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CLINICAL CASE SEMINAR |
Department of Medicine, Division of Endocrinology and Diabetes Center, University of California (C.L.-B, C.V.), San Francisco, California 94143-0573; and Institut National de la Santé et de la Recherche Médicale, Unité 561, Department of Pediatric Endocrinology, Saint Vincent de Paul Hospital, University René Descartes (C.L.S., P.B.), Paris, France
Address all correspondence and requests for reprints to: Dr. Christian Vaisse, Diabetes Center, University of California, 513 Parnassus Avenue, Box 0573, San Francisco, California 94143-0573. E-mail: vaisse{at}medicine.ucsf.edu.
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Abstract |
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Introduction |
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MSH and cocaine- and amphetamine-regulated transcript and inhibits orexigenic neurons expressing agouti-related peptide (AGRP) and neuropeptide Y (NPY). MSH and AGRP are both high-affinity ligands of the melanocortin-4 receptor (MC4R), a G protein-coupled receptor expressed in the paraventricular nucleus of the hypothalamus. MC4R transduces signal by coupling to the heterotrimeric Gs protein and activating adenylate cyclase (2), and its activation results in decreased food intake. As an MC4R agonist, MSH stimulates satiety, whereas AGRP acts as an antagonist/inverse agonist of this receptor. Human monogenic forms of obesity linked to mutations in genes involved in the leptin/melanocortin axis demonstrate the essential role of this axis in human energy homeostasis and endocrine function. Homozygous mutations in the leptin or leptin receptor (LEPR) gene cause hyperphagia and a severe early-onset obesity associated with pituitary dysfunction (3, 4). Heterozygous mutations in MC4R are implicated in 2–6% of severe obesity cases, making this the most frequent cause of severe obesity in humans yet described (5, 6, 7, 8, 9).
The relative extent to which MC4R mediates leptin effects has been well studied in rodents. Mice lacking both alleles of Mc4r (Mc4r–/– mice) develop hyperphagia and an obesity that appears later and is less severe than that of mice lacking both leptin (Lep–/–) or LEPR (LepR–/–) alleles. Mc4r–/– mice partially respond to leptin before the onset of obesity. This suggests that additional hypothalamic pathways mediate the early anorectic effects of leptin in mice. Similar experiments also indicate that MC4R does not mediate the positive effects of leptin on reproduction and growth, but might mediate the effects of leptin on the hypothalamo-pituitary-thyroid axis (10, 11).
In humans, the relative magnitude of the melanocortin pathway in mediating the effects of leptin is not known. We describe here a patient with a total loss of MC4R function. Comparison of the longitudinal clinical features of this patient with those of patients bearing null mutations in the leptin axis delineates the relative importance of leptin and MC4R signaling in long-term energy homeostasis and endocrine functions in humans.
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Materials and Methods |
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750–751GA mutation
Mutation screening was performed by direct sequencing of the coding region of MC4R (7) in 109 obese children (mean age ± SD, 12.8 ± 0.8 yr) from a previously described cohort (12). Genotyping of the 750–751GA MC4R mutation for segregation analysis was performed by PCR-restriction fragment length polymorphism (PCR-RFLP) using MC4R-AF-MC4R-XR primers (5) and the PflM1 restriction site created by the 750–751GA mutation. This study was approved by the local ethics committee, and informed consent was obtained from all participating human subjects.
Functional studies of the 750–751GA mutation
Wild-type and 750–751GA MC4Rs were cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA) (7). MSH activation as well as cell surface localization of the truncated MC4R were compared with those of the wild-type receptor as previously described (5).
Clinical and biochemical data
Growth and weight curves were obtained from the medical health records of the subjects. The z-scores were calculated using the 2000 Center for Disease Control data for weight and height (7). Fat-free mass and body fat were determined by bioelectric impedance analysis (Eugenia, Paris, France). Oral glucose tolerance tests were performed by giving 1.75 g glucose/kg body weight (maximum of 75 g) after overnight fasting. Plasma glucose and insulin levels were measured in the fasting state and 120 min after glucose ingestion. Plasma insulin was measured by RIA (12). Serum leptin, ACTH, and TSH were measured by standard immunoassays.
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Results |
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By screening 109 children presenting with a body mass index (BMI) above the 85th percentile before age 6 yr for mutations in MC4R, we detected two heterozygous carriers of the known missense mutations Ser127Leu and Val253Ile as well as one patient carrying a homozygous 2-bp deletion (750–751GA). This mutation leads to a truncation of the receptor at the sixth trans-membrane domain and the addition of 33 unrelated amino acids. Segregation of the mutation 750–751GA in the family of the proband was examined by PCR-RFLP (Fig. 1
). The patient is the third child of first cousins of north African origin. The parents and all siblings of the proband are heterozygous for the 750–751GA mutation.
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750–751GA MC4R mutant results in total loss of function
We evaluated the activity of the 750–751GA MC4R by comparing the MSH-induced cAMP production and the membrane expression of this receptor to those of the wild-type receptor. The mutant receptor does not respond to its endogenous ligand MSH (Fig. 2A) as a result of a total loss of membrane expression (Fig. 2B).
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The patient carrying a homozygous null mutation in MC4R was born at term and had a normal birth weight (3555 g), confirming that MC4R signaling is not required for intrauterine growth. Excessive weight gain started before 6 months of age.
The BMI curve of this patient shows a preserved adiposity rebound (in the absence of any dietary intervention) occurring very early at age 4 yr (Fig. 3). At age 7 yr, the patient’s BMI was 26.57 kg/m2 (z-score = 5.74), and his fat mass was 37.2% of his total body mass. The patient’s leptin level was in the normal range for the increased adiposity at 13 ng/ml or 0.73 ng/kg fat.
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) indicates a clear gene dosage effect of the 750–751GA mutation on the age of onset of obesity. The heterozygous parents were overweight, but not obese, confirming the variable expressivity of heterozygous MC4R mutations (5).
To evaluate the relative effects of lack of leptin or MC4R signaling on weight and growth in humans, we compared this patient with subjects lacking LEPR signaling (4). Despite differences in the genetic backgrounds, BMI curves indicated little difference in the very early onset of obesity, suggesting that lack of MC4R signaling could be sufficient to explain the early-onset obesity seen in LEPR-deficient patients. After age 1 yr, a lesser increase in BMI was observed in the patient lacking MC4R (Fig. 3). Interestingly, this lesser increase in BMI was accounted for not by a difference in weight, but, rather, by a difference in growth. Indeed, the most striking difference between the patient lacking MC4R and the patients lacking LEPR involves linear growth (Table 1). Although LEPR–/– patients showed an early growth deficit, the MC4R–/– patient showed accelerated growth, a feature also found in patients with heterozygous MC4R mutations. This result suggests that in humans the positive effects of leptin on linear growth are not mediated through the melanocortin pathway.
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It was recently suggested that heterozygous MC4R deficiency in subjects younger than 10 yr is associated with higher fasting insulin levels compared with those in similarly obese controls (8). We measured glucose, insulin, and C peptide levels during an oral glucose tolerance test in our 7-yr-old patient and in 39 sex-, age-, and BMI-matched controls with no MC4R mutation. Fasting and oral glucose tolerance test insulin levels were not significantly elevated in the MC4R-deficient subject (Table 2).
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Studies in rodents suggest that leptin controls the thyroid axis through the melanocortin pathway and MC4R activity (11). LEPR-deficient patients present with significant central hypothyroidism (decreased free T4 and TSH) (4), and alteration of the thyroid axis has been described for a leptin-deficient patient (3). Exploration of the thyroid axis in the homozygous MC4R-deficient patient indicates that lack of MC4R signaling does not affect the thyroid axis under basal conditions. Although we cannot exclude that dynamic tests of this axis could unmask subtle defects, our results suggest that the effect of leptin on the thyroid axis in humans is mediated through a pathway other than the melanocortin pathway.
Finally, repeated assessment of the hypothalamic-pituitary-adrenal axis did not show any anomaly (Table 3), and lipid metabolism was unremarkable (cholesterol, 4.5 mmol/liter; triglycerides, 1.36 mmol/liter) in the homozygous MC4R 750–751GA patient.
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Discussion |
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750–751GA MC4R mutation carrier described in this study is the first patient to present with a demonstrated absolute lack of residual MC4R signaling and for whom comprehensive longitudinal anthropometric data as well as biochemical measurements are available. Experiments in rodents suggest that the repression of food intake of leptin occurs through at least two independent mechanisms: activation of the anorexigenic melanocortin pathway and repression of the orexigenic NPY pathway. Indeed, genetic ablation of NPY decreases the obesity of leptin-deficient mice (13), and null mutations in the MC4R in rodents cause a later onset and less severe obesity than null mutations in leptin or LEPR (10). In contrast, MC4R- and LEPR-deficient patients present with a similar very early-onset increase in weight. This observation suggests that in humans the full extent of the effect of leptin on body weight could be accounted for by activation of the MC4R pathway.
Recent genetic experiments in mice also suggest that independent signaling pathways mediate the effect of leptin on appetite and growth, the first being mediated by LepR/Stat3 activation and melanocortin signaling through MC4R, and the second by NPY signaling (14). The striking difference observed between the effects of LEPR and MC4R deficiency on growth in patients suggests that the same pathway duality exists in humans and that leptin’s effects on growth are not mediated through melanocortin activation of the MC4R.
Finally, although glucose homeostasis could be affected later in life in our patient, our observation that complete abolition of MC4R signaling does not cause hyperinsulinemia suggests a more complex role for central melanocortin signaling in human glucose homeostasis and demonstrates that fasting hyperinsulinemia is not a clinical criteria suitable for the detection of MC4R-deficient patients.
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Footnotes |
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Abbreviations: AGRP, Agouti-related peptide; BMI, body mass index; LEPR, leptin receptor; MC4R, melanocortin-4 receptor; NPY, neuropeptide Y; RFLP, restriction fragment length polymorphism.
Received November 17, 2003.
Accepted February 3, 2004.
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References |
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-melanocyte-stimulating hormone genes in severely obese children. J Pediatr 139:204–209[CrossRef][Medline]
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30; children with BMI 
BMI <30). Age, BMI (kilograms per meter squared), z-score for BMI, and genotype are indicated for each individual when available. The proband IV:3 is indicated with an arrow. Genotyping was performed by PCR-RFLP on individuals III:6, IV:1, IV:2, IV:3, and IV:4 as described in Materials and Methods. MWM, Molecular weight marker; 
