2008 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 We made progress in characterizing our mutant mice, in terms of gene expression, feeding response, & adipocyte-specific gene expression (Project 1). A pilot study was conducted to test the effectiveness of diet behavior modification or diet behavior modification with structured aerobic exercise for preventing unhealthy weight gain & improvement in fitness, psychological state, & comorbidity risk in Hispanic children (Project 2). The body composition database was updated during the year with additional longitudinal data for African-American males, ages 8-18 years. Software for Least-Medium-Squares (LMS) modeling was obtained & installed, with preliminary testing for age-specific model of body cell mass for females. We explored designs of an instrument for longitudinal assessment of body protein & fat mass in transgenic mice. Variations in design were tested using simulation software (Project 3). We recruited over 200 minority children to determine specific barriers & facilitators to physical activity (PA). We created an extensive database & conducted preliminary analyses to determine the barriers & facilitators of PA for this group (Project 4). We have determined that deficiency of yo T cells results in suppression of proinflammatory cytokines even as long as 6 months on the high fat diet, that diet induced elevations in insulin resistance are significantly reduced, that increases in CD11c+ macrophages fails to occur even though macrophages numbers remain elevated in adipose tissue, & that mice deficient in CD11c exhibit reduced inflammation exhibited by significant reductions in proinflammatory cytokines & reduced numbers of macrophages in the adipose tissue (Project 5). 17 new subjects completed the exercise program & the two metabolic studies, which include calorimetry, stable isotope infusions, DXA & MRI. Analyses of total, visceral & intrahepatic fat as well as insulin & glucose concentrations pre & post exercise have been completed. Statistical analyses have been performed & the data have been summarized & interpreted & a manuscript is in process (Project 6). [NP107, Component 6] 4.Accomplishments 1. Identifying the Obese through VIVA LA FAMILIA: The VIVA LA FAMILIA Study was designed to identify genetic and environmental factors affecting childhood obesity and its comorbidities in the Hispanic population. Children's Nutrition Research Center researchers detected a highly significant linkage signal on a specific region of chromosome (18q) that influences sedentary activity in Hispanic children, and suggestive linkage for total activity counts, percent awake time in light or moderate activity, and carbohydrate intake. Given its role in the regulation of food intake and energy expenditure, melanocortin 4 receptor gene (MC4R) is a strong candidate gene responsible for the association for physical activity and dietary intake in Hispanic children. Through resequencing the MC4R gene and part of its flanking regions, our researchers identified seven changes in the genetic sequence of this gene, three of which are novel, nonsynonymous mutations in our cohort. In the flanking regions around MC4R, we have identified 18 genetic single nucleotide polymorphisms ranging in frequency from 0.2 to 35.9%. These findings are significant since the mapping and identification of genes that influence childhood obesity and its comorbidities ultimately will permit the identification of children who are at increased genetic risk and who will most benefit from specific therapies or lifestyle changes. [NP107, Component 6] (CNRC Project 1) 2. Increasing Physical Activity in Hispanic Children: Ethnic minorities have the highest rates of obesity, which is closely associated with decreased amounts of physical activity. Researchers at the Children's Nutrition Research Center, Houston, TX, examined specific facilitators and barriers to physical activities that were reported by minority children and their parents. Based on this information, our researchers developed a pilot program to increase the amount of physical activity these children receive. Research results show that this intervention has the possibility of not only improving physical activity but also reducing overall disease risk. In light of the current obesity epidemic, this study has the potential to inform service providers, researchers, and policy makers of effective ways to improve minority children's health. [NP107, Component 6] (CNRC Project 3) 3. Providing Preliminary Results on the Effects of Exercise on Insulin Sensitivity and Fat Distribution in Adolescents: Obesity is a national concern in children and adolescents, and a major factor is the lack of physical activity in these groups. Children's Nutrition Research Center researchers have obtained sufficient data to demonstrate that a controlled and structured 12-week exercise program training without the intent of weight loss resulted in increased fitness in both obese subjects and lean controls. Using metabolic measurements performed pre- and post-program, our researchers discovered that in obese subjects, visceral and hepatic fat accumulation decreased, particularly in subjects with hepatic steatosis (hepatic fat decreased by 28 to 60% and insulin sensitivity increased). These results demonstrate that a moderate exercise program by sedentary obese adolescents without the intent to lose weight can significantly improve metabolic parameters involved in obesity-related disease. These research findings can be used in schools and obesity clinics as a strategy to improve insulin sensitivity and potentially delay or reduce the risk of Type-2 Diabetes mellitus. [NP107, Component 6] (CNRC Project 5) 4. T cells are Necessary for Diet Induced Adipose Inflammation: Children's Nutrition Research Center researchers are aware that a high fat diet induces inflammation in the body's adipose (fat) tissue, yet the definition of the mechanisms of this inflammation is relatively unknown. CNRC researchers demonstrated that in mice a type of white blood cell called gamma delta T cell is required for the inflammation of adipose tissue and for the development of metabolic changes considered pre-diabetic. Our lab conducted a series of studies documenting that gamma delta T cells are present in adipose tissue and make up approximately one third of the lymphocytes in the tissue. Using mice that have been genetically engineered to be completely deficient in gamma delta T cells, our researchers found that the chemical and cellular changes that are landmarks of inflammation failed to occur. In contrast, adipose tissue inflammation was seen when normal mice were fed the high fat diet for 5 weeks to 6 months. The impact of this finding is in the discovery of a necessary link in the cascade leading to adipose inflammation and pre-diabetic metabolic changes. [NP107, Component 6 Prevention of Obesity & Disease] (CNRC Project 4) 5. Macrophage Activity in Adiopose Tissue: Current research has shown that the number of macrophages (a type of white blood cell) increases in the body's adipose (fat) tissues when diet-induced obesity occurs. Macrophages are complex cells that have numerous functions, which differ according to the type of inflammatory hormones to which the macrophages are exposed to. Most current research has focused on the proinflammatory (causing inflammation) roles of these cells, but the problem under investigation in our laboratory at the Children's Nutrition Research Center is to determine if some counterbalancing (i.e., antiinflammatory) function is induced in these cells by a high fat diet. Our lab has demonstrated that there are two previously unknown characteristics of adipose tissue in response to high fat diet – the expression of an inflammatory hormone called IL-13 and a cell surface protein called TREM-2. Researchers studied mice on a diet high in milk fat or corn oil for 5 weeks, adipose tissue was analyzed to collect macrophages and other fat tissue cells. TREM-2 was found to be increased on the surface of the macrophages, and IL-13 was found to be produced by cells in adipose other than macrophages. Since it his been shown that IL-13 can induce macrophages to develop anti-inflammatory functions, and TREM-2 is increased on the surface of antiinflammatory macrophages, the impact of these observations is that this is the first recognition of anti-inflammatory factors induced by high fat diets. These findings 6. TLR-4 is Involved in Diet Induced Liver Inflammation: Earlier work at the Children's Nutrition Research Center demonstrated that a combination of stress and high fat diet induces liver injury in rats and mice, and that endotoxin (a toxin released by some types of bacteria that reside in the intestines) was increased in the blood draining the intestines. Since endotoxin induces inflammation through interactions with a specific protein called toll-like receptor 4 (TLR–4) on the surface of cells, the problem to be investigated is the possible contribution of TLR-4 to the inflammation in the body's fat tissues and liver. Children's Nutrition Research Center researchers successfully demonstrated that liver injury and inflammation and adipose tissue inflammation were significantly reduced in mice genetically deficient in TLR-4 in contrast to normal mice. The impact of this finding is support for findings from other laboratories that TLR-4 is involved in the diet-induced metabolic changes predictive of type-2 diabetes. [NP107, Component 6] (CNRC Project 4) 7. Linking Adiposity to Altered Circadian Rhythms Within the Fat Cell: Researchers are concerned that individuals that work shift work and/or suffer from sleep deprivation, are more likely to become obese due to disrupted circadian (internal body clock) behavior, specifically when direct alterations occur in the circadian clock at the adipocyte (fat cell) level). Researchers at the Children's Nutrition Research Center 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. Through long term growth studies, CNRC researchers have now clearly established in our ACM colony that both male and female research animals eating standard chow are heavier and fatter than their control counterparts by 9 weeks of age (males) to 11 weeks of age (females). When challenged with high fat foods, the transgenic animals gain more weight and become more insulin resistant compared to the control animals. In young animals our research suggested that lipid metabolism within the adipocyte may be directly influenced by the circadian clock. These findings are important for circadian research and additional experiments will be conducted in older animals to characterize metabolic rate and eating behavior. [NP107, Component 6 Prevention of Obesity & Disease] (CNRC Project 6) 8. Assessing Obesity Measurement Tools: The reference technology for body composition assessment in children is dual-energy x-ray absorptiometry (DXA). Children's Nutrition Research Center researchers assessed this technology's newest software and have determined that the increases in body fatness in younger and smaller children reported recently may be an artifact of the software and represent untrue physiological increases. These inaccuracies in the software are important; for example, children examined in 2008 could appear to have increased body fatness compared to children of the same weight or age from only a few years ago. The effect of this may contribute to a false perception that children are continuing to get fatter starting at even younger ages. Additional studies will be performed to determine the accuracy of this software for future use. [NP107, Component 6] (CNRC Project 2) 6.Technology Transfer None Review Publications Lee, C.R., North, K.E., Bray, M.S., Couper, D.J., Heiss, G., Zeldin, D.C. 2007. CYP2J2 and CYP2C8 polymorphisms and coronary heart disease risk: the Atherosclerosis Risk in Communities (ARIC) study. Pharmacogenetics and Genomics. 17(5):349-358. Lee, C.R., North, K.E., Bray, M.S., Couper, D.J., Heiss, G., Zeldin, D.C. 2008. Cyclooxygenase polymorphisms and risk of cardiovascular events: The Atherosclerosis Risk in Communities (ARIC) study. Clinical Pharmacology and Therapeutics. 83(1):52-60. Conklin, B.S., Vito, R.P., Chen, C. 2007. Effect of low shear stress on permeability and occludin expression in porcine artery endothelial cells. World Journal of Surgery. 31:733-743. Hays, S.P., Ordonez, J.M., Burrin, D.G., Sunehag, A.L. 2007. Dietary glutamate is almost entirely removed in its first pass through the splanchnic bed in premature infants. Pediatric Research. 62(3):353-356. Hart Sailors, M.L., Folsom, A.R., Ballantyne, C.M., Hoelscher, D.M., Jackson, A.S., Linda Kao, W.H., Pankow, J.S., Bray, M.S. 2007. Genetic variations and decreased risk for obesity in the Atherosclerosis Risk in Communities Study. Diabetes, Obesity and Metabolism. 9(4):548-557. Butte, N.F., Puyau, M.R., Adolph, A.L., Vohra, F.A., Zakeri, I. 2007. Physical activity in nonoverweight and overweight Hispanic children and adolescents. Medicine and Science in Sports and Exercise. 39(8):1257-1266. Sun, Y., Butte, N.F., Garcia, J.M., Smith, R.G. 2008. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology. 149(2):843-850. Johnston, C.A., Tyler, C., McFarlin, B.K., Poston, W.S.C., Haddock, C.K., Reeves, R., Foreyt, J.P. 2007. Weight loss in overweight Mexican American children: A randomized controlled trial. Pediatrics. 120(6):e1450-e1457. Johnston, C.A., Tyler, C., Foreyt, J.P. 2007. Behavioral management of obesity. Current Atherosclerosis Reports. 9(6):448-453. Fullerton, G., Tyler, C., Johnston, C.A., Vincent, J.P., Harris, G.E., Foreyt, J.P. 2007. Quality of life in Mexican American children following a weight management program. Obesity. 15(11):2553-2556. Butte, N.F., Puyau, M.R., Vohra, F.A., Adolph, A.L., Mehta, N.R., Zakeri, I. 2007. Body size, body composition, and metabolic profile explain higher energy expenditure in overweight children. Journal of Nutrition. 137(12):2660-2667. Brunetti-Pierri, N., Doty, S.B., Hicks, J., Phan, K., Mendoza-Londono, R., Blazo, M., Tran, A., Carter, S., Lewis, R.A., Plon, S.E., Phillips, W.A., Smith, O.E., Ellis, K.J., Lee, B. 2008. Generalized metabolic bone disease in Neurofibromatosis type I. Molecular Genetics and Metabolism. 94:105-111. Bray, M.S., Shaw, C.A., Moore, M.W.S., Garcia, R.A.P., Zanquetta, M.M., Durgan, D.J., Jeong, W.J., Tsai, J., Bugger, H., Zhang, D., Rohrwasser, A., Rennison, J.H., Dyck, J.R.B., Litwin, S.E., Hardin, P.E., Chow, C., Chandler, M.P., Abel, E.D., Young, M.E. 2008. Disruption of the circadian clock within the cardiomyocyte influences mycardial contractile function, metabolism, and gene expression. American Journal of Physiology - Heart and Circulatory Physiology. 294(2):H1036-H1047. Kung, T.A., Egbejimi, O., Cui, J., Ha, N.P., Durgan, D.J., Essop, M.F., Bray, M.S., Shaw, C.A., Hardin, P.E., Stanley, W.C., Young, M.E. 2007. Rapid attenuation of circadian clock gene oscillations in the rat heart following ischemia-reperfusion. Journal of Molecular and Cellular Cardiology. 43(6):744-753. Bush, C.L., Pittman, S., McKay, S., Ortiz, T., Wong, W.W., Klish, W.J. 2007. Park-based obesity intervention program for inner-city minority children. Journal of Pediatrics. 151(5):513-517. Ellis, K.J., Shypailo, R.J. 2008. COHN analysis: Body composition measurements based on the associated particle imaging and prompt-gamma neutron activation analysis techniques. Journal of Radioanalytical and Nuclear Chemistry. 276(1):79-83. Petrescu, M.S., Larry, C.L., Bowden, R.A., Williams, G.W., Gagen, D., Li, Z., Smith, C.W., Burns, A.R. 2007. Neutrophil interactions with keratocytes during corneal epithelial wound healing: A role for CD18 integrins. Investigative Ophthalmology and Visual Science. 48(11):5023-5029. Gonzalez, A.L., El-Bjeirami, W., West, J.L., McIntire, L.V., Smith, C.W. 2007. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis. Journal of Leukocyte Biology. 81:686-695. Johnston, C.A., Tyler, C., Fullerton, G., Poston, W.S.C., Haddock, C.K., Mcfarlin, B., Reeves, R.S., Foreyt, J.P. 2007. Results of an intensive school-based weight loss program with overweight Mexican American children. International Journal of Pediatric Obesity. 2(3):144-152. Butte, N.F., Christiansen, E., Sorensen, T.I.A. 2007. Energy imbalance underlying the development of childhood obesity. Obesity. 15(12):3056-3066. Cai, G., Cole, S.A., Butte, N.F., Voruganti, V.S., Comuzzie, A.G. 2007. A quantitative trait locus on chromosome 13q affects fasting glucose levels in Hispanic children. Journal of Clinical Endocrinology and Metabolism. 92(12):4893-4896. Desai M.S., Mariscalco, M.M., Tawil, A., Vallejo, J.G., Smith, C.W. 2008. Atherogenic diet-induced hepatitis is partially dependent on murine TLR4. Journal of Leukocyte Biology. 83:1336-1344. Suminski, R.R., Poston, W.S.C., Foreyt, J.P., St Jeor, S. 2008. Physical activity assessed with three different methods and the Framingham Risk Score on 10-year coronary heart disease risk. Medical Science Monitor. 14(1):CR1-CR9. Cai, G., Cole, S.A., Butte, N.F., Voruganti, V.S., Comuzzie, A.G. 2008. Genome-wide scan revealed genetic loci for energy metabolism in Hispanic children and adolescents. International Journal of Obesity. 32:579-585. Hunter, C.M., Peterson, A.L., Alvarez, L.M., Poston, W.C., Brundige, A.R., Haddock, C.K., Van Brunt, D.L., Foreyt, J.P. 2008. Weight management using the internet: A randomized controlled trial. American Journal of Preventive Medicine. 34(2):119-126. Heshka, S., Ruggiero, A., Bray, G.A., Foreyt, J., Kahn, S.E., Lewis, C.E., Saad, M., Schwartz, A.V. 2008. Altered body composition in type 2 diabetes mellitus. International Journal of Obesity. 32(5):780-787. Dalton, W.T., Johnston, C.A., Foreyt, J.P., Tyler, C. 2008. Brief report: Weight dissatisfaction, weight status, and weight loss in Mexican-American children. Journal of Pediatric Psychology. 33(6):673-677. Toffolo, G., Man, C.D., Cobelli, C., Sunehag, A.L. 2008. Glucose fluxes during OGTT in adolescents assessed by a stable isotope triple tracer method. Journal of Pediatric Endocrinology & Metabolism. 21(1):31-45. Smith, W.C. 2008. Adhesion molecules and receptors. Journal of Allergy Clinical Immunology. 121: S375-S379. Li, Z., Burns, A., Rumbaut, R., Smith, W.C. 2007. Gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion. American Journal of Pathology. 171:838-845. Heinrich, K.M., Jitnarin, N., Suminski, R.R., Berkel, L., Hunter, C.M., Alvarez, L., Brundige, A.R., Peterson, A.L., Foreyt, J.P., Haddock, C.K., Poston, W.S.C. 2008. Obesity classification in military personnel: A comparison of body fat, waist circumference, and body mass index measurements. Military Medicine. 173(1):67-73.