DIABETES, VOL. 49, AUGUST 2000 1347 Soluble Leptin Receptor in Serum of Subjects With Complete Resistance to Leptin Relation to Fat Mass Najiba Lahlou, Karine Clement, Jean-Claude Carel, Christian Vaisse, Chantal Lotton, Yvette Le Bihan, Arnaud Basdevant, Yves Lebouc, Philippe Froguel, Marc Roger, and Bernard Guy-Grand Leptin resistance and obesity have been related to muta- tions of the leptin receptor gene in rodents and, recently, in a consanguineous family. The latter mutation results in a receptor lacking transmembrane and intracellular domains. Homozygous and heterozygous individuals with this mutation had serum leptin levels higher than expected, given their BMIs: 600, 670, and 526 ng/ml and 145, 362, 294, 240, and 212 ng/ml, respectively. Their serum leptin was fractionated by gel filtration: >80% was present as a high–molecular size complex vs. 7.5% in the nonmutated sister. Western blot analysis showed a band at 146 kDa reacting specifically with an antibody directed against the leptin receptor ectodomain. In 10 obese con- trol subjects, as in the mutated patients, free leptin lev- els correlated with BMI ( r = 0.70, P = 0.0011) and reflected fat mass, regardless of leptin receptor func- tioning. In the patients, bound leptin levels correlated with BMI ( r = 0.99, P = 0.0002) and were related to the num- ber of mutated alleles. These data demonstrate that the truncated receptor is secreted into blood and binds the majority of serum leptin, markedly increasing bound and total leptin. Free serum leptin was similarly correlated with BMI in the mutated and nonmutated obese individ- uals, providing evidence that the relationship between BMI and circulating free leptin is preserved in this fam- ily. This finding suggests that the leptin receptor itself may not be specifically involved in the control of leptin secre- tion, and it supports the concept of relative resistance to leptin in common obesity. Diabetes 49:1347–1352, 2000 L eptin, the ob gene product secreted by adipocytes (1), exerts its biological effects through a mem- brane receptor, a member of the cytokine receptor superfamily. The human leptin receptor (LepR) has a long extracellular domain (2), where the leptin binding domain was recently localized to residues 323–640 (3). LepR is expressed in many tissues, including the brain, where short and long isoforms of the cytoplasmic domain are suspected to play different roles (4,5). Several mouse and rat strains harbor LepR gene mutations. In the db /db mouse, the mutated gene encodes a truncated long form of the LepR protein lacking most of the cytoplasmic region (5). The mutated receptor is defective for signal trans- duction but not for leptin binding (6). In the fa/fa rat, a single base substitution affects the intracytoplasmic transport of the receptor (7) and causes leptin resistance. Both types of mutations are associated with early-onset obesity, hyperpha- gia, hyperinsulinemia, and infertility (8). Search for mutations of the LepR gene in common human obesity remained unsuc- cessful (9,10). Leptin circulates in serum both as a free form of ~16 kDa and as a form bound to a carrier protein. The balance between free and bound leptin is a potential regulator of lep- tin bioavailability (11,12). While screening a large cohort of morbidly obese patients, we identified members of a consanguineous family whose serum leptin levels were much higher than expected, given their BMI. We found a mutation in the LepR gene leading to a truncated receptor that was lacking transmembrane and cytoplasmic domains and was responsible for leptin resis- tance (13). The unexpectedly high serum leptin levels in indi- viduals with mutated LepR led us to study the circulating forms of leptin and to further characterize 1) the leptin bind- ing factor in patients’ sera and 2) the relationship between cir- culating forms of leptin and fat mass. RESEARCH DESIGN AND METHODS Control subjects. Serum samples were obtained from 1,333 lean and obese subjects (471 men and 862 women) who gave their informed consent to undergo a serum leptin assay. Their age ranged from 18 to 87 years and their BMI ranged from 14 to 82 kg/m 2 . Serum from 10 women who had common obe- sity (aged 30–62 years, BMI 40–67 kg/m 2 ) were subjected to the same chro- matographic fractionation as serum from the patients. Patients. Serum samples were obtained from the parents and from the 8 siblings (Table 1) in a consanguineous family harboring a mutation in the LepR gene (13). The proband (patient 1) was a 19-year-old girl characterized by morbid obe- sity (BMI 66 kg/m 2 ). She developed obesity as early as 3 months of age and was referred to a pediatric department at the age of 6 years for retarded growth and later for delayed puberty. Two sisters (patients 2 and 3) shared the same clini- cal phenotype and the same medical history. Both parents (patients 4 and 7) and 3 other sibs (patients 5, 6, and 8) were moderately overweight. Two other sibs (one boy and one girl [patients 9 and 10]) were normal except for mild obesity in the girl. Genetic data have been described elsewhere (13). Briefly, the 3 mor- bidly obese girls ( mut/mut subjects) were homozygous for a G→A base sub- stitution in the splice donor site of exon 16 of the gene, which resulted in the skipping of exon 16. This mutation predicts a truncated receptor of 831 amino From Unit 342, Institut National de la Santé et de la Recherche Médicale, and the Department of Biochemistry, Hôpital Saint-Vincent-de-Paul (N.L., J.-C.C., C.L., Y.L.B., M.R.); Service de Nutrition (K.C., C.V., A.B., B.G.-G.), Hôtel-Dieu; the Explorations Fonctionnelles Endocriniennes (Y.L.), Hôpi- tal Trousseau, Paris; and Centre National de la Recherche Scientifique, Equipe postulante n° 10 (P.F.), Institut Pasteur, Lille, France. Address correspondence and reprint requests to Najiba Lahlou, MD, Service de Biochimie, Hôpital Saint-Vincent-de-Paul, 82 av. Denfert- Rochereau, 75014 Paris, France. E-mail: najiba.lahlou@ svp.ap-hop-paris.fr. Received for publication 6 July 1999 and accepted in revised form 17 April 2000. ECL, enhanced chemiluminescence; LepR, leptin receptor.