2009 Annual Report 1a.Objectives (from AD-416) 1) Identify a number of genes that affect the expression of childhood obesity in Hispanic children, investigate strong positional candidate obesity-related genes, and test if weight changes and metabolic, hormonal and immunologic responses to weight changes are dependent upon genotype;. 2)Establish a reference model of body composition in children;. 3)Identify barriers and facilitators for the physical activity component of the 2005 Dietary Guidelines for Americans and relate to obesity risk in urban, African-and Mexican-American children and families;. 4)Determine the contribution of leukocytes to the composition and function of adipose tissue, investigate the influence of dietary factors on the composition of leukocytes within adipose tissue and liver, and explore how obesity can both reduce host resistance and enhance inflammatory tissue injury;. 5)To determine whether a 12-week exercise program without intent to weight loss would increase insulin sensitivity and reduce insulin secretion and glucose production from gluconeogenesis in obese adolescents, and if so, whether these changes are associated with a decrease in intramyocellular and intrahepatic fat content; and. 6)To develop a greater understanding that altered sleep patterns associated with our "24-hour" lifestyle may contribute to the accumulation of body fat, and that such altered sleep patterns may ultimately represent alterations in both the central and peripheral circadian clock mechanisms. 1b.Approach (from AD-416) 1) A systemic genomic scan and follow-up fine mapping and sequencing of positional candidate genes will be performed on 300 overweight Hispanic children and their biological parents and siblings with respect to adiposity, the regulation of food intake, energy expenditure and energy partitioning before and after weight loss.. 2)A 5-level body composition model will be determined from multiple-method body composition assessments in 1500 multi-ethnic children. The predictive accuracy of the model for the individual will be verified by longitudinal restudy.. 3)Implement an intensive physical activity intervention in urban African- and Mexican-American families that will assist in determining specific barriers/facilitators for children and families adhering to the physical activity component of the 2005 Dietary Guidelines.. 4)Animal models (murine) and human tissue will be used to characterize myeloid and lymphoid cells within adipose tissue and changes that occur in mice fed high fat diets. Phenotypic markers and in situ hybridization will be used to characterize the cell types, ultrastructural studies, and confocal microscopy will define the position of these cells in relationship to other structural components of the adipose tissue, and tissue fractionation techniques will be used to isolate these cells for function studies in vitro. Cell lines (e.g., RAW cells and 3T3 cells) will be used to study cytokine, chemokine and hormone release, adipocyte differentiation and lipid metabolism. Contributions of the immune cells to lipid metabolism in vivo will be sought in mice with disrupted or activated (e.g., with endotoxin) immune cell functions.. 5)Insulin sensitivity and secretion will be studied using stable isotopes, and intramyocellular and intrahepatic fat content will be determined using MRI.. 6)Through employing both hypothesis-generating and hypothesis-testing approaches to ascribe roles for the circadian clock within the adipocyte will increase our understanding of altered sleep patterns and how they may contribute to the accumulation of body fat. 3.Progress Report Analysis of genetic variation in clock genes as it relates to obesity, body composition, & sleeping behavior in humans is underway, & we expect several publications from this work. Using our animal model of adipocyte-specific circadian disruption, we have determined that this genetic alteration directly influences the timing of feeding & that alterations in the timing of feeding can produce obesity, even in wildtype animals. A paper describing this work is currently in preparation. (Project. 1)Childhood obesity & its associated comorbidities continue to grow in epidemic proportions in the US, particularly among minority children. Community-based programs are needed to address this serious public health problem. Forty obese Hispanic children (7-12 y) were randomized to 2 treatment groups that consisted of weekly 45-minute behavior modification & nutrition education consultations, with or without structured aerobic exercise. Changes in weight & body composition were achieved, as well as improvements in several cardiovascular-diabetes risk factors such as total cholesterol, LDL-cholesterol, triglycerides, glucose, insulin, liver enzymes & inflammatory markers. Better understanding of the impact of weight loss & body composition changes on obesity-related risk factors will facilitate meaningful goal setting in the treatment of childhood obesity. (Project. 2)Based on extensive mathematical modeling, height (stature) was identified as the most critical anthropometric parameter, followed by age, gender, & ethnicity, for defining or standardizing body compositional changes during growth. This modeling is being used to update the Z-score prediction models available on the Children's Nutrition Research Center's website. (Project. 3)We have successfully conducted the intervention phase of a randomly controlled trial examining the impact of physical activity in minority students. A preliminary analysis of our outcomes suggests this intervention has significant health benefits. We are currently on track to examine longer-term outcomes through a maintenance program. (Project. 4)We have compared diets that are matched for nutrients except for the sources & quantities of fat in studies of diet induced obesity & metabolic syndrome in a murine model. The milk fat diet, high in saturated fatty acids, induces adipose tissue inflammation & hepatic steatosis more rapidly than the corn oil diet, rich in n-6 fatty acids. We have used this model to investigate contributions of T cells, toll-like receptors (TLRs) & adhesion molecules in diet-induced pathology. (Project. 5)A total of 30 post pubertal adolescents (14 lean & 16 obese) have completed a 12-week aerobic exercise program & 12 obese post-pubertal adolescents have completed a 12-week resistance exercise program during the past year. Eight of the subjects have been studied during this time period. The results demonstrate that the aerobic exercise program increased fitness & both hepatic & peripheral insulin sensitivity; reduced intra-abdominal fat &, importantly, reduced hepatic fat. The resistance exercise primarily increased muscle mass & hepatic insulin sensitivity. (Project 6) 4.Accomplishments 1. Linking Adiposity to Altered Circadian Rhythms within the Fat Cell: Individuals who perform shift work or who experience chronic disruptions in sleep are at increased risk for the development of obesity, type 2 diabetes, and cardiovascular disease, but the mechanisms behind this are not known. Disruptions of our internal molecular body clocks, which reside both in the brain and within every cell in the body, are thought to be at least 1 link between the development of metabolic disease and disruptions in daily circadian patterns of behavior. These body clocks can be disrupted by mutations in the genes that encode components of the clock mechanisms. Children's Nutrition Research Center researchers have investigated DNA sequence variations in circadian clock genes for an association to body size and composition, and we have shown that DNA sequence variation in several of the clock genes is related to BMI, waist/hip ratio, and weight gain over time in humans. In addition to our human studies, we have developed a unique animal model, the Adipocyte Clock Mutant (ACM), in which the circadian clock transcription mechanism is disrupted in a tissue-specific manner within the adipocyte. We have now established that both male and female ACM animals eating standard chow are heavier and fatter than their control counterparts by 9 weeks of age (males) to 11 weeks of age (females) and that these ACM animals are also less glucose tolerant compared to wildtype mice and have altered lipid metabolism. Calorimetry studies have revealed that ACM animals exhibit alterations in the timing of feeding--specifically, these animals ingest a large portion of calories in the last 4 hours of their waking cycle. When these same alterations in eating behavior are presented to wildtype mice, wildtype mice also gain weight. These studies revealed something linking disturbances of the circadian clock to altered feeding patterns and obesity. (Project 6: Circadian rhythms in adipose biology & obesity) 2. Genetic Susceptibility to Obesity in Hispanic Children: Obesity is a complex disease influenced by genes and the environment. The VIVA LA FAMILIA Study was designed to identify genetic and environmental factors affecting childhood obesity and its related health risk in 1030 Hispanic children. Children's Nutrition Research Center scientists performed genetic studies to identify chromosomal regions that influence obesity, followed by statistical analysis. This year we identified and reported on two chromosome regions for energy metabolism and cytokines associated with obesity. Understanding the genetic and environmental factors contributing to childhood obesity will permit identification and targeting of children who are at increased genetic risk and who will benefit from specific therapies or lifestyle interventions. (Project 1: Genetic and environmental factors contributing to childhood obesity) 3. Effective Intervention for Weight Loss with Minorities: Few studies with the goal of improving the health of children and adolescents through weight loss actually result in a reduction of weight. Children's Nutrition Research Center researchers conducted a school-based intervention that resulted in improved weight and clinical outcomes in overweight Mexican American children. Our research team accomplished this by designing and successfully implementing a novel approach, which included a high level of intensity and parental involvement, to weight loss in an applied setting. This study provides important information about successful weight loss strategies with minority students and will potentially assist school professionals, health care providers, and policy makers in delivering effective interventions.(Project 3: Prevention of overweight in children) 4. White blood cells in Murine Fat Tissue: High-fat diets rich in fats common to the American diet induce obesity and diabetes in normal mice, and the mechanisms by which these diets cause disease are poorly understood. Children's Nutrition Research Center researchers investigated the hypothesis that white blood cells are present in fat tissues within the abdomen and cause inflammation in these tissues in response to the high-fat diet. Since there are many different types of white blood cells, it was important to first analyze the tissues to determine which types are present. Two types were found in fat tissues of lean mice, one called T cells (a type of lymphocyte) and the other called macrophages. The high-fat diets caused increases in both lymphocytes and macrophages that continued as the degree of obesity increased. The investigators then studied mice genetically engineered to be deficient in one type of T cell (the gamma delta T cell) and found that these mice were significantly protected from the inflammation and diabetic complications accompanying progressive obesity. This is the first demonstration that this subset of T cells is important to disease produced by high-fat diets. (Project 4: Nutritional influences on innate immunity) 5. Toll-like Receptors (TLRs) and Diet-Induced Obesity: High-fat diets rich in fats common to the American diet induce obesity and diabetes in normal mice. Children's Nutrition Research Center researchers investigated the hypothesis that high fat diets may cause an inappropriate activation of a portion of the body's immune system called innate immunity which is necessary for resistance to infection, but if inappropriately activated may cause tissue damage or disease. One aspect of innate immunity is a family of cell surface proteins called Toll-like Receptors, or TLRs, that serve as danger signals when tissues are invaded by infection, in which they activate the inflammatory processes that are necessary for killing the invading organisms. However, if the TLRs are falsely activated by some types of fat in the diet they may cause inflammation and tissue injury. The investigators studied mice that were genetically engineered to be deficient in either TLR2 or TLR4 to determine if either of these TLRs is involved in diet-induced inflammation in abdominal fat tissues. TLR2 deficient mice failed to develop fat tissue inflammation, diabetes, blood lipid abnormalities, and obesity, indicating that TLR2 may be important in all aspects of high-fat-diet-induced diseases; while in contrast, TLR4-deficient mice were protected from the fat tissue inflammation and diabetes but not the blood lipid abnormalities and obesity. This is the first demonstration of the contributions of TLR2 in the causation of obesity and of distinctions between two members of the TLR family. (Project 4: Nutritional influences on innate immunity) 6. Adhesion Molecules in Diet-Induced Obesity: Children's Nutrition Research Center researchers have shown that a cell surface protein known as intercellular adhesion molecule-1, or ICAM-1, is produced in fat tissue in mice during the development of diet-induced obesity. One of the functions of this protein is known to be important for the migration of white blood cells into tissue during the development of inflammation such as that caused by infection. Since it is known that high fat diets induce inflammation in fat tissues, the investigators wished to determine if ICAM-1 is important for the migration of white blood cells into fat tissue during diet-induced obesity. This was done by analyzing mice genetically engineered to be deficient in ICAM-1. It was found that in contrast to the hypothesis, the absence of ICAM-1 did not alter the accumulation of white blood cells into abdominal fat tissues of mice on a high fat diet. Others have suggested that ICAM-1 could be a therapeutic target for disruption of the inflammatory effects of obesity, but our data fail to support this concept. (Project 4: Nutritional influences on innate immunity) 7. Effects of Exercise Without Weight Loss on Metabolism and Body Composition in Adolescents: CNRC scientists seek to determine if controlled aerobic and resistance exercise programs would improve body composition and insulin sensitivity in insulin-resistant obese adolescents and lean adolescents (controls). We enlisted study participants to attend onsite sessions twice a week for 12 weeks, with each session consisting of 30 minutes of an aerobic program and a 1-hour resistance exercise program, with the participants in the aerobic exercise program performing an additional 2 sessions (30 minutes each) at their home. Energy expenditure, substrate oxidation, and an infusion study was performed to measure glucose and lipid metabolism as well as insulin sensitivity, and fat distribution was measured. We were able to show that obese sedentary adolescents were able to complete either program (attendance about 90% of the exercise sessions) all that all metabolic measurements were successfully accomplished. These results demonstrate that obese adolescents can complete both aerobic and resistance exercise programs and that exercise in itself (i.e., without the intent of weight loss) has important effects on body fat distribution and insulin sensitivity, and that aerobic exercise affects metabolically important fat compartments (i.e., visceral and hepatic fat) as well as both peripheral and hepatic insulin sensitivity. The primary effect of resistance exercise was increased muscle mass and hepatic insulin sensitivity. Since increased muscle mass is attractive for obese subjects, while the more comprehensive effects of aerobic exercise are important for the overall prevention of obesity related illness, future studies are needed determine the effects of combined aerobic and resistance exercise. (Project 5: Effects of dietary macronutrient distribution and exercise on glucose metabolism in obese adolescents) 6.Technology Transfer Number of Web Sites Managed 1 Review Publications Bray, M.S., Young, M.E. 2008. Diurnal variations in myocardial metabolism. Cardiovascular Research. 79(2):228-237. Jackson, A.S., Ellis, K.J., McFarlin, B.K., Sailors, M.H., Bray, M.S. 2009. Cross-validation of generalised body composition equations with diverse young men and women: The Training Intervention and Genetics of Exercise Response (TIGER) Study. British Journal of Nutrition. 101(6):871-878. Sunehag, A.L., Man, C.D., Toffolo, G., Haymond, M.W., Bier, D.M., Cobelli, C. 2009. beta-Cell function and insulin sensitivity in adolescents from an OGTT. Obesity. 17(2):233-239. Swinburn, B.A., Sacks, G., Lo, S.K., Westerterp, K.R., Rush, E.C., Rosenbaum, M., Luke, A., Schoeller, D.A., Delany, J.P., Butte, N.F., Ravussin, E. 2009. Estimating the changes in energy flux that characterize the rise in obesity prevalence. American Journal of Clinical Nutrition. 89(6):1723-1728. Bray, M.S., Hagberg, J.M., Perusse, L., Rankinen, T., Roth, S.M., Wolfarth, B., Bouchard, C. 2009. The human gene map for performance and health-related fitness phenotypes: The 2006-2007 update. Medical Science Sports Exercise. 41(1):34-72. Motil, K.J., Ellis, K.J., Barrish, J.O., Caeg, E., Glaze, D.G. 2008. Bone mineral content and bone mineral density are lower in older than in younger females with Rett syndrome. Pediatric Research. 64(4):435-439. Bielinski, S.J., Pankow, J.S., Bray, M.S., Kao, W.H., Folsom, A.R. 2008. Lack of association between uncoupling protein-2 Ala55Val polymorphism and incident diabetes in the atherosclerosis risk in communities study. Acta Diabetologia. 45(3):179-182. Lalani, S.R., Thakuria, J.V., Cox, G.F., Wang, X., Bi, W., Bray, M.S., Shaw, C., Cheung, S.W., Chinault, A.C., Boggs, B.A., Ou, Z., Brundage, E.K., Lupski, J.R., Gentile, J., Waisbren, S., Pursley, A., Ma, L., Khajavi, M., Zapata, G., Friedman, R., Kim, J.J., Towbin, J.A., Stankiewicz, P., Schnittger, S., Hansmann, I., Ai, T., Sood, S., Wehrens, H., Martin, J.F., Belmont, J.W., Potocki, L. 2009. 20p12.3 microdeletion predisposes to Wolff–Parkinson–White syndrome with variable neurocognitive deficits. Journal of Medical Genetics. 46(3):168-175. Maskarinec, G., Verheus, M., Steinberg, F.M., Amato, P., Cramer, M.K., Lewis, R.L., Murray, M.J., Young, R.L., Wong, W.W. 2009. Various doses of soy isoflavones do not modify mammographic density in postmenopausal women. Journal of Nutrition. 139:981-986. Wadden, T.A., West, D.S., Neiberg, R.H., Wing, R.R., Ryan, D.H., Johnson, K.C., Foreyt, J.P., Hill, J.O., Trence, D.L., Vitolins, M.Z. 2009. One-year weight losses in the Look AHEAD study: factors associated with success. Obesity. 17(4):713-722. Robinson, S.F., Bielamowicz, M.K., Rodgers, A.S., Wong, W.W., Konzelmann, K.L. 2008. Designing a bone health and soy focus group discussion guide based on the health belief model. Electronic - Journal of Extension. Available: http://www.joe.org/joe/2008february/rb3.php. Bray, M.S. 2008. Implications of gene-behavior interactions: Prevention and intervention for obesity. Obesity. 16(Suppl.3):S72-S78. Butte, N.F. 2009. Impact of infant feeding practices on childhood obesity. Journal of Nutrition. 139(2):412S-416S. Patrick, T.A., Rosner, G.L., Matthay, K.K., Moore, T.B., Bomgaars, L.R., Ellis, K.J., Renbarger, J., Berg, S.L. 2009. Impact of body composition on pharmacokinetics of doxorubicin in children: A Glaser Pediatric Research Network study. Cancer Chemotherapy and Pharmacology. 64:243-251. McFarlin, B.K., Johnston, C.A., Tyler, C., Hutchison, A.T., Kueht, M.L., Reeves, R., Foreyt, J.P. 2007. Inflammatory markers are elevated in overweight Mexican-American children. International Journal of Pediatric Obesity. 2(4):235-241.