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? Why does it matter?
The Dietary Guidelines for Americans 2005 are a potentially valuable intervention for preventing obesity and achieving healthy body weight. However, current consumer acceptance and compliance with the Guidelines is poor, and available scientific evidence has not yet demonstrated that the Guidelines are effective for obesity prevention or for promotion of a healthy phenotype in overweight individuals. The long-term goal of our research is to determine if the current dietary and physical activity guidance for Americans will achieve metabolic health and contribute to obesity prevention. The investigators will focus on three specific areas included in the Guidelines: the quality and quantity of carbohydrates in the diet, the health-promoting properties of dietary fat, and the physical activity recommendations to prevent weight gain. Comprehensive studies on the inclusion of whole grain products in the daily diet will be conducted to evaluate the acceptability and use of whole grain products, determine the optimal amount of whole grains that should be consumed, and identify a biomarker of whole grain intake. Similarly, studies will be conducted to further clarify the effects of ingesting added sugars, including sucrose and high fructose corn syrup, on satiety and energy metabolism. The investigators will conduct preliminary studies to determine how different sources of omega-3 fatty acids affect the partitioning of lipids and the formation of potentially-damaging, pro-inflammatory oxy-lipins. These compounds may play an important role in altering the risk of cardiovascular disease and affect the pathways to lipid storage or lipid combustion. Finally, a study will be conducted to evaluate the current recommendation for physical activity to prevent weight gain. The investigators will determine what barriers and facilitators exist for incorporating activity into daily lives and maintaining adequate physical activity levels, examine the effect of regular physical activity on dietary intake, energy expenditure, metabolic health, and quality of life. In conjunction with these studies, investigators will identify molecular factors that influence critical metabolic pathways in body weight regulation and determine if gene expression is responsive to dietary and physical activity interventions related to the Guidelines.

This work is relevant to all Americans who will benefit from the knowledge obtained to help achieve and maintain a healthy body weight. National surveys indicate that more than half of our population is overweight or obese, and the prevalence of obesity is escalating. Obesity is associated with increased risk of developing chronic diseases such as type 2 diabetes, vascular disease, and certain cancers, and there is increased occurrence of gallbladder disease, complications of pregnancy, sleep apnea, and psychosocial distress in obese people. Steps must be taken now to reverse the increasing incidence of obesity, otherwise future generations will face unprecedented levels of premature disease, disability and the burden of overwhelming healthcare costs. Evaluating our Dietary Guidelines as an obesity-preventive or health-promoting intervention is crucial to understanding and preventing obesity. Through this scientific understanding we will be better equipped to improve strategies to prevent adult weight gain or to improve the health of all individuals regardless of body size.

This project addresses Component 5 - Health Promoting Properties of Plant and Animal Foods, Component 6 - Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle, and Component 7 - Health Promoting Intervention Strategies for Targeted Populations of NP107, Human Nutrition.

2.List by year the currently approved milestones (indicators of research progress)
FY2006 (3rd & 4th Qtr only) 1. Complete study plans for the whole grain study and submit to the IRB. 2. Procure or build the DNA construct for the transgenic mouse model 3. Establish acyl-carnitine methods 4. Expand capabilities of analytical methodology for ALA metabolism 5. Write protocol for ALA feeding study in insulin-resistant hamster model and submit to Animal Use Committee for approval 6. Implement the ALA feeding study 7. Begin laboratory analysis of samples generated in the ALA study 8. Complete study plans for the physical activity study and submit to the IRB 9. Describe the expression patterns of AAP in the rodent 10. Describe how the diet influences the expression patterns of AAP 11. Compare the expression of AAP in obesity models 12. Determine the role of AAP in adipocyte differentiation 13. Hire SY: behavioral nutritionist 14. Conduct focus groups with food purchaser/preparer in low income households to assess knowledge and awareness, attitudes and beliefs of the Dietary Guidelines

FY2007 1. Recruit 1st cohort of subjects for whole grain study 2. Recruit 2nd cohort of subjects for whole grain study 3. 1st cohort of subjects complete the whole grain study 4. Analyses of all whole grain study samples begins 5. Complete study plans for the sugar study and submit to the IRB. 6. Implement sugar study and complete subject testing for 1/2 of subjects 7. Begin the process of establishing the working colony of transgenic mouse model 8. Maintain transgenic mouse colony 9. Conduct phenotyping of mouse colony 10. Conduct dietary challenge studies in transgenic mice 11. Establish model for muscle-oxidation 12. Complete work on acyl-carnitine methods 13. Complete laboratory analyses for the ALA study in insulin-resistant hamsters 14. Complete data analyses for ALA study 15. Plan the n-6:n-3 feeding study in insulin resistant hamsters 16. Conduct the n-6:n-3 study 17. Begin laboratory analyses for the n-6:n-3 study 18. Begin enrollment of volunteers into the physical activity study; achieve 100% of total number of subjects needed 19. Implement the physical activity study 20. Begin laboratory analyses of samples from the physical activity study 21. Complete data analysis on AAP studies 22. Prepare publications on the AAP studies 23. Continue with focus groups to assess knowledge and awareness, attitudes and beliefs of the Dietary Guidelines 24. Begin focus group discussions to determine strategies for adopting the Dietary Guidelines

FY2008 1. Complete all lab analyses for samples obtained in the whole grain study 2. Complete data analyses for the whole grain study 3. Complete all lab analyses for samples obtained in the sugar study 4. Complete data analyses for the sugar study 5. Complete data analyses for the transgenic mouse studies 6. Prepare publications on the transgenic mouse studies 7. Complete data analyses for the studies on biomarkers of -oxidation 8. Conduct acyl-carnitine and metabolomic studies in response to exercise 9. Prepare publications on the ALA hamster study 10. Complete all lab analyses for samples obtained in the n-6:n-3 study 11. Complete data analyses for the n-6:n-3 study 12. Complete all lab analyses for samples obtained in the physical activity study 13. Complete data analyses for the physical activity study 14. Prepare publications on the physical activity study 15. Complete data analyses for the focus group data

FY2009 1. Prepare publications on the whole grain study. 2. Prepare publications on the sugar study 3. Prepare publications on the n-6:n-3 study 4. Prepare publications on the results of the focus group studies

4a.List the single most significant research accomplishment during FY 2006.
We have discovered a novel expression pattern for a gene that is abundantly expressed in white adipose tissue, Tusc5. This gene is also expressed in sensory nervous system structures, suggesting cross-talk or shared function between white adipose tissue and specific neurons. Furthermore, an evaluation of polymorphisms of Tusc5 in 384 obese people uncovered rare amino acid shifts in residues otherwise highly conserved across proteins sharing some domain structure with Tusc5. This research addresses Component 6 – Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle of the NP107 (Human Nutrition) and was conducted in the laboratory of Dr. Sean H. Adams at the Western Human Nutrition Research Center, Davis, CA (genetics work in cooperation with L. Pennacchio, Lawrence Berkeley National Lab). The functional relevance of this finding is under investigation but it provides initial evidence suggesting that Tusc5 plays a role in determining adiposity and susceptibility to metabolic dysfunction related to dietary intake.

4b.List other significant research accomplishment(s), if any.
Postprandial Activation of Monocytes Associated with Meals Based on Refined or Whole Grains. We discovered that consumption of a mixed meal based on refined grains or a meal based on whole grains led to a prolonged, significant increase in the number of monocytes expressing TNF in overweight, but otherwise healthy women. Monocyte activation has been associated with development of plaque formation in major blood vessels and subsequent heart disease. The observed inflammatory response to a high carbohydrate meal, regardless of carbohydrate source, is concerning and suggests that an atherogenic milieu exists in postprandial circulation. This research addresses Component 6 of NP107 – Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle and was conducted in the laboratory of Dr. Nancy L. Keim at the Western Human Nutrition Research Center, Davis, CA in cooperation with Dr. John Rutledge of the University of California Davis School of Medicine, Department of Internal Medicine. This research addresses Component 6 of NP107, Prevention of Obesity and Disease. Soluble Epoxide Hydrolase-dependent Metabolism is a Regulator of Ischemic Injury in the Heart. We discovered that blockade of soluble epoxide hydrolase, an enzyme induced by both diabetes and starvation, is cardioprotective in a rodent model of ischemic injury. This work was performed by Dr. John Newman at the Western Human Nutrition Research Center, Davis, CA in cooperation with Dr. Darryl Zeldin at National Institutes of Environmental Health Sciences. This research addresses Component 6 of NP107, Prevention of Obesity and Disease. Peptidyl-ureas are Potent, Water Soluble Inhibitors of the Soluble Epoxide Hydrolase. Using combinatorial chemistry we discovered potent and water soluble peptidyl-ureas inhibitors of the soluble epoxide hydrolase. The suite of synthesized compounds demonstrated that with the addition of bulky side chains, the mouse enzyme is no longer a good model for the human ortholog, and provided novel structural lead compounds for pharmaceutical development. These findings were produced by Dr. John Newman in collaboration with Drs. Christophe Morisseau and Bruce Hammock of the University of California Davis, Department of Entomology. This research addresses Component 6 of NP107, Prevention of Obesity and Disease.

Lipid Phosphate Analogs are Potent Inhibitors of the Phosphotase Activity Associated with the Soluble Epoxide Hydrolase. We discovered that lipid phosphate analogs, including phosphonates, sulfates and sulfonates, are potent inhibitors of the soluble epoxide hydrolase amino terminus phosphatase activity. This invention was defined by Dr. Newman during his post-doctoral tenure in the laboratory of Dr. Bruce Hammock in collaboration with Dr. Christophe Morisseau of the University of California Davis, Department of Entomology. A patent filing for this discovery has been made (UC Case# 2005-597-1). Title: Inhibitors of phosphatase activity of the soluble epoxide hydrolase amino terminus (Filed Aug 12, 2005). These compounds will provide novel tools to investigate the role of this enzymatic activity in human metabolism. This research addresses Component 6 of NP107, Prevention of Obesity and Disease.

4c.List significant activities that support special target populations.
None.

4d.Progress report.
Identifying a Biomarker of Whole Grain Consumption: No biomarker has been identified that correlates either qualitatively or quantitatively with whole grains in the diet. To address this problem, we are comparing blood plasma metabolomic fingerprints of in women consuming a diet containing either 0 or 6 or more servings of whole grains per day. This work represents a collaboration between ARS WHNRC scientists Dr. John Newman and Dr. Nancy Keim and addresses the NP107 Component 6 - Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle.

Quantitative analysis of -linolenic acid (ALA)-derived oxylipins: Progress toward the expansion of the existing oxylipin analyses to include ALA metabolites has been slow due to a 6 month delay in instrument procurement and hardware stabilization. The established method is now fully operational and the training of technical staff in the routine use of these techniques is nearly complete. Commercially available analytical standards have been purchased for the initial expansion. Collaborations have been established with Dr. Bruce Hammock (University of California Davis, Department of Entomology) and Dr. Theodore Goodfriend (University of Wisconsin, Madison and the Veterans Administration) to produce additional ALA oxidation products. The two non-commercial ALA epoxides have been synthesized and purified to date.

ALA-feeding in Insulin-resistant hamsters: Meetings have been held with Dr. Wallace Yokoyama (USDA, ARS, WNRC - Albany, CA) to coordinate logistics and refine the experimental design related to these studies. These interactions have resulted in an estimated 20% reduction in the proposed animal number, reducing the cost of the study. Animal use protocols are currently being modified to reflect these experimental changes. All animal handling will occur under the direction of Dr. Yokoyama, and will be covered under his existing authorization.

Characterization of a Novel Candidate Gene Believed to Regulate Adipose Function (Tusc5): We have accomplished several key elements toward the goal of elucidating the biological properties of Tusc5. These include: (a) full tissue expression profiles determined in humans and mice, (b) expression pattern analysis during adipogenesis, (c) initiation of a collaboration with L. Pennacchio (Lawrence Berkeley National Lab) to determine genetic polymorphism differences for the gene comparing ~760 lean and obese persons, (d) establishment of tissue fixation parameters in situ hybridization studies of Tusc5 in sensory nerve structures and adipose tissue to better understand which cells express AAP/Tusc5, via a collaboration with T. Baumann (Oregon Health Sciences Univ.). This work addresses the NP107 Component 6 - Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. These accomplishments have confirmed the adipocyte-abundant expression of the gene, and have established key infrastructure for further analyses of cell-specific expression of Tusc5 in neuronal structures and genetic polymorphism determinations.

Determine the Role of Skeletal Muscle Fat Combustion in Determining Insulin Sensitivity Differences Following Alterations in Dietary Carbohydrate. Toward the goal of identifying markers and models of muscle fat and carbohydrate utilization, (a) conditions have been established to perform metabolite profiling studies in isolated muscle mitochondria from rodents (a preliminary dataset has been generated in collaboration with M-E. Harper, Univ. of Ottawa, and O. Fiehn, UCD), and (b) a Material Transfer Agreement is being formulated between ARS and Novartis Pharmaceuticals to obtain a cDNA vector to facilitate generation of transgenic mice with high muscle fat combustion. With respect to fructose and sugar metabolism, a plasma fructose assay has been established and validated in preparation for studies involving modulation of fructose intake in humans and animals, and a new tissue extraction and assay protocol has been worked out to determine tissue hexosamine concentration to support animal model studies examining the metabolic ramifications of fructose intake. This work addresses the NP107 Component 6 - Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. The establishment of analytical techniques and models to test metabolite profiles indicative of muscle fat and carbohydrate utilization lays the groundwork for planned studies to evaluate the interaction of these macronutrients in determining insulin sensitivity.

5.Describe the major accomplishments to date and their predicted or actual impact.
Expression Pattern & Genetic Polymorphism Studies of AAP/Tusc5. We have discovered a novel expression pattern for a gene that is abundantly expressed in white adipose tissue, Tusc5. This gene is also expressed in sensory nervous system structures, suggesting cross-talk or shared function between white adipose tissue and specific neurons. Furthermore, an evaluation of polymorphisms of Tusc5 in 384 obese people uncovered rare amino acid shifts in residues otherwise highly conserved across proteins sharing some domain structure with Tusc5. This research addresses Component 6 - Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle of the NP107 (Human Nutrition) and was conducted in the laboratory of Dr. Sean H. Adams at the Western Human Nutrition Research Center, Davis, CA (genetics work in cooperation with L. Pennacchio, Lawrence Berkeley National Lab). The functional relevance of this finding is under investigation but it provides initial evidence suggesting that Tusc5 plays a role in determining adiposity and susceptibility to metabolic dysfunction related to dietary intake.

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?
The ALA oxylipin analyses will be made available to collaborators at the University of California Davis and University of Wisconsin, Madison within the next 6 months. These techniques will be provided to the general public within 1yr, and metabolites synthesized during this period will be made available to interested parties.

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).
Health At Every Size. New Hope for Obese Americans? Agricultural Research Magazine. March 2006; pp 10-11.

Whole-Grains Foods’ Fat-Fighting Role Scrutinized. Agricultural Research Magazine. March 2006, pp 20-21.

Review Publications
Keim, N.L., Levin, R.J., Havel, P.J. Carbohydrates. Modern Nutrition in Health and Disease, 10th edition, Lippincott Williams & Williams, 2005. Pp. 62-82.