Progress 05/21/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This unit evaluates childhood obesity, specifically the regulation of energy balance and body composition. It is composed of six individual research projects; 1) Genetic and environmental factors contributing to childhood obesity; 2) Biological diversity of human growth: body composition references standards for children; 3) Prevention of overweight in children; 4) Nutrition influences on innate immunity; 5) Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents; and 6) Circadian clocks in adipose biology and obesity. This research contributes to the goals of NP107, Human Nutrition, specifically "Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease." It addresses ARS Strategic Plan Goal 4 Improve the Nation's Health, specifically
objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. Project 1: Genetic and environmental factors contributing to childhood obesity Childhood obesity in the United States has dramatically increased in the past decade according to the latest National Health and Nutrition Examination Survey (NHANES), particularly among Mexican-American children. Childhood obesity is associated with hypertension, hyperinsulinemia, steatohepatitis, and alterations in lipoprotein metabolism. Childhood obesity not only presents serious medical and psychosocial consequences to the child, but also predisposes to adult obesity and its complications. Obesity is a complex multi-factorial disease. The current surge in obesity in the United States is due to an interaction between a genetic predisposition
toward efficient energy storage and a permissive environment of readily available food and sedentary behaviors. Despite the high prevalence of obesity among Hispanic children, the etiology underlying the heightened susceptibility to childhood obesity in Hispanic populations has not been investigated. Researchers at the Children's Nutrition Research Center, Houston, TX, will assess the relative contributions of genetic and environmental (diet and physical activity) factors influencing childhood obesity, and identify several positional candidate genes throughout the genome that are linked with obesity- related phenotypes in Hispanic children. The main objective of this project is to identify a number of genes that affect the expression of childhood obesity in Hispanic children. Although this project focuses on Hispanic children, all American children should benefit from prevention and/or management strategies aimed at childhood obesity. Scientists and clinicians will benefit from an
increased understanding of the genetic and environmental causes of childhood obesity. USDA food and nutrition programs may adopt strategies to prevent childhood obesity based on this study's findings. Several genes that are linked with the quantitative variation in obesity-related phenotypes in children will be identified. Genetic polymorphisms will be identified that can be used to screen children at increased risk of developing obesity before excessive weight gain occurs. In addition, by measuring phenotypes such as those related to adiposity, the regulation of food intake, energy expenditure, and energy partitioning during growth, we will identify metabolic markers for early screening of "at risk" individuals. Effective strategies for the prevention and management of childhood obesity will be developed with the goal of reducing the staggering medical and societal costs associated with childhood obesity. Project 2: Biological diversity of human growth: body composition reference
standards for children Obesity, defined as excess body fat stores, is a major health problem in the US, as well as worldwide. Its prevalence continues to start at younger ages and often tracks into adulthood. This pattern is evident in all ethnic groups, with the largest increases in the United States occurring in minority populations. There are few analytical techniques that can measure the body's fat content. Although a number of anthropometric-based indices using weight and height have been proposed, these tend to be inaccurate for the individual. To better assess the impact of changes in the environment, i.e., modification of diet and/or physical (in)activity, improved measures of body fatness, especially for growing children, are needed. The range of 'normal' body fatness for children has not been established. Our lab continues to obtain body composition measurements in children from birth through age 18 years, using several different analytical techniques. For these data, the
percentile distributions for body composition (fat, bone, muscle, water) in the general pediatric population are to be established. This information will provide the more accurate estimates for establishing the appropriate age-, gender-, and (possibly) ethnic-dependent upper limits for normal adiposity for children. This information provides a contemporary and unique pediatric reference that is available for use by health professionals to help establish more appropriate nutritional and activity guidelines for achieving acceptable growth without accumulation of excess body fat. Nutritional scientists, dietitians, exercise specialists, and pediatricians when assessing the nutritional status of children can use this information. Project 3: Prevention of overweight in children Our goal is to develop a program that promotes sustained weight management in children and adolescents, especially one that is effective for use with a Hispanic population. We will additionally determine treatment
effects on other health outcomes as well as psychosocial functioning, and will test our objectives by conducting a randomized clinical trial in which children are assigned to either a behavioral, family-based weight management program led by an instructor or a self- help led by a parent. This project will have applications in terms of improving health outcomes in children, a central mission of ARS National Program 107 Human Nutrition. Project 4: Nutritional influences on innate immunity The major problem being addressed in this research project is the effect of high fat and high sucrose diets on adipose tissue and the liver. Obesity is a major health problem, and there is growing evidence that high fat and high sucrose diets cause systemic inflammation that contributes to many of the complications of obesity such as arterial disease. In addition, such diets are associated with fatty changes in the liver often with liver cell injury leading to cirrhosis. We are working on the
mechanisms by which obesity causes inflammatory mediators to be generated in adipose tissue and how obesity causes liver injury. Our approach is to use animals of dietary obesity, cell and molecular biology. The work is important to those attempting to treat and prevent the complications of obesity. Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents Over the past 30 years the prevalence of overweight children (6-19 years) in the USA (BMI>95th percentile) has increased 3-fold to reach 15% in 2000, with an even higher incidence in Hispanic and African American adolescents (24% in both). Concomitant with the increase in childhood overweight, the incidence of type 2 diabetes in children (0-19 years) has increased 4-fold, with predominance for African American and Hispanic children. We have demonstrated that healthy obese adolescents are significantly insulin-resistant regardless of diet, but maintain normoglycemia,
normolipidemia and appropriate substrate oxidation rates by increasing their insulin sensitivity more than 2-fold as compared with their lean counterparts. This will, however, place increased demands on their pancreatic Beta-cells, thus increasing their risk of type 2 diabetes. These results emphasize the importance of preventing obesity and treating insulin resistance. It is well-known that achieving and maintaining weight loss is very difficult, particularly in adolescents. Therefore, the primary objectives of this proposal are to determine in healthy adolescents whether a 12-week exercise program improves insulin sensitivity and glucose metabolism; whether exercise-induced metabolic effects of exercise are correlated with changes in intramyocellular, visceral, and/or hepatic lipid content; and whether ethnicity, gender, and obesity have any impact on metabolic effects of exercise. To address these questions, 96 adolescents will be studied on two occasions (just prior to the start
of a 12-week exercise program and during the last week of exercise) using state of the art stable isotope and magnetic resonance imaging (MRI) techniques. The information provided by these studies will improve our understanding of the metabolic effects of ethnicity, exercise, and obesity in adolescents, and will help in identifying factors that may increase the risk of type 2 diabetes, and in designing new treatment strategies to prevent or delay the development of type 2 diabetes in adolescents. The knowledge that is likely to be gained by these studies will benefit adolescents, particularly from minority groups, who are at high risk of becoming overweight and developing insulin resistance and type 2 diabetes in that the information will be used in developing strategies to improve insulin sensitivity, thus preventing and/or delaying type 2 diabetes. This research will provide detailed information on the effects of ethnicity, obesity, and exercise on hepatic and intramyocellular
lipid content, glucose and lipid metabolism, and insulin sensitivity and secretion in adolescents. This information is crucial in defining treatment strategies to improve insulin sensitivity and glucose metabolism in adolescents, thus reducing their risk of or delaying the onset of type 2 diabetes. The information from this research will be used by adolescents in general and those who are overweight and their parents; health care professionals taking care of obese adolescents and young patients with type 2 diabetes; policy groups designing nutritional and lifestyle guidelines for children and adolescents; school professionals, e.g., principals, school nurses, Physical Education teachers, committees planning educational programs; and organizations responsible for after school activities. Project 6: Circadian clocks in adipose biology and obesity More than two-thirds of the adults in the U.S. and more than 9 million children age 6-16 yrs are currently considered overweight or obese.
Obesity is the second leading cause of preventable death in the United States behind smoking. Abdominal obesity, in particular, is associated with insulin resistance and an atherogenic phenotype characterized by hypertension, hyperlipidemia, hyperglycemia, and a prothrombotic state. Because obesity has a multifactorial etiology, the strategies and methods for its treatment are equally numerous and varied. An estimated $30 to $50 billion is spent yearly on efforts to lose weight, and the success rate of most long-term intervention programs for weight loss is dismal, illustrating the fact that obesity is both difficult to alleviate and, even today, not well understood. Recent reports have suggested that altered sleep patterns related to our "24-hour" lifestyle are associated with increased body fat and obesity, although the physiologic mechanisms that promote the accumulation of adipose tissue are not known. This project is designed to investigate the impact of disruption of normal
intrinsic circadian rhythms, a potentially important contributor to the recent rise in human obesity, on the lipogenic and/or lipolytic function of the adipocyte. By increasing our understanding of potential mechanisms that exacerbate the susceptibility for accumulating body fat, this work is relevant for physicians, interventionists, and prevention specialists, in recognizing lifestyle and physiologic factors that may have a profound influence both on response to obesity treatments, as well as prevention of obesity onset. 2. List the milestones (indicators of progress) from your Project Plan. This research agreement is being terminated on 9/30/05, to be replaced by a new specific cooperative agreement. Research under the individual projects will continue under the new agreement. Milestones for the continuing project will be identical to those shown below. Project 1: Genetic and environmental factors contributing to childhood obesity Year 1 2005: Complete enrollment and phenotyping
of 300 families Repeat anthropometry and body composition measurements on 250 children Data entry and error checking Genotyping of first and second cohorts Preliminary quantitative trait loci (QTL) linkage analysis Year 2 2006: Repeat anthropometry and body composition measurements on 250 children Data entry and error checking Genotyping of third and fourth cohorts (780 individuals) QTL linkage analysis Publications Year 3 2007: Begin 1-y intervention trial for weight loss in overweight children Phenotyping including anthropometry and body composition of overweight children Data entry and error checking QTL linkage analysis Fine mapping and sequence positional candidate genes to identify single nucleotide polymorphisms (SNPs) Publications Year 4 2008: Continue 1-y intervention trial for weight loss in overweight children Phenotyping including anthropometry and body composition of overweight children Data entry and error checking QTL linkage analysis Fine mapping and sequence
positional candidate genes to identify SNPs Publications Year 5 2009: Complete 1-y intervention trial for weight loss in overweight children Phenotyping including anthropometry and body composition of overweight children Data entry and error checking QTL linkage analysis Bayesian quantitative trait nucleotide (BQTN) analysis Publications Project 2: Biological diversity of human growth: body composition reference standards for children Year 1 2005: Recruitment for Cross-Sectional Pediatric Database Testing of different mathematical models as they relate to the Pediatric Reference Model Statistical analysis of percentile distribution models Extension of BodPod assay to include preschool ages and testing of PeaPod instrument for infants Year 2 2006: Continue longitudinal recruitment for longitudinal databases Test prediction models with pediatric populations with known abnormal body composition Year 3 2007: Improvement of precision and accuracy of DXA software for use with infants and
toddlers Continue longitudinal recruitment Identification of growth model that best describes changes in body composition Year 4 2008: Continue longitudinal recruitment Test of predictive accuracy of model in longitudinal population Test accuracy of forward prediction of Z-model with longitudinal database and disease populations Development of magnetic resonance technique for body water assay in preterm infants Year 5 2009: Continue longitudinal recruitment Adjust mathematical parameters to optimize models Update CNRC website with latest Z-score prediction models Development of body nitrogen assay with very low dose Development of fat distribution assay for use with children Project 3: Prevention of overweight in children Year 2 2006: Program design Program development Designing materials Participant recruitment and screening Year 3 2007: Continue participant recruitment and screening Complete baseline measurements on Phase 1 participants Randomize Phase 1 participants to intensive
treatment or self-help treatment Begin treatment of Phase 1 participants Complete 6-month measurements on Phase 1 participants Begin analyses with Phase 1 6-month data Complete baseline measurements on Phase 2 participants Begin treatment of Phase 2 participants Year 4 2008: Continue treatment of Phase 2 participants Complete 12-month measurements of Phase 1 participants Begin analyses with Phase 1 12-month data Complete 6- and 12-month measurements of Phase 2 participants Complete study analyses Determine program strengths/weaknesses Revise and redesign program Participant recruitment and screening Year 5 2009: Complete baseline measurements of participants Randomize participants to intensive treatment or self-help treatment Begin treatment of participants Complete 6-month measurements on participants Begin analyses with 6-month data Continue maintenance treatment with participants Complete 12-month measurements Project 4: Nutritional influences on innate immunity Year 1 2005: Define
kinetics of macrophages in adipose depots and expression of mediators Determine the role of adhesion in RAW cell 3T3-L1 co-cultures Year 2 2006: Define kinetics of leukocytes in livers of mice on high fat diet Determine the contribution of bone marrow precursors Year 3 2007: Confirm that primary adipose macrophages modulate adipocyte function in vitro Determine the contribution of bone marrow precursors Year 4 2008: Determine the functional effects of macrophage inhibition Determine the synergy between dietary steatosis and endogenous endotoxin stimulus Year 5 2009: Determine the effects of differing lipids in the diet on macrophages in adipose tissue Determine the effects of differing lipids in the diet on synergistic liver injury Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents Year 1 2005: We anticipate that 40 subjects with a balanced recruitment (20 overweight subjects, 10 boys, 10 girls; and 20 normal
weight subjects, 10 boys, 10 girls) will have been studied. Year 2 2006: Determine 1) effect of obesity and gender at baseline; 2) effects of exercise; 3) impact of obesity and gender on exercise-induced metabolic effects; and 4) correlations between exercise-induced changes in hepatic and intramyocellular lipid content and potential exercise-induced improvements in insulin sensitivity and gluconeogenesis Year 3 2007: Study an additional 20 subjects, and write manuscripts based on the data from the first 40 subjects. Year 4 2008: Continue research studies and initiate data analysis. Year 5 2009: Complete all research studies and data analysis. Manuscript preparation Project 6: Circadian clocks in adipose biology and obesity Year 2 2006: Identify zeitgebers that regulate the adipocyte specific clocks Establish and maintain transgenic animal colony Perform microarray analysis in TG and WT animals Identify circadian clock-regulated genes Year 3 2007: Perform confirmatory Taqman assays
Detailed characterization of adipocyte function and cellularity Initiate feeding studies of WT and TG animals Characterize weight gain and body composition changes in high- and low- fat fed animals Characterize gene expression patterns of clock mechanism genes Characterize gene expression patterns of clock-regulated genes Year 4 2008: Identify timing of alterations in the circadian clock in relation to obesity onset Identify timing of alterations in clock-regulated genes in relation to obesity onset Year 5 2009: Complete genotyping of candidate genes identified in Specific Aims 1-3 in participants from the TIGER study Complete analysis of genetic data generated from this project 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Project 1: Complete enrollment and phenotyping of 300 families Milestone Fully Met 2. Project 1: Repeat anthropometry and body
composition measurements on 250 children Milestone Fully Met 3. Project 1: Data entry and error checking Milestone Fully Met 4. Project 1: Genotyping of first and second cohorts (760 individuals) Milestone Fully Met 5. Project 1: Preliminary quantitative trait loci (QTL) linkage analysis Milestone Fully Met 6. Project 2: Recruitment for Cross-Sectional Pediatric Database Milestone Substantially Met 7. Project 2: Testing of different mathematical models as they relate to the Pediatric Reference Model Milestone Substantially Met 8. Project 2: Statistical analysis of percentile distribution models Milestone Not Met Other 9. Project 2: Extension of BodPod assay to include preschool ages and testing of PeaPod instrument for infants Milestone Substantially Met 10. Project 4: Define kinetics of macrophages in adipose depots and expression of mediators Milestone Fully Met 11. Project 4: Determine the role of adhesion in RAW cell 3T3-L1 co-cultures Milestone Not Met Redirection of Research
focus due to change in priorities 12. Project 5: We anticipate that 40 subjects with a balanced recruitment (20 overweight subjects, 10 boys, 10 girls; and 20 normal weight subjects, 10 boys, 10 girls) will have been studied. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This research agreement is being terminated on 9/30/05, to be replaced by a new specific cooperative agreement. Research under the individual projects will continue under the new agreement. Milestones for the continuing project will be identical to those shown below. Project 1: Genetic and environmental factors contributing to childhood obesity Year 2 2006: Repeat anthropometry and body composition measurements on 250 children Accomplish: Anthropometry and body composition measurements
will be completed on the entire cohort of children to measure changes in weight and fat mass. Data entry and error checking Accomplish: Data will be entered and screened for possible errors Genotyping of third and fourth cohorts (780 individuals) Accomplish: Additional volunteers will be genotyped. QTL linkage analysis Accomplish: Once the genotyping is complete, variance component linkage analysis will be performed on the entire cohort to find and localize quantitative trait loci (QTLs) that influence quantitative variation in adiposity, the regulation of food intake, energy expenditure, and energy partitioning in children and to test whether QTLs have measurable pleiotropic effects across phenotypes. Publications Accomplish: Manuscripts on body composition, energy expenditure, weight gain and energy storage, physical activity, diet intake, and eating behavior will be prepared for publication. Year 3 2007: Begin 1-y intervention trial for weight loss in overweight children
Accomplish: The 1-y intervention trial for weight reduction in overweight Hispanic children will begin. Phenotyping including anthropometry and body composition of overweight children Accomplish: Anthropometry and body composition measurements will be completed on the cohort of children that are considered overweight. Data entry and error checking Accomplish: Data will be entered and screened. QTL linkage analysis Accomplish: We will pursue significant linkage signals detected in our initial VIVA LA FAMILIA genome scan by using DNA sequencing of strong positional candidate genes located in proximity to QTLs with significant LOD scores. Fine mapping/sequence positional candidate genes to identify single nucleotide polymorphisms SNP Accomplish: We will refine linkage signals by fine genetic mapping, and investigate strong positional candidate obesity-related genes. Publications Accomplish: Manuscripts on body composition, energy expenditure, weight gain and energy storage,
physical activity, diet intake, and eating behavior will be prepared for publication. Year 4 2008: Continue 1-y intervention trial for weight loss in overweight children Accomplish: We will explore additional significant linkage results in chromosomal regions that either lack clear positional candidate genes or that contain multiple reasonable positional candidates in the region by typing additional microsatellites and known SNPs within the linkage regions to use in linkage and association analyses to fine map the QTLs. Phenotyping including anthropometry and body composition of overweight children Accomplish: Anthropometry and body composition measurements will be completed on the cohort of children that are considered overweight. Data entry and error checking Accomplish: Data will be entered and screened. QTL linkage analysis Accomplish: We will explore additional significant linkage results in chromosomal regions that either lack clear positional candidate genes or that contain
multiple reasonable positional candidates in the region by typing additional microsatellites and known SNPs within the linkage regions to use in linkage and association analyses to fine map the QTLs. Fine mapping and sequence positional candidate genes to identify SNPs Accomplish: We will explore additional significant linkage results in chromosomal regions that either lack clear positional candidate genes or that contain multiple reasonable positional candidates in the region by typing additional microsatellites and known SNPs within the linkage regions to use in linkage and association analyses to fine map the QTLs. Publications Accomplish: Manuscripts on body composition, energy expenditure, weight gain and energy storage, physical activity, diet intake, and eating behavior will be prepared for publication. Project 2: Biological diversity of human growth: body composition reference standards for children Year 2 2006: Continue longitudinal recruitment for longitudinal databases
Accomplish: Approximately 100-150 children will return for body composition measurements. Test prediction models with pediatric populations with known abnormal body composition Accomplish: Following recalibration of the DXA instrument, new reference ranges for bone mineral will be established, and its impact tested in children with abnormal bone growth. Year 3 2007: Improvement of precision and accuracy of DXA software for use with infants and toddlers Accomplish: DXA scans for 1400 children may have to be reanalyzed using the newest software version, which is known to alter values for younger children. Comparison of DXA scans in infants with a criterion reference model will be completed in order to determine accuracy of DXA body fat assessment, and to develop translational algorithms between different DXA instruments and software. Continue longitudinal recruitment Accomplish: Longitudinal recruitment will continue. These data are needed in order to correctly test the accuracy of
the population-based prediction models for changes in body composition for the individual. If diet/activity interventions are proposed, it is essential that the technique used to monitor changes in fat, bone, or muscle is accurate for the individual, not simply a population-based index. Identification of growth model that best describes changes in body composition Accomplish: Data collected in the previous years for the infant, child, and adolescent reference populations will be combined into a unified model and will be made available on an interactive web-site for use by other scientists. Age- and gender-based models from 1 to 18 years, with percentile distributions at 5-10% increments will be provided. Year 4 2008: Continue longitudinal recruitment Accomplish: Targeted age groups will be determined by recruitment in previous years. Test of predictive accuracy of model in longitudinal population Accomplish: Comparison of the predicted outcomes based on measurements at prepubertal
ages in the cross-section reference population will be made with follow-up measurements the same children as adolescents. Test accuracy of z-score prediction models with longitudinal data and in pediatric disease populations with known abnormalities of body composition. Accomplish: The on-line interactive model will be tested by scientists and pediatricians worldwide using WWW access. The current on-line model for bone mineral has more than 200 registered users. Development of magnetic resonance technique for body water assay in preterm infants Accomplish: New and innovative magnetic-based techniques will continue to be developed and tested. An instrument specifically designed for use with preterm infants will be designed, built, tested with animal models, and calibrated for human use. Project 3: Prevention of overweight in children Year 2 2006: Program design Accomplish: A trial will be designed by which to compare an intensive versus a self-help weight management program for
children at risk for overweight/obesity and their families. Program development Accomplish: We will develop a behavioral, family-based weight management program. Designing materials Accomplish: Materials will be designed to assist in teaching children and their parents' lifestyle nutrition and physical activity modifications. Participant recruitment and screening Accomplish: Students will be screened for treatment. Year 3 2007: Continue participant recruitment and screening Accomplish: We will continue recruiting and screening when we begin treatment with the final, Phase 2 participants. Complete baseline measurements on Phase 1 participants Accomplish: Baseline measurements have been taken on 67 students. Randomize Phase 1 participants to intensive treatment or self-help treatment Accomplish: A total of 67 students were randomized to either intensive or self-help treatment. Begin treatment of Phase 1 participants Accomplish: A total of 67 Phase 1 participants have completed the
first 5 months of treatment. Complete 6-month measurements on Phase 1 participants Accomplish: The 67 Phase 1 participants will complete 6-month measurements. Begin analyses with Phase 1 6-month data Accomplish: We will begin analyzing 6-month data from Phase 1 participants. Complete baseline measurements on Phase 2 participants Accomplish: All baseline measurements for Phase 2 participants will be completed. Begin treatment of Phase 2 participants Accomplish: The Phase 2 participants will begin treatment in the program. Year 4 2008: Continue treatment of Phase 2 participants Accomplish: The Phase 2 participants will have completed intensive intervention and will begin maintenance sessions. Complete 12-month measurements of Phase 1 participants Accomplish: The Phase 1 participants will complete 12-month measurements. Begin analyses with Phase 1 12-month data Accomplish: We will begin analyzing 12-month data from Phase 1 participants. Complete 6- and 12-month measurements of Phase 2
participants Accomplish: The Phase 2 participants 6-month measurements and 12-month measurements will be completed. Complete study analyses Accomplish: Analyses will be conducted on data collected from both Phase 1 and 2 participants. Determine program strengths/weaknesses Accomplish: Based on the analyses performed on the 6- and 12-month data, the overall effectiveness of the program will be determined. Additionally, program adherence will be viewed in combination with the outcomes. Revise and redesign program Accomplish: Based on the strengths and weaknesses identified, the program will be made that will be modified with the intention of further enhancing weight loss/maintenance. Participant recruitment and screening Accomplish: Children will be screened and recruited to participate in the newly developed program based on modifications to the previous program. Project 4: Nutritional influences on innate immunity Year 2 2006: Define kinetics of leukocytes in livers of mice on
high-fat diet Accomplish: We have already made considerable progress in this direction studying the changes in the liver of the animals on the high fat diets. We found that accumulation of neutrophils in the livers after 3 weeks on the high-fat diet, a relatively short time period for such changes. Efforts to define lymphocytes and macrophages has demonstrated increases in monocytes but not lymphocytes. Determine the contribution of bone marrow precursors Accomplish: Using bone marrow transplants we will show the turnover of macrophages in the livers during high-fat diet. Year 3 2007: Confirm that primary adipose macrophages modulate adipocyte function in vitro Accomplish: We will isolate primary macrophages from tissues including adipose and show that in vitro they influence the 3T3-L1 cell functions. Determine the contribution of bone marrow precursors Accomplish: Bone marrow transplants will be used as in year 2006 to define whether the increase in macrophages as adipose tissue
increases is due to bone marrow derived cells or local proliferation. Year 4 2008: Determine the functional effects of macrophage inhibition Accomplish: We will have defined the mediators and sources of macrophages in adipose tissue and liver in previous years and will have a strategy for specific inhibition. That will allow assessment of effects of inhibition on adipose production of inflammatory mediators or macrophage based injury to the liver. Determine the synergy between dietary steatosis and endogenous endotoxin stimulus Accomplish: This work will involve blocking endotoxin or the receptors that recognize endotoxin. Some work in this direction is already underway, with a focus on the receptors of the TLR family. Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents Year 2 2006: Determine 1) effect of obesity and gender at baseline; 2) effects of exercise; 3) impact of obesity and gender on exercise-induced
metabolic effects; and 4) correlations between exercise-induced changes in hepatic and intramyocellular lipid content and potential exercise-induced improvements in insulin sensitivity and gluconeogenesis Accomplish : I anticipate this would be accomplished and will be instrumental in our research studies. Year 3 2007: Study an additional 20 subjects, and write manuscripts based on the data from the first 40 subjects. Accomplish: I anticipate this would be accomplished as manuscripts will be presented and recruitment of subjects will continue. Year 4 2008: Continue research studies and initiate data analysis. Accomplish: I anticipate this would be accomplished as studies will be continued and data will be obtained and evaluated. Project 6: Circadian clocks in adipose biology and obesity Year 2 2006: Identify zeitgebers that regulate the adipocyte specific clocks Accomplish: Though the basic clock mechanism has been elucidated, it is not known which neurohumoral or other types of
factors (zietgebers) may regulate it. Identification of these zeitgebers in adipose tissue will provide new insight into the molecular underpinnings of adipose accumulation and lipid metabolism and the role of the circadian clock in these processes. Establish and maintain transgenic animal colony Accomplish: Successful establishment of the adipose-specific clock "knockout" transgenic animal will allow for the investigation of the physiologic consequences of altered intrinsic circadian rhythms within the adipocyte. Perform microarray analysis in TG and WT animals Accomplish: The microarray analyses in the WT and TG animals are hoped to identify both genes that are regulated by the circadian clock as well as genes that are part of the clock mechanism itself. The use of array technologies is a valuable and efficacious approach for identifying new genes/pathways. By performing the array analyses in both TG and WT animals, the genes that are determined to have rhythmicity of expression
in WT animals can be confirmed by showing that rhythmicity is absent in TG animals. Identify circadian clock-regulated genes Accomplish: Though both central and peripheral circadian clocks are hypothesized to play critical roles in human physiology, not all genes demonstrate a circadian pattern of gene expression. Identification of the genes/pathways specifically regulated by the clock mechanism will provide new insight into the role of the intrinsic circadian clock in obesity-associated phenomena, including adipose accumulation and altered lipid metabolism. Year 3 2007 Perform confirmatory Taqman assays Accomplish: Confirmatory Taqman assays will provide further confirmation that genes identified during microarray analysis are truly regulated by or are components of the circadian clock. Taqman assays will also provide confirmation that the array approach is capable of detecting changes in gene expression that occur over the course of 24 hours. Detailed characterization of adipocyte
function and cellularity Accomplish: Experiments designed to investigate adipocyte function and cellularity will provide insight into the functional alterations that take place consequent to rhythmic and/or altered circadian gene expression. Initiate feeding studies of WT and TG animals Accomplish: Feeding studies will be critical in establishing the time course by which obesity may influence the function of the circadian clocks intrinsic to the adipocyte. Feeding studies in WT animals will address the hypothesis that obesity may produce alterations in the circadian clock, while feeding studies in the TG animals will address the hypothesis that an altered circadian clock mechanism within the adipocyte may promote the development of obesity. Characterize weight gain and body composition changes in high and low fat fed animals Accomplish: Tracking of weight gain and body composition in the TG and WT animals will be critical in assessing the time course of obesity onset. Characterize
gene expression patterns of clock mechanism genes Accomplish: Monitoring the changes that occur in the clock mechanism both prior to and following obesity onset is the central aim of this project. Year 4 2008: Identify timing of alterations in the circadian clock in relation to obesity onset Accomplish: As detailed above, identification of the exact timing changes in the clock mechanism itself will provide insight into whether alterations in the adipocyte-specific clock may precede or follow obesity onset. Identify timing of alterations in clock regulated genes in relation to obesity onset Accomplish: As detailed above, identification of the exact timing changes in the clock-regulated genes will provide insight into whether alterations in the genes downstream of the adipocyte-specific clock may precede or follow obesity onset. 4a What was the single most significant accomplishment this past year? Project 1: Genetic and environmental factors contributing to childhood obesity
CHILDHOOD OBESITY IN THE HISPANIC POPULATION To identify genetic and environmental factors influencing childhood obesity in the Hispanic population, a family-based study was performed at the Children's Nutrition Research Center, at Houston, TX, in collaboration with Southwest Foundation for Biomedical Research. Scientists have successfully phenotyped and genotyped 1030 children (ages 4 to 19 years) and 600 parents participating in the VIVA LA FAMILIA Study. Anthropometric, body composition, biochemistries, food intake, eating behavior, physical activity, and energy expenditure of 1030 Hispanic children were measured. A genetic map was constructed for use in a multipoint genome scan to find quantitative trait loci (QTLs) that influence adiposity, energy intake and expenditure by performing variance component linkage analysis. Significant genetic linkages (QTLs) for obesity-related phenotypes have been found, indicating a strong genetic contribution to obesity in Hispanic children. From
this study, researchers will gain an in-depth understanding of the risk factors and consequences of obesity among Hispanic children. 4b List other significant accomplishments, if any. Project 1: Genetic and environmental factors contributing to childhood obesity GAINING GENETIC INSIGHT ON OBESITY IN HISPANIC POPULATIONS Identification of the genetic contribution to childhood obesity in the Hispanic population is needed to gain perspective insight on the role genes play in becoming overweight. Scientists at the Children's Nutrition Research Center at Houston, TX, have genotyped a subset from the VIVA LA FAMILIA study and have performed preliminary variance component linkage analysis. Genetic markers were typed in 800 individuals, and genetic maps were constructed for use in a genome-wide scan. We have detected 11 linkage signals with LOD scores greater than 3 on chromosomes 2, 4, 8, 9, 10, 11, 12, 16, and 18. Such research findings are important as they provide researchers with an
understanding of where there may be genetic linkages for individuals that run risk of becoming obese. Project 2: Biological diversity of human growth: body composition reference standards for children IDENTIFICATION OF SKEWED DISTRIBUTION MEASUREMENTS IN INFANT COMPOSITION An acceptable calibration tool is needed to measure body composition measurements for younger children. Scientists at the Children's Nutrition Research Center in Houston, TX, have analyzed children's body composition measurements that used dual-energy x-ray absorptiometry (DXA). CNRC researchers have determined that the analysis software for a major instrument (DXA) has been designed for use with adults and adolescents and the software does not provide an acceptable calibration for younger children. Examination of the data collected to date indicated skewed distributions with a disproportionate increase at higher values, a pattern that is different among the three major ethnic groups (Caucasian, African-American,
Hispanic-American) as well as for gender. Body fat and bone mineral values were most affected, especially for younger ages. This finding will require revision of the published conclusions in the scientific literature regarding the relationship between mineralization of bone and obesity during childhood. CHILDREN'S OBESITY TREND INCREASES IN RESEARCH VOLUNTEERS An analysis of previous Children's Nutrition Research Center childhood subjects' body mass index measurements compared with current subjects is needed to learn of possible demographic trends. Scientists at the Children's Nutrition Research Center in Houston, TX, have documented that the prevalence of childhood obesity continues to increase. Findings from this year were compared with findings obtained in the mid-1990s at the CNRC, and the data indicated that the levels of obesity increased over time and is consistent or greater to the national average. Such findings draw continuing concern that obesity is still becoming a
national and local problem in today's youth. Project 4: Nutritional influences on innate immunity INFLAMMATION CELLS IN ADIPOSE TISSUE A potential source of the systemic inflammation in adipose tissue, which accompanies obesity, is needed. Children's Nutrition Research Center scientists in Houston, TX, have recognized the cell types in adipose tissue that express inflammatory mediators. Researchers used the dietary obese mouse model to conduct adipose tissue research studies. The digested fat isolate was immediately processed for analysis by flow cytometry and molecular analysis. These findings address a potential source of the systemic inflammation that accompanies obesity. Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents PRELIMINARY RESULT OF OBESITY AND EXERCISE PROGRAM ON FITNESS, INSULIN, AND FAT ACCUMULATION Scientists at the Children's Nutrition Research Center in Houston, TX, showed that the exercise
program designed for this research study is well accepted by both obese and lean subjects. The subjects have complied very well to the program both at the Wellness Center and at home, resulting in increased fitness in all subjects (average 17% increase in VO2 max). Researchers have also refined the MRI measures of intramyocellular, visceral, and hepatic fat. Preliminary data in these first subjects showed that obese subjects had about twice as much intramyocellular, hepatic and visceral fat as compared with their lean counterparts; in the obese subjects, the most significant fat reduction resulting from the exercise program occurred in the liver (25%); also insulin concentrations were about twice as high in the obese as compared with the lean subjects and decreased by about 11% in the obese with no change in the lean adolescents. Identification of an exercise program that is accepted by both subject groups is an accomplishment. 5. Describe the major accomplishments over the life of
the project, including their predicted or actual impact. Project 1: Genetic and environmental factors contributing to childhood obesity Our research project has made significant progress in understanding the environmental and genetic factors affecting childhood obesity in the Hispanic population in the U.S. Our major goal was to identify and phenotype/characterize 300 overweight Hispanic probands (ages 4-19 y) and his/her biological parents and siblings with respect to adiposity, the regulation of food intake, energy expenditure, and energy partitioning. We have completed the recruitment and phenotyping of a total of 1030 children and 631 parents from 319 Hispanic families in our VIVA LA FAMILIA study. The genotyping is complete on 800 individuals. Preliminary variance component linkage analysis has been performed on this subset. Biochemical analysis and genotyping, and construction of the 10 M genetic maps to be used in a genome-wide scan continue. A 6-month intervention trial for
weight reduction in overweight children has begun on a pilot sample to develop materials and methods for Hispanic families. Completing the large collection of phenotypic data on subjects is a major accomplishment of this research project and the future of this project. Project 3: Prevention of overweight in children Preliminary analyses have been conducted and demonstrated that participants who have completed the intensive phase of the intervention are not as heavy as the children assigned to the self-help program. We will present these preliminary results to scientists, clinicians, and public health authorities at an international meeting of obesity researchers in October 2005. These customers will gain valuable information regarding how to solve the problem of recruiting and retaining Mexican American children and their families in research. In addition, they will benefit from strategies and techniques that will be beneficial in addressing the problem of childhood obesity in Mexican
Americans. Project 4: Nutritional influences on innate immunity The project overall has addressed the problem of dietary obesity and its ability to produce systemic inflammation and liver injury. In addition to the accomplishment listed above we have found that endotoxin released from the gut contributes to the liver injury, and we have defined the contributions of adhesion molecules to the emigration of leukocytes. We are defining the cascade of events that leads to systemic inflammation and liver injury. These are major complex processes and have not been solved. We have defined characteristics of macrophages in fat. We have defined the major inflammatory cell type in the obesity liver. We have shown that stress increases liver damage in animals on high-fat diet. We have found sex differences in the liver and adipose tissue response to high-fat diet. Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents
Scientists at the Children's Nutrition Research Center in Houston, TX, showed that the exercise program designed for this study is well accepted by both obese and lean subjects. The subjects have complied very well to the program both at the Wellness Center and at home, resulting in increased fitness in all subjects (average 17% increase in VO2 max). Researchers have also refined the MRI measures of intramyocellular, visceral, and hepatic fat. Preliminary data in these first subjects showed that obese subjects had about twice as much intramyocellular, hepatic and visceral fat as compared with their lean counterparts; in the obese subjects, the most significant fat reduction resulting from the exercise program occurred in the liver (25%); also insulin concentrations were about twice as high in the obese as compared with the lean subjects and decreased by about 11% in the obese with no change in the lean adolescents. Since we are in the early stage of this project, the only customers
so far are the participating adolescents. The subjects involved in these studies are used to a sedentary life style. Virtually all of them have indicated that they have enjoyed participating in the program and that they have felt good about themselves for being able to fulfill the program. They have also indicated their intention to continue exercising after completion of the exercise. These results are important since these adolescents, particularly the obese, most often avoid participating in PE or other athletic activities. Project 6: Circadian clocks in adipose biology and obesity We have characterized the basic components of the circadian mechanism within the adipocyte and have demonstrated this mechanism to be fully functional within this cell type. This paper is in preparation. We have also successfully created a heart-specific dominant negative CLOCK transgenic mouse which shows little or no rhythmicity of the clock mechanism genes. We have just begun to characterize this
mouse, but its successful creation provides us with confidence that creating a similar animal in which the circadian clock is non-functional within the adipocyte is feasible. This research contributes to the goals of NP107, Human Nutrition, specifically "Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease." It addresses ARS Strategic Plan Goal 4 Improve the Nation's Health, specifically objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Project 2: Biological diversity of
human growth: body composition reference standards for children Our findings have been transferred to the scientific community at national and international meetings, and in publications in peer-reviewed journals. To provide wider distribution of this information, an inter- active web-site with access to z-score models for pediatric body composition is maintained. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Project 5: Effects of ethnicity, exercise and obesity on glucose metabolism and insulin sensitivity in healthy adolescents Sunehag, A. 2005. Insulin secretion and resistance in obese adolescents: Impact of dietary macronutrient intake. Mars Inc. Nutritional Research Council, January 2005, Houston, Texas. Sunehag, A. 2005. Insulin resistance in adolescents: Obesity is a more important contributor than macronutrient intake. Pediatric Research
Society, Baylor College of Medicine, March 2005, Houston, Texas. Sunehag, A. 2005. Effects of dietary fructose intake on insulin sensitivity and secretion and glucose and lipid metabolism in lean and obese adolescents. International Life Science Institute, May 2005, Washington, D.C. Sunehag, A. 2005. Glucose metabolism and insulin sensitivity were unaffected by dietary fructose intake (and glycemic index) in obese and lean adolescents. Annual Meeting for the Society of Pediatric Research, May 2005, Washington, D.C.
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