Genetic Variation in the Leptin Receptor Gene, Leptin, and Weight Gain in Young Dutch Adults

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1 See Accompayig Commetary by Jekis ad Campbell o pages Geetic Variatio i the Lepti Receptor Gee, Lepti, ad Weight Gai i Youg Dutch Adults Carolie T.M. va Rossum,* Barbara Hoebee, Marlee A. va Baak, Moica Mars, Wim H.M. Saris, ad Jacob C. Seidell* Abstract VAN ROSSUM, CAROLINE T.M., BARBARA HOEBEE, MARLEEN A. VAN BAAK, MONICA MARS, WIM H.M. SARIS, AND JACOB C. SEIDELL. Geetic variatio i the lepti receptor gee, lepti, ad weight gai i youg Dutch adults. Obes Res. 2003;11: Objective: To ivestigate the associatio betwee lepti levels, polymorphisms i the lepti receptor (LEPR) gee, ad weight gai. Research Methods ad Procedures: From two large prospective cohorts i The Netherlads ( 17,500), we compared the baselie lepti of 259 subjects who had gaied a average of 12.6 kg (rage 5.5 to 33 kg) with 277 subjects who kept stable weight (rage 2.6 to 3.1 kg) after a mea follow-up of 6.8 years. Three polymorphisms i the LEPR gee (Lys109Arg, Gl223Arg, ad Lys656As) were determied. Results: Weight gaiers had sigificatly higher baselie lepti levels tha those who kept stable weight (odds ratio 1.27, 95% cofidece iterval 1.1 to 1.5, per SD icrease i log e -trasformed lepti). Weight gaiers with the Arg109 or the Arg223 alleles had higher lepti levels compared with the ocarriers of these alleles. Oly amog me, the associatio betwee lepti ad weight gai teded to be stroger amog those with a Arg223 allele compared with those without this mutatio. Discussio: Relatively high lepti levels predict weight gai, suggestig that lepti resistace plays a role i the developmet of obesity i the geeral populatio. Higher lepti levels for those with a Lys109Arg or Gl223Arg Received for review April 30, Accepted i fial form December 10, *Departmet of Chroic Diseases Epidemiology ad Laboratory of Health Effects Research, Natioal Istitute of Public Health ad the Eviromet, Bilthove, The Netherlads; Departmet of Huma Biology, Maastricht Uiversity, Maastricht, The Netherlads; ad Departmet of Huma Nutritio ad Epidemiology, Wageige Uiversity, Wageige, The Netherlads. Address correspodece to Carolie va Rossum, Departmet of Chroic Diseases Epidemiology, Natioal Istitute of Public Health ad the Eviromet, P.O. Box 13720, BA Bilthove, The Netherlads. carolie.va.rossum@rivm.l Copyright 2003 NAASO mutatio (or a liked other marker) may imply that these subjects have a modified fuctioal lepti receptor. However, the role of these mutatios o weight gai is limited. Key words: weight gai, lepti, polymorphisms, lepti receptor, prospective study Itroductio Lepti is a hormoe that is maily produced by adipose tissue ad bids to receptors i the hypothalamus (1). The discovery of mutatios i the lepti gee ad its receptor i rodet models of obesity (e.g., ob/ob ad db/db mice) ad i rare cases of huma morbid obesity idicates that lepti fuctios as a afferet sigal i a egative feedback loop that regulates eergy balace (2 6). However, the role of lepti ad its receptor i the developmet of obesity i the geeral populatio is less clear. The etiology of commo obesity is more complex, because may geetic, metabolic, ad evirometal factors might iteract, but it remais possible that lepti or its receptor is ivolved. Particular mutatios i the lepti gee i aimal models ad humas lead to lepti deficiecy ad extreme obesity (2 6). For commo obesity, a similar, but weaker, egative associatio betwee lepti ad weight might be foud. Commo obesity, however, is characterized by high, rather tha low, levels of lepti, ad it has bee suggested that obese subjects may be lepti resistat (7). Several mechaisms may cotribute to lepti resistace. Dimiished lepti receptor sigalig due to a mutat lepti receptor might be oe of the explaatios (8). This has bee demostrated i db/db mice ad some severely obese humas who are homozygous for a mutatio i the lepti receptor, who have extremely high lepti levels (6). Such mutatios are extremely rare ad caot be resposible for obesity i the geeral populatio. However, this observatio suggests that other, less severe disruptios of the ormal lepti-sigalig pathway might be ivolved i the developmet of commo obesity (9). Therefore, it is coceivable that more commo OBESITY RESEARCH Vol. 11 No. 3 March

2 Lepti, LEPR, ad Weight Gai, va Rossum et al. mutatios i the lepti receptor (LEPR) 1 gee could modify the fuctio of the lepti receptor i such a way that they affect lepti levels i the geeral populatio. We selected, from a large populatio study, subjects who gaied weight i a follow-up period of, o average, 7 years ad subjects who kept stable weight. We ivestigated whether the baselie lepti levels predicted weight gai. I additio, we ivestigated the associatio betwee three polymorphisms i the LEPR gee ad lepti levels ad the modificatio of the associatio betwee lepti ad weight gai by these polymorphisms. Research Methods ad Procedures Subjects were selected from participats i cardiovascular moitorig projects that have bee carried out i two tows of The Netherlads, Maastricht ad Doetichem. The desig ad ratioale of the selectio of the participats has bee described i more detail elsewhere (10). I short, the baselie measuremets occurred betwee 1987 ad 1997 at the Muicipal Health Services (11). I Doetichem, a secod measuremet occurred 6 years after the baselie measuremet (1993 to 1997), agai at the Muicipal Health Service. I Maastricht, the follow-up measuremets were ascertaied i 1998 by meas of a self-admiistered questioaire. To exclude, as much as possible, other potetial iflueces o weight chage, subjects from these two cohorts ( 17,743) who were dietig, were high alcohol cosumers, were pregat, had chaged their smokig status recetly, suffered from serious illesses, ad those of 40 years ad above were excluded. Furthermore, those with a follow-up of less tha 4 years, those without a iformed coset, a blood sample, or data o weight were also excluded. From the remaiig group (19% of the origial sample), we selected high weight gaiers by takig the top decile of the distributio of average weight gai per year ( 1.4 ad 1.3 kg/year for the cohorts of Doetichem ad Maastricht, respectively). We radomly selected a equal sized group of subjects ( stable weight) whose weight remaied relatively costat (rage: 0.3 to 0.3 kg/year). The o-weight gaiers were frequecy matched for tow, sex, age, ad smokig status with the weight gaiers. Because meopause ca be a cofouder of the associatio betwee lepti ad weight gai (12,13), we also excluded i these aalyses seve female subjects who had a operatio o the womb or ovaries or did ot have a period i the last 12 moths. Data o lepti levels ad DNA polymorphisms were available for 259 weight gaiers ad 277 o-weight gaiers. Measuremets The examiatios at baselie ad the secod measuremet icluded physical examiatios, e.g., athropometric 1 Nostadard abbreviatios: LEPR, lepti receptor gee; BMI, body mass idex. measuremets, a self-admiistered questioaire, ad blood samplig. However, for those i Maastricht, the secod measuremet icluded oly a self-admiistered questioaire. Athropometric Measures. Weight at baselie was measured without shoes ad wearig light idoor clothig. For all participats i Doetichem, weight was remeasured after 6 years i the same seaso as i the iitial measuremet. For the participats from Maastricht, it is likely that subjects wore less clothig whe measured at home compared with the measuremet at the health ceter. To allow for the weight of clothig, we added 1.5 kg at the self-reported weight. This amout was based o some measuremets of the clothes by ivestigators at the health ceter. Body mass idex (BMI) was calculated as weight divided by squared height. Weight gai was defied as the differece betwee the weight at baselie ad the weight at the secod examiatio. Because the period of follow-up varied (6 years i Doetichem ad 4.0 to 11.3 years i Maastricht), we calculated the average weight gai per year. Characteristics of the Study Populatio. The questioaires at baselie ad/or follow-up provided iformatio about history of chroic diseases, alcohol cosumptio, cigarette smokig, pregacy, ad educatioal level. Smokig history was categorized ito osmokers (those who had ever smoked or stopped smokig more tha 5 years before the start of the study), smokers (subjects who reported to be a curret smoker at baselie as well as at the secod measuremet), ad the remaiig group ( subjects who had chaged their smokig habits durig the two measuremets or stopped smokig 5 years before the start of the study). This last category was a exclusio category. The educatioal level, a measure for socioecoomic status, was classified ito three categories: low (primary school, lower vocatioal/geeral educatio), medium (itermediate/ higher geeral educatio, itermediate vocatioal educatio), ad high (higher vocatioal educatio, uiversity). Lepti. Lepti cocetratios were measured i the baselie ofastig plasma samples, which had bee stored at 20 C for 6 to 14 years. These measuremets were doe i duplicate by radioimmuoassay (HL-81K kit, Lico Research Ic., St. Charles, MO). Geotypig. We determied three polymorphisms i the LEPR gee: a lysie to alaie substitutio at codo 109 (Lys109Arg), a glutamie to argiie substitutio at codo 223 (Glu223Arg), ad lysie to asparagie at codo 656 (Lys656As) (14). Geotypig was doe usig polymerase chai reactio-restrictio fragmet legth polymorphism aalyses. To icrease the power of the statistical aalyses, for each polymorphism, heterozygotes were combied with the smallest group of homozygotes. For example, subjects with the Lys109/Arg109 or the Arg109/ Arg109 geotype of the LEPR gee were combied ad compared with those with the Lys109/Lys109 geotype. 378 OBESITY RESEARCH Vol. 11 No. 3 March 2003

3 Lepti, LEPR, ad Weight Gai, va Rossum et al. Table 1. Characteristics of the study populatio [meas (SD) or percetages] High weight gai ( 126) Me Stable weight ( 134) p value High weight gai ( 133) Wome Stable weight ( 143) p value At baselie Age (years) 28.2 (5.9) 28.9 (5.6) * 30.1 (5.9) 29.9 (6.0) * Weight (kg) 79.4 (10.9) 76.6 (10.0) (11.1) 63.4 (8.1) 0.02 Height (cm) 1.82 (0.07) 1.80 (0.07) (0.07) 1.67 (0.06) 0.11 BMI (kg/m 2 ) 24.0 (3.0) 23.7 (2.8) (3.7) 22.9 (2.8) 0.12 Smokig (%) * * Socioecoomic status (%) Low Medium High At ed of follow-up Follow-up time (years) 6.9 (1.7) 7.2 (1.9) (1.5) 6.8 (1.6) 0.51 Weight (kg) 92.0 (11.6) 77.0 (10.0) * 79.1 (12.4) 63.9 (8.1) * Weight gai (kg/year) 1.86 (0.54) 0.06 (0.17) * 1.90 (0.46) 0.08 (0.17) * BMI (kg/m 2 ) 27.9 (3.3) 23.9 (2.8) * 28.0 (4.0) 23.1 (2.8) * * p Values ot give; criteria for group selectio. Data Aalyses Differeces i characteristics betwee high weight gaiers ad o-weight gaiers were aalyzed with chi-square tests ad Studet s t tests. Because lepti cocetratios ad weight gai were ot ormally distributed, these variables were atural logarithmically trasformed (log e ). The associatio betwee lepti ad weight gai was performed i three ways. First, lepti levels adjusted for baselie BMI were evaluated accordig to weight gai (yes/o) usig aalyses of covariace. The (adjusted) logarithmically trasformed lepti values were trasformed back to ormal values. Secod, logistic regressio aalyses were carried out to ivestigate the associatio betwee lepti (per SD icrease i the logarithm) ad weight gai (yes/o). I additio, amog weight gaiers, regressio aalyses were performed to ivestigate the BMI-adjusted associatio betwee lepti ad weight gai per year. meas of lepti levels accordig to each polymorphism i the LEPR gee were also calculated o the basis of aalysis of covariace. For each polymorphism separately, we also aalyzed these levels accordig to combiatios of two polymorphisms. These aalyses were performed separately i weight gaiers ad those with stable weight. Fially, we tested whether the associatio betwee lepti ad weight gai (yes/o) was modified by the geotypig of the LEPR gee, by addig the geotype ad the iteractio term betwee these geetic factors ad lepti to the logistic regressio models. All these associatios were adjusted for baselie BMI. Furthermore, all aalyses were doe separately i me ad wome ad i me ad wome combied (adjusted for sex), ad they were performed usig the statistical package SAS (versio 8.1; SAS Istitute, Cary, NC). Results The characteristics of the study populatio are show i Table 1. Subjects were, o average, 29.3 years (rage 20 to 40) old ad had a baselie BMI of 23.5 kg/m 2 (SD 3.1). Subjects with high weight gai icreased, o average, 12.6 kg (rage 5.5 to 32.9 kg) correspodig with a icrease i BMI of 4 uits, whereas those with stable weight gaied, o average, 0.5 kg (rage 2.6 to 3.1 kg) durig a mea follow-up of 6.8 years. At baselie, weight gaiers already had a higher body weight ad height (i me) compared with the oweight gaiers. BMI was ot sigificatly differet betwee the two groups (p 0.40 ad 0.12 for me ad wome, respectively). Weight gaiers did ot sigificatly differ i educatioal level from o-weight gaiers. OBESITY RESEARCH Vol. 11 No. 3 March

4 Lepti, LEPR, ad Weight Gai, va Rossum et al. Table 2. Lepti levels for weight gaiers ad those who kept stable weight Total media (rage) High weight gai, geometric mea (95% cofidece Stable weight, geometric mea (95% CI) p value Me Lepti ( g/l) 3.0 (1.1 to 15.5) 3.4 (3.1 to 3.7) 3.0 (2.7 to 3.3) 0.06 Lepti, adjusted for baselie BMI ( g/l) 3.3 (3.1 to 3.6) 3.0 (2.8 to 3.3) 0.08 Wome Lepti ( g/l) 9.7 (1.9 to 74) 10.6 (9.6 to 11.8) 9.2 (8.3 to 10.2) 0.05 Lepti, adjusted for baselie BMI ( g/l) 10.2 (9.5 to 10.9) 9.6 (8.9 to 10.3) 0.25 Me ad wome Lepti ( g/l)* 5.8 (1.1 to 74) 6.0 (5.6 to 6.5) 5.3 (4.9 to 5.6) Lepti, adjusted for baselie BMI ( g/l)* 5.8 (5.5 to 6.1) 5.4 (5.1 to 5.7) 0.04 * (Also) adjusted for sex. Lepti ad Weight Gai Lepti levels of wome were higher tha those of me (media 9.7 ad 3.0 g/l, respectively). These levels were strogly correlated with baselie BMI levels (r 0.60 ad 0.72 for me ad wome, respectively). Table 2 shows the geometric meas of the lepti levels for the weight gaiers ad those who kept stable weight. At baselie, weight gaiers had sigificatly higher lepti levels tha oweight gaiers, 3.4 vs. 3.0 ad 10.6 vs. 9.2 g/l for me ad wome, respectively. These differeces could partly be explaied by the variatio i BMI as a idicator for fat mass. For me, the differece i lepti levels remaied borderlie statistically sigificat after adjustmet for BMI. The odds ratio for weight gai accordig to oe SD icrease i the logarithm of lepti levels was 1.27 (95% cofidece iterval, 1.07 to 1.51) for the total group of subjects. After adjustmet for BMI, this was 1.28 (1.01 to 1.61) for the total group (see Table 3). Table 4 shows the associatio betwee lepti ad weight gai amog the weight gaiers. For the male weight gaiers, higher lepti levels teded to be associated with more weight gai (p 0.02), whereas amog female weight gaiers, o associatio was observed. Table 3. s for the risk of weight gai (yes/o) from baselie to follow-up for SD icrease i log e -trasformed lepti levels Me Wome Total SD of the atural logarithm (95% cofidece SD of the atural logarithm (95% cofidece (95% cofidece * Risk of weight gai per SD icrease i log e -trasformed lepti levels 0.54 Risk of weight gai per SD icrease i log e -trasformed lepti levels, adjusted for baselie BMI (0.99 to 1.63) 0.62 (1.00 to 1.61) (1.07 to 1.51) 1.32 (0.97 to 1.80) 1.23 (0.87 to 1.74) 1.28 (1.01 to 1.61) * Also adjusted for sex. 380 OBESITY RESEARCH Vol. 11 No. 3 March 2003

5 Lepti, LEPR, ad Weight Gai, va Rossum et al. Table 4. Average log e -trasformed weight gai per year from baselie to follow-up per SD icrease i log e - trasformed lepti levels amog weight gaiers Me ( 126) Wome ( 133) (95% CI) p value (95% cofidece p value Average log e -trasformed weight gai per year (0.007, 0.089) ( to 0.037) 0.90 Average log e -trasformed weight gai per year, adjusted for baselie BMI (0.002, 0.104) ( 0.063, 0.062) 0.98 Lepti ad Polymorphisms i LEPR Gee. Table 5 shows the geometric meas of lepti accordig to the polymorphisms i the LEPR gee. I additio, the cotrast i lepti betwee carriers ad ocarriers of combiatios of two mutatios is show. Statistically sigificat differeces i lepti levels were observed amog oly the weight gaiers. Carriers of the Arg109 allele or the Arg223 allele teded to have higher lepti levels compared with ocarriers of these alleles, p 0.02 ad 0.03, respectively. For carriers of the Arg109 ad Arg223 alleles, lepti levels were 10% to 20% higher compared with the ocarriers of these two alleles (6.6 vs. 5.6 g/l, p 0.02). Differeces i lepti levels accordig to the polymorphism at positio 656 of the LEPR gee were ot statistically sigificat. However, male weight gaiers with either the Arg109 allele or the Arg223 allele ad the As656 allele have (borderlie) sigificatly higher lepti levels compared with those with oe of these alleles (4.0 vs. 3.0 g/l, p 0.06; or 3.6 vs. 2.6 g/l, p 0.02). Lepti ad Polymorphism i LEPR Gee ad Weight Gai. Table 3 showed that the risk of beig a weight gaier icreases with higher baselie levels of lepti. If polymorphisms i the lepti receptor play a role i the lepti sigalig, it is likely that the associatio betwee lepti ad weight gai is modified by mutatios i the LEPR gee. However, Table 6 shows that the associatios betwee lepti ad risk for weight gai were ot clearly modified by these mutatios. Oly amog me, the associatio teded to be stroger amog those with a Arg223 allele compared with those without this mutatio. Discussio I our study populatio, lepti levels adjusted for BMI were positively associated with weight gai; those with higher lepti levels had a higher risk for gaiig weight. Furthermore, amog those who gaied weight, baselie lepti levels were higher for carriers of the Arg109 or Arg223 mutatios i the lepti receptor. The associatios were clearer amog me tha amog wome. Amog me, the associatio betwee baselie lepti ad weight gai was modified by the Gl223Arg mutatio i the LEPR gee. To appreciate these fidigs, certai aspects of the study must be cosidered. A advatage of our study was our prospective desig to study the associatio betwee lepti ad weight gai. Because lepti might affect weight gai, ad a higher weight could, i tur, ifluece lepti levels, cross-sectioal ad logitudial studies o the role of lepti i the etiology of obesity ca geerate completely opposite coclusios (15). Secodly, the issue of potetial selectio bias must be cosidered. The participatio rate i the origial cohort was 50% (11). It is ulikely, however, that there was selective participatio by baselie lepti level or geotype amog the weight gaiers ad o-weight gaiers. This is supported by the fact that the distributios of the geotypes were i Hardy-Weiberg Equilibrium. Thirdly, our fidigs could be affected by misclassificatio problems. For the subjects from Maastricht, the weights at the ed of the follow-up were self-reported. Because errors i self-reported weight icrease with the magitude of overweight, this misclassificatio was probably ot radomly distributed (16). However, we assumed that the distictio betwee weight gaiers ad those who kept stable weight ad the rakig i weight gaiers were hardly affected by this. Lepti levels were measured i ofastig blood samples that were ot take for all subjects at the same time of the day. Because lepti cocetratios i humas show a clear diural patter, ad this patter is etraied to meal timig (17), lepti levels measured i our study might be affected by this patter. However, it is ulikely that these factors differed for weight gaiers ad weight keepers, or that they were associated with the geotype of the LEPR gee. This is cofirmed i the follow-up measuremets of a subsample of 286 subjects. I this subsample, time of blood samplig or time of last eatig was ot sigificatly associated with lepti levels, or did the weight gaiers ad weight keepers differ i these factors. Because several studies foud that OBESITY RESEARCH Vol. 11 No. 3 March

6 Lepti, LEPR, ad Weight Gai, va Rossum et al. Table 5. Lepti levels ( g/l) accordig to polymorphisms i the LEPR gee, adjusted for baselie BMI Me Wome Total High weight gai Stable weight High weight gai Stable weight High weight gai Stable weight mea (95% cofidece mea (95% cofidece mea (95% cofidece mea (95% cofidece mea (95% cofidece * mea (95% cofidece * LEPR Lys109Arg Lys109/Lys (2.9 to 3.6) (2.7 to 3.3) (9.1 to 11.1) (8.3 to 10.1) (5.4 to 6.2) (5.0 to 5.7) Arg109/ (3.3 to 4.1) (2.7 to 3.3) (10.3 to 12.6) (8.4 to 10.2) (6.0 to 7.1) (5.0 to 5.8) p value LEPR Gl223Arg Gl223/Gl (2.7 to 3.5) (2.6 to 3.4) (8.7 to 11.4) (7.8 to 10.1) (5.1 to 6.2) (4.8 to 5.7) Arg223/ (3.3 to 4.0) (2.8 to 3.3) (10.0 to 11.9) (8.6 to 10.1) (6.0 to 6.8) (5.1 to 5.7) p value LEPR Lys656As Lys656/Lys (3.0 to 3.7) (2.7 to 3.1) (9.7 to 11.6) (7.7 to 9.8) (5.6 to 6.4) (4.9 to 5.7) As656/ (3.1 to 4.0) (2.9 to 3.6) (9.2 to 12.2) (8.7 to 10.3) (5.7 to 6.9) (5.0 to 5.8) p value Combiatio of two polymorphisms LEPR Lys109Arg ad Gl223Arg Lys109/Lys109 & Gl223/Gl (2.7 to 3.5) (2.6 to 3.4) (8.7 to 11.4) (7.7 to 10.0) (5.1 to 6.2) (4.7 to 5.7) Arg109/ & Arg223/ (3.3 to 4.1) (2.7 to 3.3) (10.2 to 12.6) (8.3 to 10.1) (6.1 to 7.1) (5.0 to 5.7) p value LEPR Lys109Arg ad Lys656As Lys109/Lys109 & Lys656/Lys (2.6 to 3.5) (2.4 to 3.1) (8.6 to 11.1) (8.6 to 11.4) (5.0 to 6.1) (4.8 to 5.8) Arg109/ & As656/ (3.1 to 5.2) (2.4 to 3.6) (8.5 to 14.2) (7.4 to 11.4) (5.7 to 8.2) (4.6 to 6.2) p value LEPR Gl223Arg ad Lys656As Gl223/Gl223 & Lys656/Lys (2.1 to 3.2) (2.1 to 3.0) (8.1 to 11.6) (8.2 to 12.4) (4.5 to 5.9) (4.4 to 5.8) Arg223/ & As656/ (3.1 to 4.3) (2.5 to 3.6) (9.1 to 129) (7.7 to 10.9) (5.6 to 7.2) (4.8 to 6.1) p value * Also adjusted for sex. Based o aalyses i which all four combiatios of these two polymorphisms were icluded, oly the results of these two extreme groups are show. lepti cocetratios fluctuate accordig to the phase of the mestrual cycle (18,19), ad blood samples were ot take for all wome at the same time i the cycle, it is likely that this variatio has diluted our results. This might explai the clearer fidigs i me compared with those amog wome. Furthermore, the matchig factors could have cofouded our results. Fortuately, these biases were of mior importace because similar results were foud with ad without adjustmet for these factors. At baselie, female weight gaiers had slightly higher BMI values compared with those who kept stable weight. Because persos with more fat mass have higher lepti levels, ad fat mass also idepedetly affects the risk of weight gai due to e.g., evirometal factors, we adjusted the associatios by takig BMI ito the aalyses to cotrol 382 OBESITY RESEARCH Vol. 11 No. 3 March 2003

7 Lepti, LEPR, ad Weight Gai, va Rossum et al. Table 6. for the risk of weight gai from baselie to follow-up per SD icrease i log e -trasformed lepti levels accordig to polymorphisms i the LEPR gee, adjusted for baselie BMI Me Wome Total (95% cofidece (95% cofidece (95% cofidece LEPR Lys109Arg Lys109/Lys (0.49 to 2.04) (0.66 to 2.37) (0.87 to 1.83) Arg109/ (0.98 to 2.28) (0.80 to 1.90) (0.99 to 1.70) p iteractio LEPR Gl223Arg Gl223/Gl (0.24 to 1.38) (0.46 to 2.39) (0.63 to 1.54) Arg223/ (1.11 to 2.33) (0.87 to 1.83) (1.06 to 1.74) p iteractio LEPR Lys656As Lys656/Lys (0.84 to 3.48) (0.61 to 2.04) (1.05 to 2.14) As656/ (0.72 to 1.78) (0.79 to 2.09) (0.86 to 1.54) p iteractio Combiatios of two polymorphisms LEPR Lys109Arg ad Gl223Arg Lys109/Lys109 & Gl223/Gl (0.56 to 1.36) (0.78 to 1.99) (0.75 to 1.42) Arg109/ & Arg223/ (0.98 to 2.09) (0.93 to 1.85) (1.06 to 1.76) p iteractio LEPR Lys109Arg ad Lys656As Lys109/Lys109 & Lys656/Lys (0.79 to 2.06) (0.65 to 1.73) (0.84 to 1.65) Arg109/ & As656/ (0.66 to 3.60) (0.44 to 1.92) (0.65 to 1.94) p iteractio LEPR Gl223Arg ad Lys656As Gl223/Gl223 & Lys656/Lys (0.44 to 2.08) (0.46 to 1.70) (0.57 to 1.54) Arg223/ & As656/ (0.79 to 2.76) (0.67 to 1.92) (0.85 to 1.88) p iteractio p Iteractio, test of p value of the iteractio term betwee lepti ad the specific geotype. * Also adjusted for sex. Subjects with other geotypes were excluded from these aalyses. for the cofoudig. BMI is ot a perfect measure for the amout of body fat percetage, but i Dutch adults they are highly correlated (r 0.75) (20). Therefore, it is possible that the observed differeces betwee weight gaiers ad weight keepers are still partly caused by residual cofoudig of body fat percetage. Although this cofoudig may have affected the associatio betwee lepti ad weight gai, it is ulikely that it has affected our coclusio that lepti predicts weight gai. I additio, it remais possible that our associatio ca be explaied by other ukow ad cofoudig factors that ifluece both weight gai ad lepti. Rodet models of obesity (e.g., ob/ob mice) ad some huma cases of severe obesity are lepti deficiet due to a rare mutatio i the lepti gee (2,5). Treatmet with lepti results i a decrease i food itake ad body weight (21 24). Therefore, we expected that for commo obesity, a similar, but weaker, egative associatio betwee lepti ad OBESITY RESEARCH Vol. 11 No. 3 March

8 Lepti, LEPR, ad Weight Gai, va Rossum et al. weight would be observed. However, we foud o evidece for the hypothesis that relatively low lepti levels predispose to weight gai. I cotrast, we observed that those with higher lepti levels teded to have a icreased risk for gaiig weight compared with those with low levels. Our fidig is i cotrast with a study i 36 obese Pima Idias; those with relatively low lepti levels were more proe to gai weight compared with those with higher lepti levels (25). Also, Lidroos et al. foud a egative associatio betwee lepti ad weight gai, but oly i a small subgroup of 24 very obese Swedes without a history of paretal obesity (26). However, other prospective studies i more moderately obese populatios foud o associatio (27 29) or also a positive (30,31) associatio, as i our study, betwee lepti ad weight gai. There are several possible explaatios for these differeces. First, it is possible that the associatio betwee lepti ad weight gai is modified by the degree of obesity. Secodly, i the study i Pima Idias, adjustmets were made for more accurate measures of body fat percetage (assessed by hydrodesitometry) tha BMI. Thirdly, other populatio characteristics that were differet across studies may explai the icosistecies. Because db/db mice ad some humas with lepti resistace due to a defect i their lepti receptor are severely obese ad have high plasma lepti levels (6), we expected a similar, but weaker, associatio betwee lepti ad weight gai i our populatio. Our observatio that weight gaiers had sigificatly higher baselie lepti cocetratios tha weight keepers is cosistet with this hypothesis that lepti resistace cotributes to weight gai i a geeral white populatio. Some other studies reported lepti levels accordig to mutatios i the LEPR gee (32 34). Oly Chago et al. also reported higher lepti levels after adjustmet for fat mass amog middle aged me with a Arg223 allele (34). Most of the other associatios foud were ot idepedet of BMI ad were performed i very small groups, i overweight ad obese, owhite, or postmeopausal wome, which may explai the icosistecy i these results (33). Our fidigs of icreased lepti levels i carriers of the Arg109 allele or the Gl223 allele of the LEPR gee compared with those i the ocarriers of these alleles suggest that for these variats, the sigalig of lepti is impaired. However, so far as we kow, o studies have bee performed o the affected fuctioality of this protei due to these mutatios or other liked markers. Whether its fuctioality is ideed impaired eeds further ivestigatio. A defective lepti receptor due to mutatios i the gee is oe of the explaatios for a positive associatio betwee lepti ad weight gai. Arch et al. suggested that i most cases, lepti cocetratios are raised ot oly because of geetic variability, but also because of other opposig forces that promote obesity (35). Our results support this idea, because weight gaiers differed i some aspects of dietary habits ad physical activity from o-weight gaiers (10), ad weight gaiers had higher lepti levels. It is likely that variatio i geetic factors ad lifestyle factors, such as physical activity ad metabolic factors (36 38), explais the icosistecy i the reported associatios betwee lepti ad weight gai. Nevertheless, our data suggest that persos with the Arg109 allele or the Gl223 allele are more proe to this ifluece of other lepti-ifluecig factors. Seeig the differet associatios foud amog me betwee lepti ad weight gai across the geotypes, it is likely that these polymorphisms i the LEPR gee have some small impact o weight gai. That this is a small impact is ot surprisig because weight gai is iflueced by several factors ad pathways. However, i the same study populatio, a weak associatio betwee these polymorphisms ad weight gai was observed amog wome for oly the Lys109Arg geotype; wome with the Lys109/Lys109 polymorphism had a lower risk to gai weight (10). These cotrastig fidigs suggest that the role of these polymorphisms o weight gai is very complicated ad ot yet clear. Also, meta-aalyses have show that there is o clear evidece that these polymorphisms are associated with BMI (39). They also argue that these alleles ca still have a impact o weight, but these effects ca be compesated by other (geetic) factors ad pathways. Therefore, further studies o the ifluece of these alleles o itermediate pheotypes istead of weight could help to elucidate the role of these alleles o the developmet of obesity. Thus, we coclude that i white subjects, relatively high lepti levels seem to predict subsequet weight gai. Furthermore, Lys109Arg or Gl223Arg polymorphisms i the LEPR gee are associated with higher lepti levels, suggestig that these polymorphisms (or markers that are i likage disequilibrium with these polymorphisms) lead to lepti resistace. However, the role of these mutatios o weight gai is limited. Ackowledgmets The Cardiovascular Disease Risk Factor Moitorig Study (Peilstatios project) ad the Moitorig Project o Risk Factors for Chroic Diseases (MORGEN project) were fiacially supported by the Miistry of Health, Welfare, ad Sport of The Netherlads ad the Natioal Istitute of Public Health ad the Eviromet. C. T. M. va Rossum is supported by the Dutch Scietific Orgaizatio (NWO). The authors would like to thak the epidemiologists ad field workers of the Muicipal Health Services i Doetichem ad Maastricht for their importat cotributio to the data collectio for this study. The authors wish to thak G.L. Oberma-de Boer for coordiatig the Peilstatios project. The project steerig committee of the MORGEN project cosisted of H.B. Bueo de Mesquita, H.A. Smit, 384 OBESITY RESEARCH Vol. 11 No. 3 March 2003

9 Lepti, LEPR, ad Weight Gai, va Rossum et al. W.M.M. Verschure, ad J.C. Seidell (project leader). The project leader of the follow-up measuremets i Maastricht was A.J. Schuit. Logistics maagemet was provided by A. Jase ad J. Steebrik, ad data maagemet was provided by A. Blokstra, P. Steiberger ad A. va Kessel. Furthermore, we gratefully ackowledge the group of C. Bouchard, K. Wichers, ad H. Hodemaekers, who performed the polymerase chai reactio aalyses; ad J. Sede, who performed the measuremets of the lepti levels. Refereces 1. Friedma JM, Halaas JL. Lepti ad the regulatio of body weight i mammals. Nature. 1998;395: Zhag Y, Proeca R, Maffei M, Baroe M, Leopold L, Friedma JM. Positioal cloig of the mouse obese gee ad its huma homologue. Nature. 1994;372: Lee GH, Proeca R, Motez JM, et al. Abormal splicig of the lepti receptor i diabetic mice. Nature. 1996;379: Che H, Charlat O, Tartaglia LA, et al. 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