Co-occurrence of Two Partially Inactivating Polymorphisms of MC3R Is Associated With Pediatric-Onset Obesity
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糖尿病学杂志 2005年第9期
1 Unit on Growth and Obesity, National Institutes of Health, Bethesda, Maryland
2 Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Development, Rockville, Maryland
3 Pediatric Department, University of Insubria, Varese, Italy
4 Nutrition Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Both human linkage studies and MC3R knockout mouse models suggest that the MC3R may play an important role in energy homeostasis. Here we show that among 355 overweight and nonoverweight children, 8.2% were double homozygous for a pair of missense MC3R sequence variants (Thr6Lys and Val81Ile). Such children were significantly heavier (BMI and BMI SD score: P < 0.0001), had more body fat (body fat mass and percentage fat mass: P < 0.001), and had greater plasma leptin (P < 0.0001) and insulin concentrations (P < 0.001) and greater insulin resistance (P < 0.008) than wild-type or heterozygous children. Both sequence variants were more common in African-American than Caucasian children. In vitro expression studies found the double mutant MC3R was partially inactive, with significantly fewer receptor binding sites, decreased signal transduction, and less protein expression. We conclude that diminished MC3R expression in this double MC3R variant may be a predisposing factor for excessive body weight gain in children.
Both human linkage studies and knockout mouse models suggest that MC3R (the melanocortin 3 receptor gene) plays an important role in energy homeostasis (1eC5). Mice in whom both alleles of the MC3R have been inactivated have greater fat mass and reduced lean mass, while heterozygotes have normal body composition (3). Three human coding sequence variants have been reported in the MC3R (6eC10): a heterozygous missense sequence variant Ile183Asn (I183N) that inactivates MC3R function (11,12), identified in two individuals (10), and two other variants, Thr6Lys and Val81Ile, that individually do not affect signal transduction (12) and have been reported not to be associated with body weight (6eC9). Because most single gene mutations affecting body weight become manifest during childhood, we performed an MC3R mutation screening and association analysis in a large cohort of African-American and Caucasian overweight and normal weight children and then performed functional studies for the sequence variants that were identified.
RESEARCH DESIGN AND METHODS
A total of 355 children aged 5eC18 years (mean age 10.7 ± 3.1 years) were recruited from the community for metabolic studies at the National Institutes of Health. By design, the study population was enriched for obesity: 190 were overweight, with BMI 95th percentile for age and sex, and 165 were nonoverweight (BMI 5th to 94th percentiles). No subject had significant medical illness, and none were taking medications known to affect body weight. The National Institute of Child Health and Human Development institutional review board approved the clinical protocol. Signed consent and assent forms were obtained from parents and children.
Phenotypic data.
BMI-SD score, body adiposity, and body circumferences as well as metabolic parameters were obtained as previously described (13eC15). The homeostasis model assessment for insulin resistance index (HOMA-IR) (16) was estimated using the formula HOMA-IR = [fasting insulin (e蘒/ml) x fasting glucose (mmol/l)]/22.5.
Mutation screening and genotyping.
After PCR amplification of genomic DNA, we directly sequenced the human MC3R (L06155), including a 336 bp of proximal promoter and the entire coding region, in 47 extremely overweight (BMI 41.8 ± 10.5 kg/m2) and 31 lean (BMI 16.8 ± 2.2 kg/m2) children. Restriction enzyme fragment length polymorphism analysis of identified variants was used for genotyping the entire cohort.
Construction and expression of MC3R.
Wild type, Thr6Lys, and Val81Ile single mutants and Thr6Lys+Val81Ile double mutant MC3R (i.e., MC3R sequence altered to introduce both mutations into the same gene) were constructed in pcDNA3.1/V5-His TOPO vectors (Invitrogen, Carlsbad, CA) without expressing the V5 or His tag. Wild type and the Thr6Lys+Val81Ile double mutant were also constructed with a C-terminus enhanced green fluorescence protein (EGFP) vector, pEGFP-N2 (BD Biosciences, San Jose, CA). Constructs were transiently expressed in HEK-293 or LVIP2.0Zc cells. LVIP2.0Zc is a reporter cell line containing -galactosidase driven by a CRE-dependent promoter (17). Transfections were performed in Opti-MEM I Reduced Serum Medium (Invitrogen, Carlsbad, CA) using Lipofectamine Plus reagent (Invitrogen).
Receptor binding assay.
Ligand binding studies were carried out in HEK-293 cells exposed to 100,000 cpm of [125I] (Nle4,D-Phe7) -melanocyte stimulating hormone (NDP -MSH) (Amersham, Piscataway, NJ) and increasing concentrations of cold NDP -MSH (Peninsula, San Carlos, CA) in the range of 10eC10eC10eC6 mmol/l. After 4 h of incubation at room temperature, membrane-bound counts per minute were determined in a gamma counter.
-Galactosidase activity measurement and cAMP assay.
Ligand-stimulated receptor activity was determined by -galactosidase activity in transfected LVIP2.0Zc reporter cells. -galactosidase activity was measured using a -galactosidase enzyme assay system (Promega, Madison WI). Ligand-stimulated intracellular cAMP accumulation was also directly measured in transfected HEK293 cells incubated with NDP -MSH in the presence of 1 mmol/l 3-Isobutylmethylxanthine (Sigma, St. Louis, MO).
Confocal imaging, fluorescence-assisted cell sorting (FACS) and Western analysis.
EGFP-tagged wild-type and double mutant MC3R were transiently transfected in HEK293 cells and studied 48 h posttransfection. For confocal imaging, cells were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature and washed with PBS. Images were captured using a Zeiss 510 confocal microscope. For FACS, trypsin-treated cells were pelletted by centrifugation and then resuspended in 1x PBS to a concentration of 1 x 106 cells/ml. Sorting data were collected with a FACS calibur flow cytometer and analyzed with Cell Quest software (Becton Dickinson, San Jose, CA). For Western analysis, whole cell lysates were separated on 10eC20% SDS-PAGE gels, electrotransferred onto nitrocellulose membranes, and immunoblotted with a mouse monoclonal antieCgreen fluorescent protein antibody (Clontech, Palo Alto, CA).
Data analysis.
Comparisons of the phenotypes by genotype were evaluated by using ANCOVA, accounting for age, height, race, and sex. Because of the multiple comparisons performed, a more conservative P < 0.0125 was accepted as significant for ANCOVAs. GraphPad Prism 4.0 (GraphPad Software, San Diego, CA) was used for calculation of Scatchard analysis data.
RESULTS
By sequencing, we found two previously identified missense sequence variants: Thr6Lys and Val81Ile. Among the 355 children studied, the two variants showed marked linkage disequilibrium (x2 = 522.4, P < 0.0001). Therefore, the genotypes were categorized based on combination of the two variants (Table 1). African-American children were more likely to carry the variant alleles (P < 0.0001 vs. Caucasians): 15.8% (vs. 1.7% of Caucasians) were homozygous for both polymorphisms. Overweight children had higher frequencies of the variants than normal weight children (x2 = 21.31, P < 0.001). Children homozygous for both variants were identified only among children who were at risk for overweight (BMI 85th percentile, 7.9%) or who were considered overweight (BMI 95th percentile, 13.7%).
Children who were homozygous for both MC3R variants had significantly greater BMI-SD score, fat mass, body circumference measurements, insulin, and leptin compared with wild-type or heterozygous children (Table 2). Analysis by race showed greater BMI-SD score and dual-energy X-ray absorptiometry body fat mass in African-American children who were double-homozygous for the two MC3R alleles. There were only three Caucasian children who were double-homozygous for the variants; trends of associations were also found between body composition and the MC3R variants in Caucasians. MC4R was also sequenced for each child who was double homozygous for these MC3R variants (data not shown). No function-altering mutations were found.
To study the functional consequences of these MC3R variants, we studied ligand binding and melanocyte-stimulating hormone (MSH)-stimulated cAMP generation of single and double mutant MC3R variants. By Northern analysis (data not shown), mRNA expression was similar for wild-type and mutant receptors. Saturation curves for binding of 125I NDP -MSH (Fig. 1A) suggested the double mutant Thr6Lys and Val81Ile MC3R bound 60% less [125I] NDP -MSH than the wild-type MC3R (P < 0.001). Scatchard analysis revealed a similar binding affinity for variant MC3Rs (Fig. 1B). However, the level of surface expression, as reflected by estimated Bmax, was significantly lower for the double mutant MC3R (56.9 pmol/l) compared with the wild-type MC3R (137.7 pmol/l). Functional activity of the mutants was in accord with their cell surface expression: the Val81Ile and Thr6Lys single mutant MC3Rs exhibited a dose-dependent pattern of response to NDP-MSH not significantly different from wild-type MC3R. However, the Val81Ile+Thr6Lys double mutant MC3R, studied using two cAMP measurement systems (Fig. 1C and D), showed significantly reduced intracellular cAMP generation. Consistent with the receptor affinity data, the half-maximal effective NDP -MSH concentration to stimulate cAMP was similar for all constructs (Val81Ile+Thr6Lys 0.11 ± 0.15 vs. wild type 0.14 ± 0.13 nmol/l).
To study protein expression and localization of mutant MC3Rs, we studied MC3R variants tagged with EGFP. Similar to the untagged receptors, cAMP generation was markedly reduced in the EGFP-tagged double mutant MC3R (Figs. 2A and B). By confocal microscopy, both wild-type and double mutant EGFP-tagged MC3Rs were expressed in the plasma membrane (Fig. 2C). However, by FACS (Fig. 2D), the double mutant had significantly less total EGFP signal, suggesting a reduced receptor protein content. This was confirmed by Western blot analysis of the EGFP-tagged receptor (Fig. 2E).
DISCUSSION
We found that the co-occurrence of two previously identified variants in MC3R was associated with impaired cAMP generation in vitro and with greater BMI, greater body fat mass, and higher plasma levels of insulin and leptin in African-American and Caucasian children. To our knowledge, although others have examined the relationships with BMI (6eC9) and the functional consequences (8,12) of these two polymorphisms separately, the present report is the first investigation to study body composition for those who are double homozygotes and to study signal transduction in the double variant MC3R. The association we found between high body weight and double homozygosity in children is consistent with findings that both MC3R alleles must be impaired for increased body adiposity to be observed in MC3R knockout animal models. The failure to observe a relationship between these variants and BMI in prior studies may be due to the fact that large clinical samples enriched with double homozygotes have not previously been examined. One study (6) of 24 obese adults and 27 control subjects reported 12% of African-American women were homozygous for these polymorphisms but failed to find an association with body weight. It is possible that insufficient numbers of African Americans were previously studied to show the impact of the presence of both MC3R variants or, since some nonobese adults are reported to be double homozygotes (6), that these variants are more associated with the development of overweight during childhood than adulthood. It is also theoretically possible that the strong association observed in the present study could be due to linkage disequilibrium with another nearby gene locus on chromosome 20 or could have occurred by chance.
Thr6Lys and Val81Ile are found, respectively, in the NH2-terminal extracellular part and the first transmembrane helix of MC3R protein. Mutations in either location might be predicted to affect melanocortin receptor function, especially the first transmembrane region that is believed to be involved in the binding of melanocortin peptides (18). The complete binding curves in our study show that the double mutant had significantly less total ligand binding capacity in spite of preserved binding affinity, similar to findings with some MC4R variants reported to be associated with obesity (19). The reduced binding in our experiments was consistent with the decreased MSH-stimulated cAMP production by the double mutant receptor, shown by direct cAMP measurements and indirectly using cAMP-responsive reporter cells. The mechanism of the lower activity of the double mutant MC3R appears to involve less receptor protein expression, as suggested by both FACS and Western blot analysis. The lower receptor protein levels for this mutant did not depend on transfection efficiency or mRNA levels (data not shown) but appears due to posttranslational events. It remains unclear whether the lower protein expression is due to decreased synthesis or increased degradation. Although the double mutation led to lower protein expression, membrane localization of this receptor appeared to be similar to that of the wild-type receptor by confocal microscopy; thus, intracellular retention, hypothesized to be the cause of the inactivity of some MC4R variants (20), appears unlikely.
The mechanism for increased adiposity among MC3R-deficient mice appears to be increased feeding efficiency, such that the same energy intake results in greater storage of calories as fat (11). Further studies should examine whether co-occurrence of these MC3R polymorphisms affects energy balance or efficiency.
Limitations of the present study include evaluation of only one cohort of children and the small number of Caucasians homozygous for these variants. The limitations of in vitro assays for receptor function should also be noted, as results potentially may differ in vivo. Finally, receptor function was evaluated only using an analog of -MSH and not with -MSH, which is also a potent agonist for the MC3R (21). However, prior studies do not suggest marked differences in response of the MC3R to - versus -MSH (22).
In summary, double homozygocity for MC3R sequence variants Thr6Lys and Val81Ile is particularly prevalent in African-American children and is associated with adiposity and insulin resistance in African-American and Caucasian children, and in vitro partially inactivates the MC3R. Double homozygocity for this MC3R mutant may contribute to the greater prevalence of overweight and insulin resistance in African Americans.
ACKNOWLEDGMENTS
This study was funded by grant HD-000641 from the National Institute of Child Health and Human Development, National Institutes of Health (J.A.Y.) and the National Center on Minority Health and Health Disparities, National Institutes of Health (J.A.Y.).
J.A.Y. and N.G.S. are commissioned officers in the U.S. Public Health Service, Department of Health and Human Services.
DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescence protein; FACS, fluorescence-assisted cell sorting; MSH, melanocyte-stimulating hormone; NDP -MSH, (Nle4,D-Phe7) -melanocyte-stimulating hormone
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2 Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Development, Rockville, Maryland
3 Pediatric Department, University of Insubria, Varese, Italy
4 Nutrition Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Both human linkage studies and MC3R knockout mouse models suggest that the MC3R may play an important role in energy homeostasis. Here we show that among 355 overweight and nonoverweight children, 8.2% were double homozygous for a pair of missense MC3R sequence variants (Thr6Lys and Val81Ile). Such children were significantly heavier (BMI and BMI SD score: P < 0.0001), had more body fat (body fat mass and percentage fat mass: P < 0.001), and had greater plasma leptin (P < 0.0001) and insulin concentrations (P < 0.001) and greater insulin resistance (P < 0.008) than wild-type or heterozygous children. Both sequence variants were more common in African-American than Caucasian children. In vitro expression studies found the double mutant MC3R was partially inactive, with significantly fewer receptor binding sites, decreased signal transduction, and less protein expression. We conclude that diminished MC3R expression in this double MC3R variant may be a predisposing factor for excessive body weight gain in children.
Both human linkage studies and knockout mouse models suggest that MC3R (the melanocortin 3 receptor gene) plays an important role in energy homeostasis (1eC5). Mice in whom both alleles of the MC3R have been inactivated have greater fat mass and reduced lean mass, while heterozygotes have normal body composition (3). Three human coding sequence variants have been reported in the MC3R (6eC10): a heterozygous missense sequence variant Ile183Asn (I183N) that inactivates MC3R function (11,12), identified in two individuals (10), and two other variants, Thr6Lys and Val81Ile, that individually do not affect signal transduction (12) and have been reported not to be associated with body weight (6eC9). Because most single gene mutations affecting body weight become manifest during childhood, we performed an MC3R mutation screening and association analysis in a large cohort of African-American and Caucasian overweight and normal weight children and then performed functional studies for the sequence variants that were identified.
RESEARCH DESIGN AND METHODS
A total of 355 children aged 5eC18 years (mean age 10.7 ± 3.1 years) were recruited from the community for metabolic studies at the National Institutes of Health. By design, the study population was enriched for obesity: 190 were overweight, with BMI 95th percentile for age and sex, and 165 were nonoverweight (BMI 5th to 94th percentiles). No subject had significant medical illness, and none were taking medications known to affect body weight. The National Institute of Child Health and Human Development institutional review board approved the clinical protocol. Signed consent and assent forms were obtained from parents and children.
Phenotypic data.
BMI-SD score, body adiposity, and body circumferences as well as metabolic parameters were obtained as previously described (13eC15). The homeostasis model assessment for insulin resistance index (HOMA-IR) (16) was estimated using the formula HOMA-IR = [fasting insulin (e蘒/ml) x fasting glucose (mmol/l)]/22.5.
Mutation screening and genotyping.
After PCR amplification of genomic DNA, we directly sequenced the human MC3R (L06155), including a 336 bp of proximal promoter and the entire coding region, in 47 extremely overweight (BMI 41.8 ± 10.5 kg/m2) and 31 lean (BMI 16.8 ± 2.2 kg/m2) children. Restriction enzyme fragment length polymorphism analysis of identified variants was used for genotyping the entire cohort.
Construction and expression of MC3R.
Wild type, Thr6Lys, and Val81Ile single mutants and Thr6Lys+Val81Ile double mutant MC3R (i.e., MC3R sequence altered to introduce both mutations into the same gene) were constructed in pcDNA3.1/V5-His TOPO vectors (Invitrogen, Carlsbad, CA) without expressing the V5 or His tag. Wild type and the Thr6Lys+Val81Ile double mutant were also constructed with a C-terminus enhanced green fluorescence protein (EGFP) vector, pEGFP-N2 (BD Biosciences, San Jose, CA). Constructs were transiently expressed in HEK-293 or LVIP2.0Zc cells. LVIP2.0Zc is a reporter cell line containing -galactosidase driven by a CRE-dependent promoter (17). Transfections were performed in Opti-MEM I Reduced Serum Medium (Invitrogen, Carlsbad, CA) using Lipofectamine Plus reagent (Invitrogen).
Receptor binding assay.
Ligand binding studies were carried out in HEK-293 cells exposed to 100,000 cpm of [125I] (Nle4,D-Phe7) -melanocyte stimulating hormone (NDP -MSH) (Amersham, Piscataway, NJ) and increasing concentrations of cold NDP -MSH (Peninsula, San Carlos, CA) in the range of 10eC10eC10eC6 mmol/l. After 4 h of incubation at room temperature, membrane-bound counts per minute were determined in a gamma counter.
-Galactosidase activity measurement and cAMP assay.
Ligand-stimulated receptor activity was determined by -galactosidase activity in transfected LVIP2.0Zc reporter cells. -galactosidase activity was measured using a -galactosidase enzyme assay system (Promega, Madison WI). Ligand-stimulated intracellular cAMP accumulation was also directly measured in transfected HEK293 cells incubated with NDP -MSH in the presence of 1 mmol/l 3-Isobutylmethylxanthine (Sigma, St. Louis, MO).
Confocal imaging, fluorescence-assisted cell sorting (FACS) and Western analysis.
EGFP-tagged wild-type and double mutant MC3R were transiently transfected in HEK293 cells and studied 48 h posttransfection. For confocal imaging, cells were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature and washed with PBS. Images were captured using a Zeiss 510 confocal microscope. For FACS, trypsin-treated cells were pelletted by centrifugation and then resuspended in 1x PBS to a concentration of 1 x 106 cells/ml. Sorting data were collected with a FACS calibur flow cytometer and analyzed with Cell Quest software (Becton Dickinson, San Jose, CA). For Western analysis, whole cell lysates were separated on 10eC20% SDS-PAGE gels, electrotransferred onto nitrocellulose membranes, and immunoblotted with a mouse monoclonal antieCgreen fluorescent protein antibody (Clontech, Palo Alto, CA).
Data analysis.
Comparisons of the phenotypes by genotype were evaluated by using ANCOVA, accounting for age, height, race, and sex. Because of the multiple comparisons performed, a more conservative P < 0.0125 was accepted as significant for ANCOVAs. GraphPad Prism 4.0 (GraphPad Software, San Diego, CA) was used for calculation of Scatchard analysis data.
RESULTS
By sequencing, we found two previously identified missense sequence variants: Thr6Lys and Val81Ile. Among the 355 children studied, the two variants showed marked linkage disequilibrium (x2 = 522.4, P < 0.0001). Therefore, the genotypes were categorized based on combination of the two variants (Table 1). African-American children were more likely to carry the variant alleles (P < 0.0001 vs. Caucasians): 15.8% (vs. 1.7% of Caucasians) were homozygous for both polymorphisms. Overweight children had higher frequencies of the variants than normal weight children (x2 = 21.31, P < 0.001). Children homozygous for both variants were identified only among children who were at risk for overweight (BMI 85th percentile, 7.9%) or who were considered overweight (BMI 95th percentile, 13.7%).
Children who were homozygous for both MC3R variants had significantly greater BMI-SD score, fat mass, body circumference measurements, insulin, and leptin compared with wild-type or heterozygous children (Table 2). Analysis by race showed greater BMI-SD score and dual-energy X-ray absorptiometry body fat mass in African-American children who were double-homozygous for the two MC3R alleles. There were only three Caucasian children who were double-homozygous for the variants; trends of associations were also found between body composition and the MC3R variants in Caucasians. MC4R was also sequenced for each child who was double homozygous for these MC3R variants (data not shown). No function-altering mutations were found.
To study the functional consequences of these MC3R variants, we studied ligand binding and melanocyte-stimulating hormone (MSH)-stimulated cAMP generation of single and double mutant MC3R variants. By Northern analysis (data not shown), mRNA expression was similar for wild-type and mutant receptors. Saturation curves for binding of 125I NDP -MSH (Fig. 1A) suggested the double mutant Thr6Lys and Val81Ile MC3R bound 60% less [125I] NDP -MSH than the wild-type MC3R (P < 0.001). Scatchard analysis revealed a similar binding affinity for variant MC3Rs (Fig. 1B). However, the level of surface expression, as reflected by estimated Bmax, was significantly lower for the double mutant MC3R (56.9 pmol/l) compared with the wild-type MC3R (137.7 pmol/l). Functional activity of the mutants was in accord with their cell surface expression: the Val81Ile and Thr6Lys single mutant MC3Rs exhibited a dose-dependent pattern of response to NDP-MSH not significantly different from wild-type MC3R. However, the Val81Ile+Thr6Lys double mutant MC3R, studied using two cAMP measurement systems (Fig. 1C and D), showed significantly reduced intracellular cAMP generation. Consistent with the receptor affinity data, the half-maximal effective NDP -MSH concentration to stimulate cAMP was similar for all constructs (Val81Ile+Thr6Lys 0.11 ± 0.15 vs. wild type 0.14 ± 0.13 nmol/l).
To study protein expression and localization of mutant MC3Rs, we studied MC3R variants tagged with EGFP. Similar to the untagged receptors, cAMP generation was markedly reduced in the EGFP-tagged double mutant MC3R (Figs. 2A and B). By confocal microscopy, both wild-type and double mutant EGFP-tagged MC3Rs were expressed in the plasma membrane (Fig. 2C). However, by FACS (Fig. 2D), the double mutant had significantly less total EGFP signal, suggesting a reduced receptor protein content. This was confirmed by Western blot analysis of the EGFP-tagged receptor (Fig. 2E).
DISCUSSION
We found that the co-occurrence of two previously identified variants in MC3R was associated with impaired cAMP generation in vitro and with greater BMI, greater body fat mass, and higher plasma levels of insulin and leptin in African-American and Caucasian children. To our knowledge, although others have examined the relationships with BMI (6eC9) and the functional consequences (8,12) of these two polymorphisms separately, the present report is the first investigation to study body composition for those who are double homozygotes and to study signal transduction in the double variant MC3R. The association we found between high body weight and double homozygosity in children is consistent with findings that both MC3R alleles must be impaired for increased body adiposity to be observed in MC3R knockout animal models. The failure to observe a relationship between these variants and BMI in prior studies may be due to the fact that large clinical samples enriched with double homozygotes have not previously been examined. One study (6) of 24 obese adults and 27 control subjects reported 12% of African-American women were homozygous for these polymorphisms but failed to find an association with body weight. It is possible that insufficient numbers of African Americans were previously studied to show the impact of the presence of both MC3R variants or, since some nonobese adults are reported to be double homozygotes (6), that these variants are more associated with the development of overweight during childhood than adulthood. It is also theoretically possible that the strong association observed in the present study could be due to linkage disequilibrium with another nearby gene locus on chromosome 20 or could have occurred by chance.
Thr6Lys and Val81Ile are found, respectively, in the NH2-terminal extracellular part and the first transmembrane helix of MC3R protein. Mutations in either location might be predicted to affect melanocortin receptor function, especially the first transmembrane region that is believed to be involved in the binding of melanocortin peptides (18). The complete binding curves in our study show that the double mutant had significantly less total ligand binding capacity in spite of preserved binding affinity, similar to findings with some MC4R variants reported to be associated with obesity (19). The reduced binding in our experiments was consistent with the decreased MSH-stimulated cAMP production by the double mutant receptor, shown by direct cAMP measurements and indirectly using cAMP-responsive reporter cells. The mechanism of the lower activity of the double mutant MC3R appears to involve less receptor protein expression, as suggested by both FACS and Western blot analysis. The lower receptor protein levels for this mutant did not depend on transfection efficiency or mRNA levels (data not shown) but appears due to posttranslational events. It remains unclear whether the lower protein expression is due to decreased synthesis or increased degradation. Although the double mutation led to lower protein expression, membrane localization of this receptor appeared to be similar to that of the wild-type receptor by confocal microscopy; thus, intracellular retention, hypothesized to be the cause of the inactivity of some MC4R variants (20), appears unlikely.
The mechanism for increased adiposity among MC3R-deficient mice appears to be increased feeding efficiency, such that the same energy intake results in greater storage of calories as fat (11). Further studies should examine whether co-occurrence of these MC3R polymorphisms affects energy balance or efficiency.
Limitations of the present study include evaluation of only one cohort of children and the small number of Caucasians homozygous for these variants. The limitations of in vitro assays for receptor function should also be noted, as results potentially may differ in vivo. Finally, receptor function was evaluated only using an analog of -MSH and not with -MSH, which is also a potent agonist for the MC3R (21). However, prior studies do not suggest marked differences in response of the MC3R to - versus -MSH (22).
In summary, double homozygocity for MC3R sequence variants Thr6Lys and Val81Ile is particularly prevalent in African-American children and is associated with adiposity and insulin resistance in African-American and Caucasian children, and in vitro partially inactivates the MC3R. Double homozygocity for this MC3R mutant may contribute to the greater prevalence of overweight and insulin resistance in African Americans.
ACKNOWLEDGMENTS
This study was funded by grant HD-000641 from the National Institute of Child Health and Human Development, National Institutes of Health (J.A.Y.) and the National Center on Minority Health and Health Disparities, National Institutes of Health (J.A.Y.).
J.A.Y. and N.G.S. are commissioned officers in the U.S. Public Health Service, Department of Health and Human Services.
DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescence protein; FACS, fluorescence-assisted cell sorting; MSH, melanocyte-stimulating hormone; NDP -MSH, (Nle4,D-Phe7) -melanocyte-stimulating hormone
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