Jack A. Yanovski, MD, PhD, Head, Unit on Growth and Obesity
Ja Shin Koo, PhD, Postdoctoral Fellow
Marian Tanofsky-Kraff, PhD, Postdoctoral Fellow
Joan Han, MD, Clinical Fellow, ETP ¹
Cong Ning, MD, PhD, Clinical Fellow, ETP ¹
Mary Roberts, MD, Clinical Fellow, ETP ¹
Erica Taylor, MD, Clinical Fellow, CRTP ³
Lisa Yanoff, MD, Clinical Fellow, ETP ¹
Areeg El-Gharbawy, MD, Clinical Fellow, GTP ²
Diane Adler Wailes, MS, Biologist
Deborah Glasofer, BA, Graduate Student
Jennifer Gustafson, BS, Postbaccalaureate Fellow
Carolyn Menzie, BS, Postbaccalaureate Fellow
Lisa Ranzenhofer, BS, Postbaccalaureate Fellow
Margaret Rutledge, BS, Postbaccalaureate Fellow
Abby Fleisch, BS, Medical Student, CRTP ³
Jennifer McDuffie, PhD, Volunteer

The prevalence of overweight and obesity in children and adults has tripled during the past 30 years. The alarming rise in body weight has likely occurred because the current environment affords easy access to calorie-dense foods and requires less voluntary energy expenditure. However, this environment leads to obesity only in those individuals whose body weight regulatory systems are not able to control body adiposity with sufficient precision in our high-calorie/low-activity environment, which suggests that some subgroups in the United States have a uniquely high susceptibility to weight gain under prevailing environmental conditions. Our research is primarily directed at increasing our understanding of the metabolic and behavioral factors involved in determining body weight regulation and body composition during childhood, with a special emphasis on minority populations. The ongoing research program prospectively evaluates risk factors for the development of obesity and its complications in children, studies the effects of medications on body weight and obesity-related co-morbid conditions in children, examines potential methods for modulating thermogenesis in children and adults, and seeks the genetic and environmental factors important for the markedly greater incidence of obesity and its co-morbid conditions in some U.S. minority populations.

Molecular studies of factors important for childhood body weight regulation

Koo, Adler-Wailes, Ning, Han, El-Gharbawy, Tanofsky-Kraff, Gustafson, Ranzenhofer, Taylor, Fleisch, Yanovski J; in collaboration with Aguilera, Farooqi, Leibel, O'Rahilly, Westphal

To identify gene variants that, with different frequencies, affect body composition in African American and Caucasian children, we have been examining polymorphisms in genes involved in the leptin signaling pathway. Genes include those encoding proopiomelanocortin (POMC), POMC processing enzymes, the melanocortin receptors 3, 4, and 5, neuropeptide Y and its receptors, brain-derived neurotrophic factor (BDNF), and the type 2 neurotrophic receptor TrkB. We have previously studied genes important for energy expenditure, such as those encoding the mitochondrial uncoupling proteins, and genes potentially involved in cortisol metabolism that may affect intra-abdominal adipose tissue, such as 11-beta-hydroxysteroid dehydrogenase. We are currently studying a variant melanocortin 3 receptor (MC3R) that is associated with adiposity in children and appears to have functional significance for MC3R signal transduction. We found two missense sequence variants--Thr6Lys and Val81Ile--that showed marked linkage disequilibrium in 355 children. Therefore, we characterized the genotypes based on a combination of the two variants. African American children were more likely to carry the variant alleles such that 15.8 percent of African Americans (versus 1.7 percent of Caucasians) were homozygous for both polymorphisms. Among both African American and Caucasian children, those homozygous for both MC3R alleles had significantly greater BMI-SD score, fat mass, and body circumference measurements and higher plasma levels of insulin and leptin than "wild-type" or heterozygous children.

To study the functional consequences of the MC3R missense variants, we transiently expressed both single- and double-variant MC3R receptors in HEK293 cells. Saturation curves for binding of 125-I-NDP-alpha-MSH showed that the doubly mutant (Thr6Lys and Val81Ile) MC3R bound approximately 60 percent less 125-I-NDP-alpha-MSH than the wild-type receptor. The Kd for the single- or double-variant MC3R did not differ significantly from the wild type. However, the level of surface expression was significantly lower for the doubly mutant MC3R than for the wild-type MC3R. The Val81Ile+Thr6Lys doubly mutant MC3R exhibited significantly lower maximal intracellular cAMP generation than wild-type MC3R. We then transiently expressed MC3R--GFP fusion proteins in LVIP2.0Zc cells, finding decreased protein expression. This is the first report of a significant relationship between these MC3R variants and pediatric obesity. Homozygocity for the doubly mutant MC3R may contribute to the greater prevalence of overweight, particularly in African Americans. Our ongoing studies attempt to understand the mechanisms by which these sequence alterations may affect body weight. We are developing transgenic knockin mice expressing the human wild-type and human doubly mutant MC3R.

We have also recently investigated the BDNF-TrkB pathway with regard to body mass in children. We measured serum BDNF in 328 overweight children, age 3--19 years. BDNF was lower in overweight children, and BMI, BMI-Z, and body fat were all negatively associated with BDNF. The data suggest that some obese individuals with low serum BDNF for age and platelet count may have mutations that alter BDNF function. In collaboration with Sadaf Farooqi and Stephen O'Rahilly, we found that individuals with heterozygous TrkB mutations do not have unusually high circulating BDNF, and we identified a child with hyperphagia and severe obesity associated with functional loss of one copy of the BDNF gene. This child had a de novo chromosomal inversion, 46,XX,inv(11)(p13p15.3), a region encompassing the BDNF gene. The patient's genomic DNA was heterozygous for a common coding polymorphism in BDNF, but we observed monoallelic expression in peripheral lymphocytes. The child's serum BDNF was markedly lower than in age- and BMI-matched control subjects; the child's haploinsufficiency for BDNF was associated with increased ad libitum food intake and severe early onset obesity. These findings provide direct evidence for the role of BDNF in human energy homeostasis. We are undertaking an initiative to assess the role of BDNF haploinsufficiency as a cause of obesity in patients with syndromes resulting from deletions in the vicinity of 11p14.1, where the human BDNF gene is found. Using a comparative genomic hybridization approach that will be verified with in situ hybridization and PCR-based assays, we will examine genotype-phenotype relationships in patients with 11p deletion syndromes.

El-Gharbawy AH, Adler-Wailes DC, Mirch MC, Theim KR, Ranzenhofer L, Tanofsky-Kraff M, Yanovski JA. Serum brain derived neurotrophic factor concentrations in lean and overweight children and adolescents. J Clin Endocrinol Metab2006;91:3548-52.

Physiology, metabolism, and psychology of childhood body weight regulation

Tanofsky-Kraff, Ning, Roberts, Han, Taylor, Fleisch, Glasofer, Gustafson, Rutledge, Ranzenhofer, McDuffie, Yanovski J; in collaboration with Cox, Field, Gorbach, Hubbard, Levine, Malley, Reynolds, Salaita, Schoeller, Sebring, Troendle, Walsh, Wilfley, Yanovski S

Our studies are directed at understanding the physiological, psychological, and metabolic factors that place children at risk for undue weight gain. We have examined how best to measure psychopathology, insulin sensitivity, changes in body composition, and energy expenditure in children. We found that African American children exhibit greater insulin secretion, a greater prevalence of acanthosis nigricans, and less insulin sensitivity, but less visceral adipose tissue than Caucasian children of similar body composition. These results imply that the relationship between visceral fat and the complications of obesity may differ in African Americans and Caucasians. The susceptibility to weight gain in African American children may also result from differences in metabolic efficiency; we found that resting energy expenditure is approximately 90 kcal/d less in African American than in Caucasian normal-weight and overweight boys and girls; we verified the results in overweight children, developing new equations for predicting resting energy expenditure in African American and Caucasian children. Our studies suggest that the differences are not explained by differences in the hormone leptin but rather are driven by variations in body composition and can primarily be explained by the greater appendicular lean body mass observed in African Americans, which is relatively metabolically inactive at rest. Leptin, however, is an important predictor of weight gain in children; longitudinal studies show that leptin levels are related to weight gain. Recent investigations have also documented a greater prevalence of fractures and musculoskeletal complaints in significantly overweight children as well as a greater degree of valgus misalignment of the lower extremities, which may contribute to difficulties with exercise and thus to further weight gain in overweight youth. Our investigations have also found marked effects of obesity on adolescents' quality of life.

Our evaluations concentrating on binge eating behaviors in children suggest that such behaviors are also associated with adiposity in children. We have found that binge eating and dieting behaviors may predict future weight gain in children at risk for overweight. Over an observation period of about four years, children reporting binge-eating behaviors gained, on average, 15 percent more fat mass than non--binge-eating children. Our recent data also suggest that children who engage in binge eating consume more calories during meals. Actual intake during buffet meals averaged 400 kcal more in children with binge eating; despite their greater intake, however, such children reported shorter-lived satiety than children without binge-eating episodes. The ability to consume large quantities of palatable foods, especially when coupled with decreased subsequent satiety, may play a role in the greater weight gain among binge-eating children. Our data also suggest that interventions targeting disordered eating behaviors may be useful in preventing excessive fat gain in children prone to obesity. In two ongoing protocols aimed at determining the factors that are most important for the development of the complications of obesity in children, we study normal-weight African American and Caucasian children and adolescents, already obese African American and Caucasian children , and non-obese African American and Caucasian children of obese parents. We examine body composition, metabolic rate, insulin sensitivity, glucose disposal, and genetic factors believed to regulate metabolic rate and body composition as well as psychological and behavioral factors, such as propensity to engage in binge eating. The longitudinal study will follow the children into adulthood. In a third protocol, we study food consumption of children during meals in order to elucidate differences in the caloric and macronutrient content of meals and the circulating hormones related to hunger and satiety in those who either endorse binge-eating behaviors or report no such behaviors. We hypothesize that differences in these factors predict the development of obesity in the populations studied and may be of great importance in developing rational approaches for preventing and treating obesity in the diverse U.S. population. A new pilot clinical protocol will examine the effects of a targeted interpersonal therapy intervention on body weight change in adolescents who endorse binge-eating behaviors.

Treatment of children and adolescents with co-morbid conditions associated with obesity

Tanofsky-Kraff, Yanoff, Roberts, Adler-Wailes, Ranzenhofer, Rutledge, Gustafson, Menzie, Taylor, Fleisch, McDuffie, Yanovski J; in collaboration with Booth, Calis, Drinkard, Hubbard, Krakoff, Reynolds, Rolls, Salaita, Sebring

Given the rapid increase in the prevalence of obesity, treatments for obesity in childhood are urgently needed. In three ongoing clinical protocols, we are studying approaches for the control of body weight in children. We have completed a pilot study demonstrating that severely overweight adolescents can lose weight when enrolled in a comprehensive weight management program that includes the gastrointestinal lipase inhibitor orlistat as an adjunct to a behavioral modification program, although such a program may demonstrate greater efficacy in Caucasian versus African American children. We also found evidence that one mechanism through which orlistat may affect body weight is by changing the hedonic value of dietary fat. In an ongoing placebo-controlled randomized trial for which 190 of the 220 required participants have been recruited, we will determine whether the use of orlistat improves the weight loss of African American and Caucasian children and adolescents with obesity-related co-morbidities. In the course of these studies, we evaluated cardiorespiratory fitness in significantly overweight children. We hypothesized that most of the difficulty with sustained exercise experienced by severely overweight adolescents occurs because the metabolic costs of moving excess mass result in the use of a high proportion of the adolescents' total oxygen reserve. We compared results from a maximal cycle ergometry fitness test in 129 severely overweight adolescents with BMI 41.5±9.7 kg/m2 and 34 non-overweight adolescents with BMI 20.1±2.9 kg/m2. We compared oxygen uptake (VO2) at three times: during a four-minute period of unloaded cycling (ULVO2), at the lactate threshold estimated by gas exchange (LTVO2), and at maximal exertion (VO2 max); we measured heart rate at rest and at VO2 max. Subjects also completed a 12-minute walk/run performance test to obtain distance traveled (D12) and heart rate. Absolute LTVO2 and VO2 max and LTVO2 as a percentage of VO2 max did not differ in overweight and non-overweight children during the cycle test. However, absolute ULVO2 was significantly greater in overweight adolescents. Resting heart rate before initiating the cycle test was significantly greater in overweight than in non-overweight adolescents. However, maximal heart rate during the cycle test was significantly lower in overweight adolescents. During the walk-run test, mean D12 was significantly shorter for overweight than for non-overweight adolescents. D12 was negatively related to BMI-SD and ULVO2. Thus, overweight and non-overweight adolescents had similar absolute VO2 at the lactate threshold and maximal exertion, suggesting that overweight adolescents are more limited by the increased cardiorespiratory effort required to move their larger body mass than by cardiorespiratory deconditioning. The higher percentage of oxygen consumed during submaximal exercise indicates that overweight adolescents are burdened by the metabolic cost of their excess mass. Their greater oxygen demand during an unloaded task predicted poorer performance during sustained exercise. The data suggest that exercise prescriptions for overweight adolescents should account for limited exercise tolerance imposed by excess body mass, focusing on activities that keep demands below lactate threshold so that exercise can be sustained. A second study examines the mechanism by which metformin may affect the body weight of younger children with hyperinsulinemia, which potentially puts such children at risk for later development of type 2 diabetes. To date, we have recruited 90 of the 100 children required for this study.

Environmental factors affecting weight gain

Yanoff, Menzie, Han, Roberts, Yanovski J; in collaboration with Gorbach, Levine, Salaita, Sebring, Yanovski S

We are interested in assessing the impact of the environment on body weight gain. To determine the role of seasonal variation in weight change, we prospectively studied 200 U.S. adults, following them longitudinally. We found that the only time when weight changes significantly is during the fall-winter holiday season between Thanksgiving and New Year's Day. Such a study has implications for individuals attempting to control their weight. Recent clinical initiatives that study modifiable factors affecting body weight examine how to augment non-exercise activity thermogenesis in children and methods to improve body heat dissipation during physical activity in adults.

We have also studied the impact of chemotherapy on weight gain in women with breast cancer, finding redistribution of body mass from lean tissues to fat mass during the first year after chemotherapy. We also found that development of estrogen deficiency due to chemotherapy is a risk factor for weight change in women treated for breast cancer. Because the treatment of HIV with protease inhibitors is also associated with development of visceral obesity, we studied the effects of protease inhibitors on lipolysis and lipogenesis in vitro.

Given that some epidemiological studies have suggested that those with greater calcium intake gain less weight over time, we are interested in the role played by dietary calcium in determining body weight change. One hypothesis for how calcium intake may affect body adiposity holds that calcium alters plasma concentrations of calcitropic hormones such as 1,25 dihydroxy Vitamin D. 1,25 dihydroxy Vitamin D has been found in vitro to stimulate lipogenesis. Others have hypothesized that deficient calcium intake stimulates parathyroid hormone and promotes conversion of 25 hydroxy Vitamin D to 1,25 dihydroxy Vitamin D, thereby stimulating lipogenesis and leading to greater body weight in humans. We determined that 1,25 dihydroxy Vitamin D concentrations are not increased in overweight and obese adults; rather, Vitamin D deficiency (and lower 1,25 dihydroxy Vitamin D concentrations) is observed among severely obese adults. Our studies have led us to examine the prevalence of Vitamin D deficiency in obese African Americans and white Americans. Because both obesity and African American race have been associated with a higher risk of Vitamin D deficiency and secondary hyperparathyroidism, we hypothesized that the risk of hypovitaminosis D would therefore be extraordinarily high in obese African American adults. In a study of 379 African American and white adults whose BMI ranged from 19.9--58.2 kg/m2, obese African American subjects had lower mean 25(OH)D than obese whites, non-obese African Americans, and non-obese whites. The prevalence of hypovitaminosis D increased with increasing BMI and was greater in African Americans than whites within all BMI categories examined. Among subjects with BMI greater than 35 kg/m2, three times as many African Americans had hypovitaminosis D as whites. iPTH was negatively correlated with 25(OH)D, suggesting that those with hypovitaminosis D had clinically important Vitamin D deficiency with secondary hyperparathyroidism. One-third of African Americans met the criteria for secondary hyperparathyroidism as compared with about 10 percent of whites. These findings have clinical implications; physicians should consider routinely supplementing such patients with Vitamin D or screening them for hypovitaminosis D.

A recently completed protocol examined the impact of dietary calcium supplementation on body weight on 340 subjects enrolled between March 2002 and December 2003. Their mean dietary intake of calcium at baseline was 882±391 mg per day; 23 percent reported dietary calcium intake of less than 600 mg per day. Comparing subjects randomized to take calcium (1,500 mg per day) with those given a placebo over the two-year study interval, we found no significant differences in body weight change, BMI change, or body fat mass. Calcium carbonate supplementation thus did not significantly improve body composition of overweight and obese adults over a two-year interval.

¹ Endocrine Training Program
² Genetics Training Program
³ Clinical Research Training Program

Collaborators

Greti Aguilera, MD, Developmental Endocrinology Branch, NICHD, Bethesda, MD
Sarah Booth, PhD, Human Nutrition Research Center on Aging at Tufts University, USDA, Boston, MA
Karim Calis, Pharm D, Pharmacy Department, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Christopher Cox, PhD, The Johns Hopkins University, Baltimore, MD
Bart Drinkard, PT, Rehabilitation Medicine Department, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
I. Sadaf Farooqi, MD, Cambridge Institute of Medical Research, Cambridge, UK
Alison Field, DSc, Channing Laboratory, Brigham and Women's Hospital, Boston, MA
Alexander Gorbach, PhD, Division of Bioengineering and Physical Science, Office of Research Services, NIH, Bethesda, MD
Van S. Hubbard, MD, PhD, Division of Nutritional Research Coordination, NIDDK, Bethesda, MD
Jonathan Krakoff, MD, Clinical Diabetes and Nutrition Section/Phoenix Epidemiology and Clinical Research Branch, NIDDK, Phoenix, AZ
Rudolph L. Leibel, MD, Columbia University College of Physicians and Surgeons, New York, NY
James Levine, MD, PhD, Mayo Clinic, Rochester, MN
James D. Malley, PhD, Mathematical and Statistical Computing Laboratory, CIT, Bethesda, MD
Stephen O'Rahilly, MD, Cambridge Institute of Medical Research, Cambridge, UK
James Reynolds, MD, Nuclear Medicine, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Barbara J. Rolls, PhD, Pennsylvania State University, University Park, PA
Christine Salaita, MS, RD, Nutrition Department, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Dale A. Schoeller, PhD, University of Wisconsin, Madison, WI
Nancy Sebring, MEd, RD, Nutrition Department, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
James Troendle, Ph.D., Biometry and Mathematical Statistics Branch, NICHD, Bethesda, MD
B. Timothy Walsh, PhD, Columbia University College of Physicians and Surgeons, New York, NY
Heiner Westphal, MD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD
Denise E. Wilfley, PhD, Washington University School of Medicine, St. Louis, MO
Susan Z. Yanovski, MD, Obesity and Eating Disorders Program, NIDDK, Bethesda, MD

For further information, contact yanovskj@cc1.nichd.nih.gov.