Differential epigenomic and transcriptomic responses in subcutaneous adipose tissue between low and high responders to caloric restriction1–3 Luigi Bouchard, Re´mi Rabasa-Lhoret, May Faraj, Marie-E`ve Lavoie, Jonathan Mill, Louis Pe´russe, and Marie-Claude Vohl ABSTRACT weight loss responses to caloric restriction show considerable Background: Caloric restriction is recommended for the treatment interindividual variability (7). Studies of genetically identical of obesity, but it is generally characterized by large interindividual monozygotic twins have been particularly useful in disentangling variability in responses. The factors affecting the magnitude of the role of environmental and heritable factors in determining the weight loss remain poorly understood. Epigenetic factors (ie, heri- degree of weight loss. It has been shown that within-pair changes table but reversible changes to genomic function that regulate gene in body fat variability after a caloric deficit is significantly lower expression independently of DNA sequence) may explain some of than between-pair variability, which suggests that genetic factors the interindividual variability seen in weight-loss responses. have an important influence on an individual’s response to caloric Objective: The objective was to determine whether epigenetics and deficit (8, 9). However, the concordance between twin pairs was gene expression changes may play a role in weight-loss responsiveness. not complete, which suggests that environmental factors or other Design: Overweight/obese postmenopausal women were recruited DNA sequence–independent mechanisms may be involved. for a standard 6-mo caloric restriction intervention. Abdominal sub- It has been suggested that monozygotic twin discordance for cutaneous adipose tissue biopsy samples were collected before (n = complex traits such as body weight could be accounted for by 14) and after (n = 14) intervention, and the epigenomic and tran- epigenetic factors (10, 11). Epigenetics refers to the heritable, but scriptomic profiles of the high and low responders to dieting, on the reversible, regulation of various genomic functions, including basis of changes in percentage body fat, were compared by using gene transcription, that are mediated principally through changes microarray analysis. in DNA methylation and chromatin structure (12). The epigenetic Results: Significant DNA methylation differences at 35 loci were regulation of cellular functions is a normal and essential process found between the high and low responders before dieting, with 3 in cell development and differentiation, and epigenetic factors are regions showing differential methylation after intervention. Some of subjected to reprogramming in response to both stochastic and these regions contained genes known to be involved in weight con- environmental stimuli (12). Such changes can be mitotically trol and insulin secretion, whereas others were localized in known imprinted genomic regions. Differences in gene expression profiles were observed only after dieting, with 644 genes being differen- 1 From the Nutraceuticals and Functional Foods Institute (LB, LP, and M- tially expressed between the 2 groups. These included genes likely CV), the Department of Preventive Medicine (LP), and the Department of to be involved in metabolic pathways related to angiogenesis and Food Science and Nutrition (M-CV), Universite´ Laval, Laval, Canada; the cerebellar long-term depression. Department of Medicine, Universite´ de Montre´al, Chicoutimi Hospital, Sa- Conclusions: These data show that both DNA methylation and gene guenay, Canada (LB); the Department of Nutrition (RR-L, MF, and M-EL), expression are responsive to caloric restriction and provide new the Montreal Diabetes Research Centre (RR-L and MF), Universite´ de Mon- tre´al, Montreal, Canada; and the Institute of Psychiatry, SGDP Research insights about the molecular pathways involved in body weight loss Centre, King’s College London, London, United Kingdom (JM). as well as methylation regulation during adulthood. Am J Clin 2 Supported by the Canadian Institute of Health Research through the Nutr 2010;91:309–20. MONET project (Montreal-Ottawa New Emerging Team; OHN-63279 and MOP62976) and the Quebec New Emerging Team (OHN 63276). LB was INTRODUCTION funded by the Laval University Merck Frosst/Canadian Institute of Health Research Chair in Obesity and the Heart and Stroke Foundation of Canada/ In 2005, 1.1 billion adults and 10% of children were over- Sanofi-Aventis research fellowship awards. RRL was supported by the Fonds weight or obese worldwide (1). Obesity is defined as an excessive de la Recherche en Sante´ du Que´bec and held the chair for clinical research accumulation of fat resulting from a long-term imbalance be- J-A de Se`ve at IRCM (Montreal Institute for Clinical Research). MF was the tween energy intake and expenditure (2). It has a dramatic effect recipient of the Canadian Institute of Health Research New Investigator on an individual’s health, with musculoskeletal, pulmonary, and Award. M-EL was supported by a scholarship from the Fonds de la Re- ´ ´ psychosocial-related problems, and is associated with an in- cherche en Sante du Quebec. 3 Address correspondence to M-C Vohl, Lipid Research Center, 2705 creased risk of morbidity and mortality attributable to cardio- Laurier Boulevard, (TR93), Que´bec City, PQ, Canada G1V 4G2. E-mail: vascular diseases, diabetes, and certain forms of cancer (1, 3). [email protected]. Interestingly, only a moderate loss of initial body weight Received May 18, 2009. Accepted for publication November 2, 2009. provides significant metabolic improvements (4–6). However, First published online November 25, 2009; doi: 10.3945/ajcn.2009.28085. Am J Clin Nutr 2010;91:309–20. Printed in USA. Ó 2010 American Society for Nutrition 309 310 BOUCHARD ET AL stable and enduring, producing long-term changes to gene ex- adipose tissue biopsy collections were preceded by a 4-wk pression, but can also be short-lived and rapidly reversed (13). weight-stability period (within 62 kg), verified on a weekly Indeed, such dynamic epigenetic changes have the potential to basis at our research unit. Also, the subjects were instructed not offer a mechanism by which cellular metabolism can be rapidly to exercise and to eat a high-carbohydrate diet for the 3 d before regulated independently of long-term, irreversible evolutionary the biopsy procedure. Fasting baseline and postintervention mutagenesis. Cytosine methylation (Cmet), occurring at position subcutaneous adipose tissue biopsy samples were obtained from 5 of the cytosine pyrimidine ring in CpG dinucleotides, is the the periumbilical level at both sides of the body by needle bi- best understood epigenetic modification. The methylation of opsy under local anesthesia (20 mg xylocaine/mL) (18–20). CpG sites disrupts the binding of transcription factors and at- One hundred thirty-seven women were recruited and com- tracts methyl-binding proteins that are associated with gene si- pleted the 6-mo weight-loss program. Baseline and post- lencing and chromatin compaction (14). Because CpG intervention adipose tissue biopsy samples were available for 29 dinucleotide methylation is associated with the regulation of of these women. Consent for biopsy was an optional part of the gene expression, altered DNA methylation could explain in- larger study. There was no other criteria to be met to be included terindividual phenotypic differences (15). in the biopsy subsample. Fourteen women were further selected We report here the results of the first comprehensive analysis of for the current study based on their response to the caloric re- epigenomic and transcriptomic responses in subcutaneous adi- striction. Women who lost 3% of their body fat were consid- pose tissue after a caloric restriction intervention in overweight ered “high responders,” whereas those who lost ,3% of their and obese women. We found that although both DNA methyl- body fat were considered “low responders” to the caloric re- ation and gene expression differences existed between the high striction. Low (n = 7) and high (n = 7) responders were matched and low responders to dietary restriction after the intervention, for baseline age, BMI, percentage body fat, resting blood only epigenetic differences exist before dieting. These data pressure, fasting blood lipids, glucose and insulin concen- suggest that the epigenetic profile has the potential to differentiate trations, and changes in fat-free mass (Tables 1 and 2). between good and poor responders to caloric restriction. Anthropometric and metabolic measurements SUBJECTS AND METHODS Standardized procedures were used to measure body weight, height, waist girth, resting blood pressure, and blood lipid, Study population and experimental design glucose, and insulin concentrations, as previously described (16, The sample used in this study was a subsample of a larger 17). Total energy expenditure was measured by using the doubly weight-loss study (n = 137) aimed at exploring the effect of labeled water technique (21), and resting metabolic rate was a caloric restriction intervention on body composition, energy measured by indirect calorimetry (16, 17). Total fat mass and fat- expenditure, insulin sensitivity, and metabolic, inflammatory, free mass were measured by dual-energy X-ray absorptiometry hormonal, and psychosocial profiles in overweight and obese (16, 17). postmenopausal women. The recruitment for the larger study began in May 2003. The