Co-occurrence of two partially inactivating polymorphisms of MC3R is associated with pediatric-onset obesity

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.

Figures

FIG. 1

In vitro studies of the MC3R variants. A: Saturation curve of NDP α-MSH binding to mutant or wild-type MC3R in transfected HEK-293 cells (data from four independent experiments, n = 12). B: Scatchard analysis plotted from the data shown in Fig. 1B. C: NDP α-MSH–stimulated β-galactosidase activity in transfected LVIP2.0ZC cells with the variants or wild-type MC3R (data from four independent experiments, n = 12). D: NDP α-MSH–stimulated intracellular cAMP generation in transfected HEK-293 cells with variant or wild-type MC3R (data from three independent experiments, n = 9). Ligand binding studies were carried out in HEK-293 cells 48 h post transfection in 24-well plates. After washing with PBS, cells were treated with 500 μl of binding buffer (Dulbecco’s modified Eagle’s media [DMEM] with 25 mmol/l HEPES, 100 kIU/ml of aprotinin, and 0.1% of BSA), containing ~100,000 cpm of [125I] NDP α-MSH and increasing concentrations of cold NDP α-MSH in the range of 10−10–10−6 mmol/l. After 4 h of incubation at room temperature, cells were washed with ice-cold PBS containing 0.1% BSA and then lysed in 0.4 ml of 0.1N NaOH. For β-galactosidase activity studies, transfected LVIP2.0ZC cells were cultured in 24-well plates and incubated with NDP α-MSH in DMEM with 100 kIU/ml of aprotinin and 0.1% of BSA. After 6 h, cells were washed with PBS and then lysed in 200 μl of 1 × Reporter Lysis Buffer (Promega, Madison, WI). For cAMP measurements, HEK-293 cells cultured in 24-well plates were incubated with NDP α-MSH in the presence of 1 mmol/l 3-Isobutylmethylxanthine (Sigma, St. Louis, MO) in DMEM with 100 kIU/ml aprotinin and 0.1% BSA. After 30 min, cells were washed with PBS and then lysed in 400 μl of 0.1N HCL. HCL extracts were assayed for intracellular cAMP using a direct cAMP enzyme immunosorbent assay kit (Assay Designs, Ann Arbor, MI).

FIG. 2

In vitro studies of MC3R variants tagged with EGFP. A: NDP α-MSH–stimulated β-galactosidase activity in transfected LVIP2.0ZC cells with the Thr6Lys+Val81Ile double mutant (DM) or wild-type MC3R (Wt). B: NDP α-MSH–stimulated intracellular cAMP generation in transfected HEK-293 cells with DM or Wt. C: Localization of the DM and Wt MC3R by confocal imaging; images were captured using a Zeiss 510 confocal microscope equipped with a 488 nm laser for excitation and a LP505 emission filter and a Plan-Apochromat 63×/1.4 oil immersion objective (Carl Zeiss, Jena, Germany). D: Quantitation of MC3R-EGFP protein expression by fluorescence-assisted cell sorting. Representative sorting of MC3R-EGFP signal cells for Wt and DM (top panels) and the average fluorescent intensity from three independent experiments (bottom panel). E: Immunoblotting analysis with a mouse monoclonal anti–green fluorescent antibody (Clontech, Palo Alto, CA) at a dilution of 1:2,000. Representative blot (top panel) and quantitation from three independent experiments (bottom panel).