weight loss during leptin treatment, and the difference in the response of Lepob/Lepob Mc4r–/– vs. Lepob/Lepob mice, was primarily due to changes in fat mass (Fig. 5D). It was not possible to measure fat mass and fat-free mass of the mice in experiment 1 due to the presence of metal in the osmotic pumps. Fat mass data were used to calculate differences in the amount of stored energy lost for Lepob/Lepob Mc4r–/– vs. Lepob/Lepob mice, with fat mass assumed to contain 37.6 kJ/g and fat-free mass 16.7 kJ/g. Lepob/Lepob mice lost 171 ± 9 kJ of stored energy, whereas Lepob/Lepob Mc4r–/– mice lost 104 ± 10 kJ, for a difference of 67 kJ. Lepob/Lepob mice consumed 92 ± 7 kJ, and Lepob/Lepob Mc4r–/– mice consumed 142 ± 12 kJ. The difference of +50 kJ consumed by Lepob/Lepob Mc4r–/– mice accounts for approximately 75% (50/67 kJ) of the extra stored energy lost by Lepob/Lepob mice. If the small reduction of fat-free mass (0.3–0.9 g) with leptin treatment represents a reduction in water retention and is not used in the calculation, the difference attributable to food intake increases to 88%. The remaining balance (7–17 kJ, or 1.4–3.4 kJ/d; 2–6% of normal daily energy expenditure for Lepob/Lepob and Mc4r–/– mice) is presumably due to differences in total energy expenditure and/or substrate oxidation.
Blood chemistries
In experiment 1, serum leptin levels of 1.4 ng/ml (SD, 1.6 ng/ml; range, 0.5–6.2 ng/ml) were observed after leptin infusion, which is within physiological range and compatible with our own unpublished and recently published data for lean mice (29). There was no significant difference in leptin between the three strains. Fasting blood glucose and insulin levels after 14 d of leptin treatment were not significantly different (data not shown). There was also no significant difference in liver weight, triglyceride content, or expression of genes involved in lipogenesis that are increased in the obese, insulin-resistant state (data not shown). Serum-free fatty acid and triglyceride levels were also not significantly different after 14 d of leptin treatment (data not shown).
After the completion of experiment 2, Lepob/Lepob Mc4r–/– and Lepob/Lepob mice were allowed 14 d for body weight and daily food intake to normalize and were then administered a second round of sc injections of either leptin (0.5 mg/kg twice per day) or diluent (0.9% saline) for 2 d (total of four injections). Food intake was significantly reduced by leptin treatment in Lepob/Lepob mice but not Lepob/Lepob Mc4r–/– mice after 24 h (data not shown). After 48 h of injections, however, daily food intake was significantly reduced in both groups by leptin treatment (data not shown).
Serum insulin levels, under basal (saline injection) or leptin treatment conditions, were not significantly different between genotypes, with leptin injections reducing insulin levels by 80% in both Lepob/Lepob Mc4r–/– and Lepob/Lepob mice (Fig. 6A). Blood glucose levels were also not significantly different (Fig. 6B). Serum corticosterone levels were reduced by 50% in Lepob/Lepob Mc4r–/– compared with Lepob/Lepob mice; however, leptin injections still reduced corticosterone levels similarly (40–50%), irrespective of Mc4r genotype (Fig. 6C) (two-way ANOVA; genotype, P < 0.05; treatment, P < 0.05). These data indicate that the regulation of the hypothalamo-pituitary-adrenal axis by leptin is still functional in Lepob/Lepob mice with or without functional MC4Rs.
Discussion
Overall, these findings are consistent with a previous report demonstrating an additive effect on leptin and MC4R insufficiency on body weight (25). Moreover, compared with obese Lepob/Lepob mice, obese Lepob/LepobMc4r–/– mice exhibit a modest resistance to the effects of leptin on food intake but exhibit a normal response in the neuroendocrine systems regulated by leptin (pancreas and adrenal gland). Adrenalectomized Lepob/Lepob Ay/a mice also exhibit an increase in body weight compared with Lepob/Lepob mice, although body composition data were not reported (25). Because impaired MC4R function increases body weight in the absence of leptin, the response of hypothalamic melanocortin neurons to leptin-independent signals regulating energy homeostasis appears to retain a significant function in preventing additional increases in adiposity in Lepob/Lepob mice. Recent data suggest that POMC neurons respond to a milieu of hormonal and neurotransmitter factors, as well as metabolites such as glucose and fatty acids (37, 38). Either alone or in combination, these other inputs into melanocortin neurons appear to prevent additional gains in adiposity of Lepob/Lepob mice.
A second important observation was the reduced efficacy of leptin injections to cause weight loss in Lepob/LepobMc4r–/– mice. This appears to be primarily (70–80%) due to differences of food intake. The differences in weight loss of Lepob/LepobMc4r–/– mice compared with Lepob/Lepob mice that are attributable to metabolism (20–30%) do not appear to involve major defects in the ability of leptin to suppress insulin or corticosterone secretion, at least as determined in response to acute injections. It should also be noted that significant differences in the efficacy of leptin treatment to reduce food intake and weight loss were only observed with leptin injections and not with infusion of leptin by osmotic pump. This discrepancy possibly indicates a resistance of Lepob/LepobMc4r–/– mice to pharmacological doses of leptin that result in large fluctuations in serum concentrations of the hormone, with no significant difference in their response to a stable increase of leptin within a physiological range.
Analysis of double heterozygotes and double knockouts revealed an additive effect of MC4R and leptin insufficiency on fat mass. Food intake and RER of double knockouts was not significantly different from Lepob/Lepob mice, suggesting that increased fat mass was due to an imbalance of energy expenditure. The increased body mass of Lepob/Lepob and Mc4r–/– mice results in a significant 40% increase in 24-h total energy expenditure compared with lean controls, with a net increase of +15.0 ± 2.4 and +15.0 ± 3.0 kJ/d, respectively. This increase in 24-h energy expenditure of severely obese mice is comparable with previous reports (17, 23, 31) and likely represents the greater metabolic demands associated with the large (70–100%) increase in total body mass. When compared with the increase observed for Lepob/Lepob mice (15.0 kJ/d), the increase in 24-h total energy expenditure was attenuated by 50% for Lepob/LepobMc4r+/– and Lepob/LepobMc4r–/– mice (7.5 ± 2.3 and 8.2 ± 3.0 kJ/d, respectively), both having similar body mass and carcass composition. The leptin-independent inputs into melanocortin neurons that prevent additional weight gain of Lepob/Lepob mice might thus act by regulating energy expenditure.
The mechanism explaining the differences in the energy expenditure of Lepob/Lepob mice lacking one or both copies of the Mc4r gene is not clear. One possible candidate explaining differences of energy expenditure is BAT. Both leptin and melanocortin agonists stimulate sympathetic nervous activity in brown adipose depots when administered intracerebroventricularly (39). The increase in BAT thermogenesis in response to increased sympathetic activity involves uncoupling protein 1 (Ucp1), an integral membrane protein found in the inner membrane of mitochondria (40). However, a reduction in BAT thermogenesis also appears to be unlikely, because Lepob/LepobUcp1–/– exhibit no difference in body weight and energy expenditure when compared with Lepob/Lepob mice (41).
The reduced energy expenditure to Lepob/Lepob mice lacking one or both functional Mc4r genes can be interpreted in two ways. The first is that loss of MC4R results in an additional reduction in the energy expenditure in the already hypometabolic Lepob/Lepob mice. In other words, both leptin and MC4Rs contribute to the maintenance of normal energy expenditure, both independently and as components of a common central pathway. An alternative interpretation is that Mc4r–/– and Lepob/Lepob mice are hypermetabolic, and that both leptin and MC4Rs are required for an increase in basal metabolic rate that is associated with severe obesity. The observation that resting energy expenditure adjusted for fat-free mass is significantly increased in Lepob/Lepob mice, and not in Lepob/Lepob mice with only one or no copies of a functional Mc4r gene, is consistent with the second interpretation. It is, however, difficult to reconcile this conclusion with current dogma, as well as the reduction of POMC mRNA and the increased AgRP and NPY gene expression, that is observed in the hypothalamus and that, at least for NPY, stimulate the development of obesity in Lepob/Lepob mice (10, 11, 12, 13).
The results of this study, and from previous experiments using Lepob/Lepob Ay/a mice (25), suggest that MC4R retains a significant function to regulate energy balance in situations of severe obesity and leptin deficiency. In the rat central nervous system, the distribution of AgRP-immunoreactive fibers projecting from the ARC does not completely overlap with that of POMC neurons. In other words, although AgRP mRNA expression is significantly increased in the hypothalamus of leptin-deficient models (12), this might not prevent MSH action in all parts of the central nervous system that regulate energy homeostasis. Moreover, the severe obesity observed in Lepob/Lepob mice is associated with increased levels of cytokines in the circulation that could affect energy expenditure through hypothalamic melanocortin neurons (42, 43). Finally, the observation that hypercorticosteronemia is attenuated in Lepob/Lepob Mc4r–/– mice also suggests a dynamic role for MC4Rs in the metabolic phenotype of Lepob/Lepob mice. Mc4r mRNA has been colocalized with corticotropin-releasing factor mRNA in the paraventricular nucleus, with intracerebroventricular administration of melanotan-II increasing corticotropin-releasing factor mRNA and serum corticosteroid levels (44).
The results of the current study suggest that, although MC4R might have an important role as a downstream effector in the thermogenic and metabolic response to leptin, considerable redundancy exists, allowing leptin to bypass the genetic obstruction due to loss of the Mc4r gene. The regulation of energy balance by leptin involves several hypothalamic neuropeptides, in addition to -MSH and MC4R, to regulate energy balance, including NPY, melanin-concentrating hormone, and galanin-like peptide (16, 45, 46, 47, 48). Any one or all of these neuropeptides might be able to compensate for the loss of -MSH/MC4R in the regulation of energy balance by leptin. In addition, a second melanocortin receptor expressed in the hypothalamus, the melanocortin-3 receptor, is also involved in the regulation of energy balance (49, 50, 51) and might also be able to compensate for loss of MC4R function.
A postnatal surge in leptin during the first week of life is important for the normal development of -MSH- and AgRP-immunoreactive fiber projections from the ARC to the paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamic area (52). Lepob/Lepob mice exhibit a marked reduction in arcuate projections to these areas, with leptin treatment during the neonatal period partially restoring normal development and resulting in a reduction of food intake in adult Lepob/Lepob mice (52). The results of the present study suggest that, despite the reduction in the density of hypothalamic -MSH- and AgRP-immunoreactive fibers projecting from the ARC to paraventricular nucleus, MC4Rs retain a significant function in the prevention of increased adiposity in adult Lepob/Lepob mice.
In summary, these results suggest a complex interaction between leptin and the MC4Rs in energy homeostasis. MC4Rs are required for the full effect of exogenously administered leptin on weight loss, primarily due to impaired inhibition of food intake. Moreover, the results of these experiments using a complete Mc4r knockout support previous data from Lepob/Lepob Ay/a mice suggesting that leptin-independent mechanisms for the regulation of MC4R activity are significant for normal energy homeostasis. The results from the current experiment also suggest that energy expenditure is a possible mechanism involved in preventing additional increases in adiposity of obese hyperphagic leptin-deficient mice by the MC4R.
Acknowledgments
Thanks to M. Josephine Babin, Diana Albarado, Sneha Patel, Emily Fontenot, Emily Meyer, and Dr. Jennifer McClaine for technical assistance and to Profs. Leslie Kozak and Eric Ravussin and Dr. Robert Koza for comments on this manuscript and data analysis. Mc4r–/– mice were kindly provided by Dennis Huszar (Millennium Pharmaceuticals, London, UK) and Roger Cone (Vollum Institute and The Center for the Study of Weight Regulation and Associated Disorders, Oregon Health and Science University, Portland, OR).
Footnotes
This work was supported by grants from the American Diabetes Association, The Pennington Biomedical Research Foundation, The Louisiana Board to Regents, and National Institutes of Health Grant DK068330 (to A.A.B.).
Abbreviations: AgRP, Agouti-related peptide; ARC, arcuate nucleus of the hypothalamus; BAT, brown adipose tissue; Lep, leptin; MC4R, melanocortin-4 receptor; NMR, nuclear magnetic resonance; NPY, neuropeptide Y; ob, obese mutation; POMC, proopiomelanocortin; RER, respiratory exchange ratio; WT, wild type.
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Abstract
Melanocortin-4 receptors (MC4Rs) are involved in the regulation of food intake, sympathetic nervous activity, and adrenal and thyroid function by leptin. The role of MC4Rs in regulating energy balance by leptin was investigated using double heterozygote or homozygous leptin (Lepob) and Mc4r gene mutant mice. Double heterozygous or homozygous mutants were generated by crossing MC4R knockout (Mc4r–/–) mice, backcrossed onto C57BL/6J, with B6.V-Lepob mice. Energy expenditure was measured using indirect calorimetry. The effect of leptin on food intake, weight loss, insulin, and corticosterone was compared for Lepob/LepobMc4r–/– mice and Lepob/Lepob mice. Double heterozygous and homozygous mutants exhibited an additive effect on fat mass. The 2-fold increase in body weight associated with severe obesity of Lepob/Lepob mice was associated with a significantly higher 24 h total and resting energy expenditure. The effect of obesity on energy expenditure was attenuated by 50% in Lepob/Lepob Mc4r+/– and Lepob/Lepob Mc4r–/– mice. Loss of MC4Rs did not affect basal food intake of Lepob/Lepob mice but was associated with partial leptin resistance in terms of food intake and weight loss. Leptin suppression of insulin and corticosterone in Lepob/Lepob mice were not significantly affected by Mc4r genotype. These results suggest a complex interaction between the Lep and Mc4r genes in energy homeostasis and suggest that MC4Rs retain significant anti-obesity function in the obese leptin-deficient state. Increased adiposity with double mutations may involve a reduction in energy expenditure. MC4Rs might have a modest role in the regulation of energy balance by exogenously administered leptin, primarily effecting food intake.
Introduction
LEPTIN, AN ADIPOCYTOKINE produced predominantly by adipose tissue, has been strongly implicated in the regulation of energy homeostasis (1). The obese (ob) mutation in the leptin gene (Lep), described in 1950 (2), is recessive and results in extreme obesity, hypercorticosteronemia, and type II diabetes (2, 3). The Lep gene encodes a 167 amino acid secreted protein that normalizes appetite, reduces body weight, and restores insulin sensitivity of leptin-deficient mice when administered peripherally or intracerebroventricularly (4, 5, 6, 7). The B6.V-Lepob strain carries a CT mutation, converting codon 105 from arginine to a stop codon, resulting in a truncated inactive protein (1). Serum leptin levels fall rapidly with caloric restriction, providing a signal of negative energy balance (3). Lepob/Lepob mice exhibit many of the behavioral and neuroendocrine abnormalities associated with fasting, including reduced gonadal and thyroid function, and increased adrenal activity (3). Most, if not all, of the regulation of food intake and metabolism by leptin are observed when recombinant protein is applied into the third ventricle (6).
In the arcuate nucleus of the hypothalamus (ARC), neurons expressing proopiomelanocortin (POMC) or that coexpress neuropeptide Y (NPY) and agouti-related peptide (AgRP) are involved in the regulation of energy balance by leptin (8). POMC and AgRP/NPY neurons project from the ARC to hypothalamic and extrahypothalamic sites within the brain involved in the regulation of neuroendocrine function, autonomic nervous activity, and ingestive behavior (9). The two groups of neurons have opposing effects on feeding and metabolism. Leptin deficiency is associated with a modest reduction in Pomc expression and an increase in the expression of AgRP and Npy mRNA (10, 11, 12, 13). Mice heterozygous or homozygous for a null Pomc gene and humans with Pomc mutations are obese and hyperphagic (14). Conditional knockout of the long form of the leptin receptor in POMC neurons also results in a modest increase in body weight (15). Conversely, double Lepob/Lepob Npy knockout mice exhibit an attenuated obese phenotype associated with reduced food intake compared with Lepob/Lepob mice (16).
Melanocyte-stimulating hormones (MSHs), derived from the posttranslational processing of POMC, regulate appetite and energy expenditure primarily through the melanocortin-4 receptor (MC4R) (17, 18). The stimulation of renal sympathetic nervous activity by -MSH and by leptin is impaired in heterozygous and abolished in homozygous Mc4r mutant mice (19). MC4Rs might also be involved in the stimulation of brown adipose tissue (BAT) thermogenesis by leptin (20). Mice and humans bearing null mutations of the Mc4r gene or overexpressing agouti, an antagonist of the MC4R, exhibit an obese phenotype correlating with the degree of functional impairment (21, 22). The obese phenotype of Mc4r mutant mice comprises hyperinsulinemia, hyperleptinemia associated with delayed peripheral and hypothalamic leptin resistance, accelerated longitudinal growth, as well as obesity and hyperphagia that is sensitive to dietary fat content (18, 21, 23).
Mice with impaired MC4R action attributable to gene deletion or expression of agouti protein, an antagonist of the melanocortin-1 receptors and MC4Rs, rapidly develop resistance to centrally and peripherally administered leptin (6, 18, 21, 24). Boston et al. (25) reported that leptin sensitivity is restored in lethal yellow (Ay/a) mice when backcrossed onto the B6-V.Lepob strain, with a partial leptin resistance still observed in males. In the same study, overexpression of agouti in adrenalectomized Lepob/Lepob mice had an additive effect on obesity. These results suggest that MC4R regulation of energy balance involves leptin-dependent and -independent functions and that residual MC4R activity functions to prevent additional weight gain of Lepob/Lepob mice. The interpretation of these experiments are confounded, however, by recent observations of MC4R-independent stimulation of adipogenesis by agouti (26, 27). Moreover, intracerebroventricular administration of the potent nonselective melanocortin receptor agonist melanotan-II to Ay/a mice still reduces food intake, mediated through MC4Rs (18), suggest significant retention of MC4R function (28). To further investigate the contribution of MC4Rs to the regulation of energy balance by leptin, we crossed the null Mc4r gene onto the B6-V.Lepob strain. Overall, our results are consistent with MC4Rs being involved in both leptin and leptin-independent pathways in the regulation of energy intake and expenditure.
Materials and Methods
Animal husbandry
The Pennington Biomedical Research Center Institutional Animal Care and Use Committee reviewed and approved these experiments. Mc4r–/– mice backcrossed 12+ generations onto the B6 background were genotyped as described previously (29). Lepob/+ Mc4r+/– heterozygotes were generated by crossing B6.V-Lepob/Lep+ mice (The Jackson Laboratory, Bar Harbor, ME) with Mc4r–/– mice. Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice were then produced by mating Lepob/Lep+ Mc4r+/– mice and genotyped as described previously (29). For the leptin-treatment experiments, male and female Lepob/Lep+ Mc4r–/– mice were crossed to produce Lepob/Lepob Mc4r–/– mice; age-matched Lepob/Lepob controls were purchased from The Jackson Laboratory. Mice were housed on a 12-h light, 12-h dark cycle (lights on, 0600–1800 h), and food intake was measured in mice housed in wire-mesh caging fed low-fat diets (low-fat diet 12450B, 10% kJ/fat, Research Diets, New Brunswick, NJ) at 1000 h and adjusted for spillage. Fat mass and fat-free mass were measured using nuclear magnetic resonance (NMR) (Bruker Mice Minispec NMR Analyzer; Bruker Optics, Billerica, MA) (30). Unless otherwise stated, all data presented are from studies using female mice.
Indirect calorimetry
Indirect calorimetry was performed using a 16 chamber Oxymax system (Columbus Instruments, Columbus, OH), as described previously (29). Chambers were housed in a temperature-controlled incubator set at 28 C. Mice were acclimated 3–7 d to housing in metabolic chambers. Data are presented as total 24-h energy expenditure (in kilojoules per day) or resting energy expenditure (kilojoules per day, mean of the lowest 10% of value assumed to be represent periods with minimal activity). Energy expenditure data were also adjusted for fat-free mass measured by NMR. The respiratory exchange ratio (RER), also known as the respiratory quotient, is the ratio of CO2 produced to O2 consumed. RER is inversely related to fat oxidation and positively related to carbohydrate utilization. That is, an increased RER value is an indicator that the animal is using carbohydrates as the preferred fuel substrate.
Blood chemistries
Blood samples were taken between 1000 and 1200 h from mice that had been fasted for 24 h (experiment 1) or 4 h (experiment 3). Insulin (CrystalChem, Downer’s Grove, IL) and corticosterone (Diagnostic Systems Laboratories, Webster, TX) levels were measured using commercially available assays.
Leptin treatment
Three experiments examining the response of Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/+ mice were performed. In experiment 1, 6-month-old Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice were continuously infused with leptin (10 μg/d; R & D Systems, Minneapolis, MN) using osmotic minipumps (Alzet; Durect, Cupertino, CA). Mice were anesthetized using isoflurane, with pumps soaked in sterile PBS for 4 h, and then implanted beneath the skin on the dorsal surface
In experiment 2, Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/+ mice were treated with leptin, supplied by Dr. A. Parlow (National Hormone and Pituitary Program), administered by ip injection to minimize the stress of surgical implantation of osmotic pumps. At approximately 16 wk of age, mice were treated with two ip injections of leptin per day (0900 and 1700 h) at a dose of 1 mg/kg·d (i.e. 0.5 mg/kg per injection, twice daily) for 5 d, after a 2-d lead-in period whereby 0.9% saline was injected ip at the same times. NMR analysis was performed before and after leptin treatment; food intake and body weight were measured daily throughout the experiment. The response of Lepob/Lepob Mc4r–/– mice to leptin injections was compared with that of female age-matched Lepob/Lepob mice (The Jackson Laboratory).
For the third experiment investigating neuroendocrine hormones, mice from experiment 2 were allowed to recover for 2 weeks, such that body weight and daily food intake had returned to baseline. The mice were then treated with either leptin ip as described for experiment 2 or 0.9% saline, for 2 d. On the final day, food was removed at 0700 h, and animals were given a final dose of leptin or saline at 0900 h and then killed at 1100 h.
Statistics
All data are presented as mean ± SE. Sigmastat software (SPSS, Chicago, IL) was used for statistical analysis. Normality of data distribution was analyzed by Kolmogorov-Smirnov test. Analysis of two or multiple groups of data used either Student’s t test or ANOVA, respectively, for normally distributed data. Food intake data were analyzed using two-way ANOVA (genotype and treatment) with repeated measures (treated and untreated). Least significant difference or Games-Howell post hoc tests were performed after ANOVA analysis for data exhibiting homogeneous or nonhomogeneous variance, respectively. Data were separated into different gender groups before analysis. Pearson correlations were performed for normally distributed data. Statistical significance was assumed for P values < 0.05.
Results
Body weight and food intake of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice
Fat mass was significantly increased in double heterozygotes compared with single heterozygotes and wild-type (WT) mice (genotype effects on fat mass and percentage body fat, P < 0.001; on fat-free mass, not significant) (Fig. 1, A and C). Between 4 and 10 wk of age, double heterozygotes gained 3-fold more fat mass compared with WT littermates and 2- to 2.5-fold more compared with single heterozygotes (Fig. 1, B and D). A 20% increase in fat mass, with no change of fat-free mass, was also observed for female Lepob/LepobMc4r–/– mice compared with Lepob/Lepob controls between 4 and 5 months of age (Fig. 1, E and F) but was not associated with a significant increase in body weight (Fig. 1G). Basal food intake of Lepob/Lepob Mc4r–/– and Lepob/Lepob Mc4r+/– mice was not significantly different from Lepob/Lepob littermates (food intake: Lepob/Lepob, 80 ± 9 kJ/d, n = 7; Lepob/Lepob Mc4r+/–, 78 ± 6 kJ/d, n = 12; Lepob/Lepob Mc4r–/–, 75 ± 7 kJ/d, n = 9).
Energy expenditure and physical activity of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice
Increased fat mass of Lepob/Lepob Mc4r–/– mice in the absence of hyperphagia might indicate reduced fatty acid oxidation and/or energy expenditure. RER was, however, not significantly affected by Mc4r genotype (representative RER data from one of three experiments: Lepob/Lepob, 0.97 ± 0.01; Lepob/Lepob Mc4r+/–, 0.96 ± 0.005; Lepob/Lepob Mc4r–/–, 0.96 ± 0.01; n = 4–6 per group).
The almost 2.0-fold gain in body weight of Lepob/Lepob and Mc4r–/– mice compared with WT mice was associated with a significant increase in total and resting energy expenditure, as reported previously (17, 23, 29, 31) (Fig. 2, A and D). Analysis of the net increase in total or resting energy expenditure, per animal or adjusted for fat-free mass, indicated that the increased energy expenditure associated with severe obesity was attenuated by 50% in Lepob/Lepob Mc4+/– and Lepob/LepobMc4r–/– mice compared with Lepob/Lepob mice (7–8 kJ/d compared with 15 kJ/d) (Table 1). The differences of 24-h energy expenditure of Lepob/LepobMc4r–/– or Lepob/LepobMc4r+/– mice as separate groups were still not significantly different. However, pooling data from Lepob/Lepob mice that were either heterozygous or homozygous for the null Mc4r allele, both of which would be expected to exhibit a phenotype based on studies demonstrating gene-dosage effects of melanocortin mutants on obesity (19, 21, 32) indicated that energy expenditure of Lepob/LepobMc4r(+/– or –/–) mice was significantly (P < 0.05) lower compared with Lepob/Lepob mice.
The interpretation of energy expenditure from lean and obese subjects is difficult because of the heterogeneity in metabolism of tissues and organs (33, 34, 35, 36). Although body weight or estimates of surface area (body weight to the power 0.75) are often used to normalize data, this is not accurate, because neither accounts for variation in body composition. Although not ideal, fat-free mass is the best predictor of resting expenditure in humans (35). For experiments comparing a combination of Lep and Mc4r mutant genes, a similar pattern of changes in energy expenditure was observed whether energy expenditure was expressed per animal (Figs. 2, A and D, 3A) or adjusted for fat-free mass (Fig. 2, B and C, and Table 1). When compared between Lepob/Lepob mice of different Mc4r genotypes, adjustment for body mass also yielded similar results (Fig. 2, E and F, and data not shown). The differences of energy expenditure between Lepob/Lepob Mc4r–/– and Lepob/Lepob mice thus appear to be due to changes in resting or basal metabolic rate and are not due to behavioral differences in spontaneous locomotory movements (Fig. 3, B and C).
Lepob/Lepob mice lacking MC4R are partially resistant to the effects of bi-daily leptin injections on food intake and weight loss
There was no significant difference in food intake of Lepob/Lepob Mc4r–/–, Lepob/Lepob Mc4r+/–, and Lepob/Lepob Mc4r+/+ mice treated with leptin by osmotic pump. In experiment 2, leptin was delivered by bi-daily ip injections (two injections of 0.5 mg/kg) for 5 d to Lepob/Lepob Mc4r–/– mice (n = 6) and Lepob/Lepob mice (n = 7). Injections of approximately 25 μg are likely to produce significantly elevated spikes in circulating leptin levels and should thus be considered as a pharmacological, rather than physiological, method of treatment. Basal food intake of Lepob/Lepob Mc4r–/– mice was not significantly different from Lepob/Lepob controls (Fig. 4, B and C). However, the inhibition of food intake by bi-daily leptin injections was significantly impaired in Lepob/Lepob Mc4r–/– mice compared with Lepob/Lepob controls (Fig. 4), with Lepob/Lepob Mc4r–/– mice consuming approximately 50% more food during 5 d of leptin treatment than Lepob/Lepob mice (142 ± 12 vs. 92 ± 7 kJ). Analysis by two-way ANOVA with repeated measures indicated a significant effect of leptin treatment on food intake (P < 0.001) that was dependent on Mc4r genotype (genotype-treatment interaction, P < 0.05).
Leptin treatment, irrespective of the method of delivery or genotype, resulted in a significant reduction of body weight (Fig. 5, A, C, and D). Weight loss associated with leptin injections was more rapid when compared with leptin infusion (12% in 5 d of injections compared with 9–10% in 14 d for leptin infusion). This might be due to differences in the dose used (10 vs. 45–50 μg/d) or the age of mice (6–7 months for mice implanted with osmotic pump, 3 months for mice given injections). Weight loss of Lepob/Lepob Mc4r–/– during leptin treatment was 60% of that observed for Lepob/Lepob mice; however, the difference was statistically significant for experiment 2 only (Fig. 5, A and C). In experiment 1, weight loss correlated with cumulative food intake over the 14 d of leptin treatment (Fig. 5B). When data from all three groups of experiment 1 was pooled (n = 18), approximately 80% of the difference in weight loss could be explained by differences in food intake (R2 = 0.781; P < 0.001). Similar significant correlations were also observed within each genotype (data not shown). When the data from both groups used for experiment 2 was pooled, differences in cumulative food intake over the 5 d of injection accounted for approximately 70% of the variation in weight loss (R2 = 0.682; P < 0.01).
In experiment 2, weight loss during leptin treatment, and the difference in the response of Lepob/Lepob Mc4r–/– vs. Lepob/Lepob mice, was primarily due to changes in fat mass (Fig. 5D). It was not possible to measure fat mass and fat-free mass of the mice in experiment 1 due to the presence of metal in the osmotic pumps. Fat mass data were used to calculate differences in the amount of stored energy lost for Lepob/Lepob Mc4r–/– vs. Lepob/Lepob mice, with fat mass assumed to contain 37.6 kJ/g and fat-free mass 16.7 kJ/g. Lepob/Lepob mice lost 171 ± 9 kJ of stored energy, whereas Lepob/Lepob Mc4r–/– mice lost 104 ± 10 kJ, for a difference of 67 kJ. Lepob/Lepob mice consumed 92 ± 7 kJ, and Lepob/Lepob Mc4r–/– mice consumed 142 ± 12 kJ. The difference of +50 kJ consumed by Lepob/Lepob Mc4r–/– mice accounts for approximately 75% (50/67 kJ) of the extra stored energy lost by Lepob/Lepob mice. If the small reduction of fat-free mass (0.3–0.9 g) with leptin treatment represents a reduction in water retention and is not used in the calculation, the difference attributable to food intake increases to 88%. The remaining balance (7–17 kJ, or 1.4–3.4 kJ/d; 2–6% of normal daily energy expenditure for Lepob/Lepob and Mc4r–/– mice) is presumably due to differences in total energy expenditure and/or substrate oxidation.
Blood chemistries
In experiment 1, serum leptin levels of 1.4 ng/ml (SD, 1.6 ng/ml; range, 0.5–6.2 ng/ml) were observed after leptin infusion, which is within physiological range and compatible with our own unpublished and recently published data for lean mice (29). There was no significant difference in leptin between the three strains. Fasting blood glucose and insulin levels after 14 d of leptin treatment were not significantly different (data not shown). There was also no significant difference in liver weight, triglyceride content, or expression of genes involved in lipogenesis that are increased in the obese, insulin-resistant state (data not shown). Serum-free fatty acid and triglyceride levels were also not significantly different after 14 d of leptin treatment (data not shown).
After the completion of experiment 2, Lepob/Lepob Mc4r–/– and Lepob/Lepob mice were allowed 14 d for body weight and daily food intake to normalize and were then administered a second round of sc injections of either leptin (0.5 mg/kg twice per day) or diluent (0.9% saline) for 2 d (total of four injections). Food intake was significantly reduced by leptin treatment in Lepob/Lepob mice but not Lepob/Lepob Mc4r–/– mice after 24 h (data not shown). After 48 h of injections, however, daily food intake was significantly reduced in both groups by leptin treatment (data not shown).
Serum insulin levels, under basal (saline injection) or leptin treatment conditions, were not significantly different between genotypes, with leptin injections reducing insulin levels by 80% in both Lepob/Lepob Mc4r–/– and Lepob/Lepob mice (Fig. 6A). Blood glucose levels were also not significantly different (Fig. 6B). Serum corticosterone levels were reduced by 50% in Lepob/Lepob Mc4r–/– compared with Lepob/Lepob mice; however, leptin injections still reduced corticosterone levels similarly (40–50%), irrespective of Mc4r genotype (Fig. 6C) (two-way ANOVA; genotype, P < 0.05; treatment, P < 0.05). These data indicate that the regulation of the hypothalamo-pituitary-adrenal axis by leptin is still functional in Lepob/Lepob mice with or without functional MC4Rs.
Discussion
Overall, these findings are consistent with a previous report demonstrating an additive effect on leptin and MC4R insufficiency on body weight (25). Moreover, compared with obese Lepob/Lepob mice, obese Lepob/LepobMc4r–/– mice exhibit a modest resistance to the effects of leptin on food intake but exhibit a normal response in the neuroendocrine systems regulated by leptin (pancreas and adrenal gland). Adrenalectomized Lepob/Lepob Ay/a mice also exhibit an increase in body weight compared with Lepob/Lepob mice, although body composition data were not reported (25). Because impaired MC4R function increases body weight in the absence of leptin, the response of hypothalamic melanocortin neurons to leptin-independent signals regulating energy homeostasis appears to retain a significant function in preventing additional increases in adiposity in Lepob/Lepob mice. Recent data suggest that POMC neurons respond to a milieu of hormonal and neurotransmitter factors, as well as metabolites such as glucose and fatty acids (37, 38). Either alone or in combination, these other inputs into melanocortin neurons appear to prevent additional gains in adiposity of Lepob/Lepob mice.
A second important observation was the reduced efficacy of leptin injections to cause weight loss in Lepob/LepobMc4r–/– mice. This appears to be primarily (70–80%) due to differences of food intake. The differences in weight loss of Lepob/LepobMc4r–/– mice compared with Lepob/Lepob mice that are attributable to metabolism (20–30%) do not appear to involve major defects in the ability of leptin to suppress insulin or corticosterone secretion, at least as determined in response to acute injections. It should also be noted that significant differences in the efficacy of leptin treatment to reduce food intake and weight loss were only observed with leptin injections and not with infusion of leptin by osmotic pump. This discrepancy possibly indicates a resistance of Lepob/LepobMc4r–/– mice to pharmacological doses of leptin that result in large fluctuations in serum concentrations of the hormone, with no significant difference in their response to a stable increase of leptin within a physiological range.
Analysis of double heterozygotes and double knockouts revealed an additive effect of MC4R and leptin insufficiency on fat mass. Food intake and RER of double knockouts was not significantly different from Lepob/Lepob mice, suggesting that increased fat mass was due to an imbalance of energy expenditure. The increased body mass of Lepob/Lepob and Mc4r–/– mice results in a significant 40% increase in 24-h total energy expenditure compared with lean controls, with a net increase of +15.0 ± 2.4 and +15.0 ± 3.0 kJ/d, respectively. This increase in 24-h energy expenditure of severely obese mice is comparable with previous reports (17, 23, 31) and likely represents the greater metabolic demands associated with the large (70–100%) increase in total body mass. When compared with the increase observed for Lepob/Lepob mice (15.0 kJ/d), the increase in 24-h total energy expenditure was attenuated by 50% for Lepob/LepobMc4r+/– and Lepob/LepobMc4r–/– mice (7.5 ± 2.3 and 8.2 ± 3.0 kJ/d, respectively), both having similar body mass and carcass composition. The leptin-independent inputs into melanocortin neurons that prevent additional weight gain of Lepob/Lepob mice might thus act by regulating energy expenditure.
The mechanism explaining the differences in the energy expenditure of Lepob/Lepob mice lacking one or both copies of the Mc4r gene is not clear. One possible candidate explaining differences of energy expenditure is BAT. Both leptin and melanocortin agonists stimulate sympathetic nervous activity in brown adipose depots when administered intracerebroventricularly (39). The increase in BAT thermogenesis in response to increased sympathetic activity involves uncoupling protein 1 (Ucp1), an integral membrane protein found in the inner membrane of mitochondria (40). However, a reduction in BAT thermogenesis also appears to be unlikely, because Lepob/LepobUcp1–/– exhibit no difference in body weight and energy expenditure when compared with Lepob/Lepob mice (41).
The reduced energy expenditure to Lepob/Lepob mice lacking one or both functional Mc4r genes can be interpreted in two ways. The first is that loss of MC4R results in an additional reduction in the energy expenditure in the already hypometabolic Lepob/Lepob mice. In other words, both leptin and MC4Rs contribute to the maintenance of normal energy expenditure, both independently and as components of a common central pathway. An alternative interpretation is that Mc4r–/– and Lepob/Lepob mice are hypermetabolic, and that both leptin and MC4Rs are required for an increase in basal metabolic rate that is associated with severe obesity. The observation that resting energy expenditure adjusted for fat-free mass is significantly increased in Lepob/Lepob mice, and not in Lepob/Lepob mice with only one or no copies of a functional Mc4r gene, is consistent with the second interpretation. It is, however, difficult to reconcile this conclusion with current dogma, as well as the reduction of POMC mRNA and the increased AgRP and NPY gene expression, that is observed in the hypothalamus and that, at least for NPY, stimulate the development of obesity in Lepob/Lepob mice (10, 11, 12, 13).
The results of this study, and from previous experiments using Lepob/Lepob Ay/a mice (25), suggest that MC4R retains a significant function to regulate energy balance in situations of severe obesity and leptin deficiency. In the rat central nervous system, the distribution of AgRP-immunoreactive fibers projecting from the ARC does not completely overlap with that of POMC neurons. In other words, although AgRP mRNA expression is significantly increased in the hypothalamus of leptin-deficient models (12), this might not prevent MSH action in all parts of the central nervous system that regulate energy homeostasis. Moreover, the severe obesity observed in Lepob/Lepob mice is associated with increased levels of cytokines in the circulation that could affect energy expenditure through hypothalamic melanocortin neurons (42, 43). Finally, the observation that hypercorticosteronemia is attenuated in Lepob/Lepob Mc4r–/– mice also suggests a dynamic role for MC4Rs in the metabolic phenotype of Lepob/Lepob mice. Mc4r mRNA has been colocalized with corticotropin-releasing factor mRNA in the paraventricular nucleus, with intracerebroventricular administration of melanotan-II increasing corticotropin-releasing factor mRNA and serum corticosteroid levels (44).
The results of the current study suggest that, although MC4R might have an important role as a downstream effector in the thermogenic and metabolic response to leptin, considerable redundancy exists, allowing leptin to bypass the genetic obstruction due to loss of the Mc4r gene. The regulation of energy balance by leptin involves several hypothalamic neuropeptides, in addition to -MSH and MC4R, to regulate energy balance, including NPY, melanin-concentrating hormone, and galanin-like peptide (16, 45, 46, 47, 48). Any one or all of these neuropeptides might be able to compensate for the loss of -MSH/MC4R in the regulation of energy balance by leptin. In addition, a second melanocortin receptor expressed in the hypothalamus, the melanocortin-3 receptor, is also involved in the regulation of energy balance (49, 50, 51) and might also be able to compensate for loss of MC4R function.
A postnatal surge in leptin during the first week of life is important for the normal development of -MSH- and AgRP-immunoreactive fiber projections from the ARC to the paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamic area (52). Lepob/Lepob mice exhibit a marked reduction in arcuate projections to these areas, with leptin treatment during the neonatal period partially restoring normal development and resulting in a reduction of food intake in adult Lepob/Lepob mice (52). The results of the present study suggest that, despite the reduction in the density of hypothalamic -MSH- and AgRP-immunoreactive fibers projecting from the ARC to paraventricular nucleus, MC4Rs retain a significant function in the prevention of increased adiposity in adult Lepob/Lepob mice.
In summary, these results suggest a complex interaction between leptin and the MC4Rs in energy homeostasis. MC4Rs are required for the full effect of exogenously administered leptin on weight loss, primarily due to impaired inhibition of food intake. Moreover, the results of these experiments using a complete Mc4r knockout support previous data from Lepob/Lepob Ay/a mice suggesting that leptin-independent mechanisms for the regulation of MC4R activity are significant for normal energy homeostasis. The results from the current experiment also suggest that energy expenditure is a possible mechanism involved in preventing additional increases in adiposity of obese hyperphagic leptin-deficient mice by the MC4R.
Acknowledgments
Thanks to M. Josephine Babin, Diana Albarado, Sneha Patel, Emily Fontenot, Emily Meyer, and Dr. Jennifer McClaine for technical assistance and to Profs. Leslie Kozak and Eric Ravussin and Dr. Robert Koza for comments on this manuscript and data analysis. Mc4r–/– mice were kindly provided by Dennis Huszar (Millennium Pharmaceuticals, London, UK) and Roger Cone (Vollum Institute and The Center for the Study of Weight Regulation and Associated Disorders, Oregon Health and Science University, Portland, OR).
Footnotes
This work was supported by grants from the American Diabetes Association, The Pennington Biomedical Research Foundation, The Louisiana Board to Regents, and National Institutes of Health Grant DK068330 (to A.A.B.).
Abbreviations: AgRP, Agouti-related peptide; ARC, arcuate nucleus of the hypothalamus; BAT, brown adipose tissue; Lep, leptin; MC4R, melanocortin-4 receptor; NMR, nuclear magnetic resonance; NPY, neuropeptide Y; ob, obese mutation; POMC, proopiomelanocortin; RER, respiratory exchange ratio; WT, wild type.
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