associated with food and alcohol intake in obese people

(2000) 24, 920±924 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo ]>5-HT 2A receptor gene polymorphism is associated with food and alcohol intake in obese people R Aubert 1, D Betoulle 1, B Herbeth 2, G Siest 2 and F Fumeron 1 * 1 Human Nutrition Laboratory, Xavier Bichat Medicine School, BP416, 75870 Paris Cedex 18, France; and 2 Centre for Preventive Medicine, 54501 Vandoeuvre-leÁs-Nancy Cedex, France OBJECTIVE: To test the association between a polymorphism of the 5-HT 2A receptor gene, 1438G=A, and energy and nutrients intake, including alcohol. SUBJECTS: Two hundred and seventy six unrelated overweight subjects (180 women, 96 men) were recruited from the Nutrition Department of Bichat Hospital in Paris on the basis of 120% of ideal body weight (body mass index, BMI ˆ 33.3 4.8 kg=m 2 ). A second overweight sample (31 women, 49 men) was drawn from the Stanislas Family Study, composed of volunteers for a free health examination in Nancy (BMI ˆ 29.6 3.1 kg=m 2 ). MEASUREMENTS: Energy and nutrients intake were assessed using the diet history method in Paris and the 3-day record method in Nancy. We analyzed the polymorphism by PCR followed by MspI digestion. Statistical differences between genotypes were assessed by using the non-parametric Kruskal ± Wallis test. RESULTS: In the whole overweight population, the A allele was associated with lower energy intake 10.3 2.8, 9.9 2.8, 9.3 2.9 MJ=day for GG, GA and AA genotypes respectively (P < 0.05). This association was signi cant in the patient sample from Paris and in the overweight male volunteers from Nancy. Allele A-related lowering in energy intake was due to a trend to lower intakes in all the main nutrients. The A allele was also associated with a lower alcohol consumption: 18.4 19.7, 15.3 21.2 and 12.3 17.5 g=day for GG, GA and AA genotypes, respectively (P < 0.05). CONCLUSIONS: These data indicate that a gene polymorphism may in uence food and alcohol intake in overweight humans. This could be explained by the role of the serotonergic system as a determinant of food intake. (2000) 24, 920±924 Keywords: 5-HT 2A receptor; genetic polymorphism; obesity; food intake; alcohol Introduction The relationship between energy intake and obesity is somewhat controversial. Previously, we have shown that higher intake was associated with higher body mass index (BMI), 1 however not all obese people are hyperphagic. Serotonin (5-hydroxy-tryptamine, 5-HT) is a key mediator in the control of food intake. Serotonin reduces food intake and is probably involved in the etiology of anorexia nervosa and in weight regulation. 2 Drugs that increase serotonergic transmission exert anorectic activity. An association between a polymorphism of the 5-HT receptor (5- HT 2A ) gene promoter, 1438G=A, and anorexia nervosa has been reported in three studies, 3±5 but was not found in two other ones. 6,7 This polymorphism could in uence food intake in the general population. The serotonergic system is also related to variations in *Correspondence: F Fumeron, Human Nutrition Laboratory, Xavier Bichat Medicine School, BP416, 75870 Paris Cedex 18, France. E-mail: fumeron@bichat.inserm.fr Received 16 August 1999; revised 5 January 2000; accepted 1 March 2000 alcohol intake, and a recent study reported an association between this polymorphism and alcohol dependence. 8 We investigated the effects of this 5-HT 2A polymorphism in order to look for an association with energy and nutrient intake, including alcohol, in two obese groups, one from a Nutrition outpatient clinic and the other from the general population. Subjects and methods Two-hundred and seventy-six unrelated overweight subjects (180 women and 96 men) were recruited from the Nutrition Department of Bichat Hospital in Paris on the basis of 120% of ideal body weight, ie, a BMI 25.8 kg=m 2 for women and 26.4 kg=m 2 for men (BMI ˆ 33.3 4.8 kg=m 2 ). The second sample (237 unrelated subjects) was drawn from the Stanislas Family Study, composed of volunteers for a free health examination in Nancy area (East of France). The Stanislas Family Study is a longitudinal survey designed to investigate the combined role of genetic and environmental factors in cardiovascular diseases. 9 In this sample, 80 subjects (49 men and 31 women) met the overweight criteria (BMI ˆ 29.6 3.1 kg=m 2 ).

The spontaneous intakes of energy and nutrients were assessed using the diet history method in Paris as described in Fricker et al 1 and the 3-day record method in Nancy as described in Vauthier et al. 10 In Paris, the questionnaire contained 450 questions covering successively breakfast, lunch, dinner, snacks and nibbling. If the patients consumed an unlisted food, he or she mentioned it in answers to open questions. A dietician then reviewed the questionnaire with the patient and cross-checked the reliability and coherence of the information on amounts and frequencies. 1 In Nancy, the subjects completed a 3-day food consumption diary on two contiguous week days and one weekend day assigned at random. Participants were given formal instruction on keeping accurate records without changing their normal eating habits, and the completed diaries were carefully reviewed by the dietician. 10 We analyzed the 1438 G=A polymorphism of the 5-HT 2A receptor gene by PCR ampli cation followed by MspI digestion as described by Collier and colleagues. 3 Statistical differences between genotypes were assessed by the non-parametric Kruskal ± Wallis test. All statistics were performed using the SYSTAT computer program. All subjects gave their informed consent and the protocol was approved by the Bichat Hospital Ethics Committee and the Scienti c Ethics Committee of Nancy. Results Characteristics of the obese subjects from the two populations are shown in Table 1. Genotype frequencies were not different between the two samples. Patients from the Nutrition Department of Bichat Hospital had on average a signi cantly higher body mass index than subjects from Nancy. In the obese group from the Nutrition Department, the A allele was associated with lower energy intake (P < 0.05), subjects with GA genotype having intermediate intake (Figure 1). A-related lowering of energy intake was due to lower intakes of all the main nutrients, although only the decrease in carbohydrates was signi cant (P < 0.05) (Figure 2). These variations in diet were not associated with differences either in BMI or in plasma lipid concentrations (data not shown). In Nancy, both in the whole sample (n ˆ 237) from the general population (data not shown) and in the obese group, including males and females (Figure 1), no association was found between the 5-HT 2A polymorphism and energy and macronutrient intake. However, when looking on the male overweight subsample, the A allele was associated with lower Table 1 Characteristics of the subjects Obese from the Nutrition Department of Bichat Hospital (Paris) Obese from the Centre for Preventive Medicine (Nancy) n subjects (M=F) 276 (96=180) 80 (49=31) Age (y) 40.7 11.0 42.8 5.5 BMI (kg=m 2 ) 33.3 4.8 29.6 3.1 Energy intake (MJ=day) 10.2 2.9 8.9 2.4 Genotype frequency (%) GG 28.3 32.5 GA 51.8 47.5 AA 19.9 20.0 Values are expressed as mean s.d. 921 Figure 1 Energy intake according to the 5-HT 2A receptor gene polymorphism. Values are expressed as means s.d. P probability by the non-parametric Kruskal ± Wallis test.

922 energy intake (Figure 1) and lower alcohol consumption (Figure 3). In the whole overweight group, when pooling subjects from Paris and Nancy, the association between energy intake and the 5-HT 2A polymorphism was still signi cant (Table 2). This difference corresponds to lower intakes of all the main nutrients. The alcohol consumption is higher in GG subjects, median in GA and lower in AA (P < 0.05). The difference in energy intake was still signi cant after removal of energy contribution of alcohol. Discussion These data show that the 5-HT 2A 1438 G=A polymorphism is associated with energy intake in overweight people, intake being lower in A carriers than in G carriers. We found this relationship in a sample of patients from the Nutrition Department and con rmed this result in an independent sample of overweight subjects from the general population. The trend is found in both groups, however it is signi cant only for the two more numerous samples, ie women in the Paris group and men in the Nancy group. Moreover the effect of genotypes in men from Nancy looks recessive=dominant although it appears codominant in Paris. The differences between Nancy and Paris could be due to different factors: the different methods used for assessing food intake, the difference in the recruitment (nutrition clinic vs health center), or both. Mean energy intake was higher in the Paris group than in the Nancy group, which could be explained by: 1) a difference in BMI Ð the Paris group was heavier, and we have previously reported that in another Figure 2 Carbohydrate intake according to the 5-HT 2A receptor gene polymorphism in the obese group from the Nutrition Department in Paris. Values are expressed as means s.d. P probability by the non-parametric Kruskal ± Wallis test. Figure 3 Alcohol intake according to the 5-HT 2A receptor gene polymorphism in the obese men from the Health Centre in Nancy. Values are expressed as means s.d. P probability by the non-parametric Kruskal ± Wallis test.

Table 2 Food intake according to the 5-HT 2A receptor gene polymorphism in the whole overweight group (Centre for Preventive Medicine and Nutrition Department) 923 Genotypes P GG GA AA (Kruskal ± Wallis) n (M=F) 104 (44=60) 181 (73=108) 71 (28=43) Age (y) 41.5 10.0 41.4 10.0 40.0 10.4 NS BMI (kg=m 2 ) 32.3 4.1 32.5 5.0 32.8 4.8 NS Energy intake (MJ=day) 10.3 2.8 9.9 2.8 9.3 2.9 0.028 Proteins (g=day) 95.0 22.6 89.9 23.8 87.5 22.3 NS (0.056) Percentage of energy intake 15.8 3.0 15.6 3.4 16.2 2.5 NS Fats (g=day) 107.7 33.1 102.8 31.4 96.9 32.9 NS (0.098) Percentage of energy intake 39.2 5.3 39.2 5.7 39.3 5.7 NS Polyunsaturated (g=day) 12.8 5.4 13.3 5.6 13.0 6.2 NS Percentage of energy intake 4.7 1.8 5.1 1.9 5.3 2.2 NS Monounsaturated (g=day) 38.9 13.6 36.4 12.7 33.8 12.9 NS (0.070) Percentage of energy intake 14.0 2.5 13.8 2.6 13.7 3.2 NS Saturated (g=day) 47.3 18.1 44.7 15.8 41.1 16.4 NS (0.059) Percentage of energy intake 16.9 3.3 17.0 3.6 16.6 3.5 NS Carbohydrates (g=day) 244.0 84.1 235.4 86.6 220.2 79.4 NS Percentage of energy intake 39.2 7.2 39.7 7.3 39.9 7.6 NS Alcohol (g=day) 18.4 19.7 15.3 21.2 12.3 17.5 0.047 Percentage of energy intake 4.9 5.1 4.3 5.7 3.5 4.6 NS Values are expressed as mean s.d. large sample of Paris obese subjects, there is a signi cant positive relationship between BMI and energy intake. 1 2) a difference in food assessment methods Ð it has been shown that food intake assessed by dietary history yield higher values than record methods. 11 Nevertheless, we think that the result is reliable because the general trend was the same, although observed in different samples with different methods. A familial aggregation in total energy and macronutrient intake has been already documented, 12,13 but this report is the rst to indicate that a gene polymorphism may partly determine food intake in humans. In mice, two genetic loci have been shown to in uence sucrose consumption. 14 The lower energy intake in overweight people carrying the A allele is concordant with higher frequency of this allele in anorexic patients. Some authors regard anorexia nervosa as a primary eating disorder; for others, it is a possible alternative expression of mood disorder leading to decrease in food intake. 15 The serotonergic system is involved in both kinds of disorders. In our study, we cannot distinguish between these two serotonergic effects. Nevertheless, the speci c involvement in food intake of the 5-HT 2A receptors, besides the above genetic effects, has been shown in rats by the use of speci c agonists. In rats, 5- HT 2A agonists decrease the NPY stimulated food intake. 16 Our results show that genetic variation at the 5- HT 2A receptor locus is associated with differences in alcohol intake. It should be noted that this receptor has been repeatedly involved in high alcohol consumption models by studies showing that 5-HT 2A receptor antagonists inhibited alcohol intake in strains of alcohol-preferring rats. 17,18 The A allele has recently been shown to be less frequent in Japanese alcoholics. 8 Our data show that the effects of the 5- HT 2A polymorphism can be seen also in a general population. The effects on food intake and on alcohol consumption seem to be independent since the differences in food intake according to the 5-HT 2A polymorphism were still signi cant after removing the energy contribution of alcohol. The associations described here are signi cant only at the P < 0.05 level. Therefore they would no longer be signi cant after correction for the number of comparisons, such as Bonferroni's. Since they are described for the rst time, they need to be con rmed in other populations. The A-related differences in food intake were not related to differences in BMI or biological variables. This nding supports the results of Hinney and colleagues, 6 that the 5-HT 2A polymorphism is not associated with obesity. A genotype effect on the clinical characteristics may have arisen during the obesity onset period, and may be not visible in this crosssectional study of weight stable patients. Such effects might be seen in longitudinal studies. The Stanislas cohort, for which data are collected every 5 y, will be investigated in this respect. Acknowledgements We are indebted to the preclinic staff and laboratory department of the Centre for Preventive Medicine of Vandoeuvre-leÁs-Nancy, to the staff of the Nutrition Department of Bichat Hospital Paris, France and to the participating subjects who made this study possible. We are grateful to the public institutions and private companies who support the Stanislas cohort: University of Nancy, INSERM, CNRS, Nancy district, Beckman, BioMeÂrieux, Daiichi, Johnson and Johnson, Merck, Roche and Sebia.

924 References 1 Fricker J, Fumeron F, Clair D, Apfelbaum M. A positive correlation between energy intake and body mass index in a population of 1312 overweight subjects. Int J Obes 1989; 13: 673 ± 681. 2 Leibowitz SF. The role of serotonin in eating disorders. Drugs 1990; 39(Suppl 3): 33 ± 48. 3 Collier DA, Arranz MJ, Li T, Mupita D, Brown N, Treasure J. Association between 5-HT 2A gene promoter polymorphism and anorexia nervosa. Lancet 1997; 350: 412. 4 Sorbi S, Nacmias B, Tedde A, Ricca V, Mezzani B, Rotella CM. 5-HT 2A promoter polymorphism in anorexia nervosa. Lancet 1998; 351: 1785. 5 Enoch MA, Kaye WH, Rotondo A, Greenberg BD, Murphy DL, Goldman D. 5-HT 2A promoter polymorphism 1438G=A, anorexia nervosa, and obsessive-compulsive disorder. Lancet 1998; 351: 1785 ± 1786. 6 Hinney A, Ziegler A, NoÈthen MM, Remschmidt H, Hebebrand J. 5-HT 2A receptor gene polymorphisms, anorexia nervosa, and obesity. Lancet 1997; 350: 1324 ± 1325. 7 Campbell DA, Sundaramurthy D, Markham AF, Pieri LF. Lack of association between 5-HT 2A gene promoter polymorphism and susceptibility to anorexia nervosa. Lancet 1998; 351: 499. 8 Nakamura T, Matsushita S, Nishiguchi N, Kimura M, Yoshino A, Higuchi S. Association of a polymorphism of the 5HT2A receptor gene promoter region with alcohol dependence. Mol Psychiat 1999; 4: 85 ± 88. 9 Siest G, Visvikis S, Herbeth B, Gueguen R, Vincent-Viry M, Sass C, Beaud B, Lecomte E, Steinmetz J, Locuty J, Chevrier P. Objectives, design and recruitment of a familial and longitudinal cohort for studying gene ± environment interactions in the eld of cardiovascular risk: the Stanislas cohort. Clin Chem Lab Med 1998; 36: 35 ± 42. 10 Vauthier JM, Lluch A, Lecomte E, Artur Y, Herbeth B. Family resemblance in energy and macronutrient intakes: the Stanislas Family Study. Int J Epidemiol 1995; 25: 1030 ± 1037. 11 Black AE, Goldberg GR, Jebb SA, Livingstone MBE, Cole TJ, Prentice AM. Critical evaluation of energy intake data using fundamental principles of energy physiology: 2. Evaluating the results of published surveys. Eur J Clin Nutr 1991; 45: 583 ± 599. 12 PeÂrusse L, Bouchard C. Genetics of energy intake and food preferences. In: Bouchard C (ed). The genetics of obesity. CRC Press: Boca Raton, FL, 1994, pp 125 ± 134. 13 Reed DR, Bachmanov AA, Beauchamp GK, Tordoff MG, Price RA. Heritable variation in food preferences and their contribution to obesity. Behav Genet 1997; 27: 373 ± 387. 14 Bachmanov AA, Reed DR, Ninomiya Y, Inoue M, Tordoff MG, Price RA, Beauchamp GK. Sucrose consumption in mice: major in uence of two genetic loci affecting peripheral sensory responses. Mamm Genome 1997; 8: 545 ± 548. 15 Gorwood P, Bouvard M, Mouren-SimeÂoni MC, Kipman A, AdeÁs J. Genetics and anorexia nervosa: a review of candidate genes. Psychiatr Genet 1998; 8: 1 ± 12. 16 Currie JP, Coscinna DV. 5-Hydroxytryptaminergic receptor agonists: effects on neuropeptide Y potentiation of feeding and respiratory quotient. Brain Res 1998; 803: 212 ± 217. 17 Lankford MF, Bjork AK, Myers RD. Differential ef cacy of serotonergic drugs FG5974, FG5893, and amperozide in reducing alcohol drinking in P rats. Alcohol 1996; 13: 399 ± 404. 18 Maurel S, De Vry J, De Beun R, Schreiber R. 5-HT2A and 5- HT2C=5-HT1B receptors are differentially involved in alcohol preference and consummatory behavior in caa rats. Pharmacol Biochem Behav 1999; 62: 89 ± 96.