2005 Annual Report 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 research unit addresses nutrient - gene interactions and investigates these interactions through two individual research projects:. 1)Natural products and nuclear receptors: PPARs; and. 2)Maltase-glucoamylase, regulator of starch digestion. This research addresses ARS Human Nutrition National Program (NP107), Component 2: Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease, Priority Objective B. Mechanism of Action - Identify and fully characterize mechanisms of action for beneficial effects of known nutrients and other potentially beneficial dietary chemicals; measure the size of the effects associated with specific amounts of the chemical or nutrient component in question. It conforms to ARS Strategic Plan Goal 4 Improve the Nation's Nutrition and 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. Metabolic disorders such as obesity and diabetes have reached epidemic proportions in the United States and a priority objective of the ARS Human Nutrition National Program (NP107) is to characterize the mechanism of action of nutrients and other agents with potentially beneficial effects on such disorders. Project 1: Natural products and nuclear receptors: PPARs Important metabolic regulatory functions have been identified for several new members of the nuclear hormone receptor superfamily, including the PPARs, LXRs, FXR, CAR, and PXR. All of these receptors are relatively promiscuous, with each recognizing both endogenous and exogenous ligands that often share little or no structural similarity. Thus, we hypothesize that these receptors are potential targets and mediators of the beneficial effects of nutrients and other natural products. Two broad objectives are proposed to test this hypothesis. The first is to identify nutritional products and other natural products that regulate the activity of specific nuclear hormone receptors, and define the active agents that modulate receptor functions. The second is to characterize the effects of such novel receptor ligands at the levels of receptor function and regulation of expression of target genes in appropriate tissues. Successful completion of these objectives will lead to the identification of new therapeutic approaches to metabolic disorders. Project 2: Maltase-glucoamylase, regulator of starch digestion Genes ultimately regulate the digestion and absorption processes. Additional research will focus on the interactions of nutrients and genes in these processes, especially the regulation of genes as they contribute to the development of diet associated degenerative diseases, such as diabetes and atherosclerosis. Our research objective is to determine the mechanisms by which dietary starch interacts with the gene expressing maltase-glucoamylase (MGA). Maltase-glucoamylase is the gate-keeping enzyme that determines human small intestinal starch digestion into glucose. The function and regulation of maltase-glucoamylase is under investigation in those subjects that are deficient in starch digestion. New discoveries in our lab have shown that both Mgamme and Mgamso are differentially spliced in a variable region. These spliced forms appear to alter substrate binding regions but not catalytic amino acids. This non-allelic variability exists in the gene as paralogous duplications. We are preparing expression constructs of these spliced forms to determine the function of the variable regions. We have also begun mouse feeding studies that test whether types of food starches can alter the splicing of Mgamme and Mgamso and to identify the foods and other natural products that regulate splicing. The function of the protein domains in the Mgamme and Mgamso will be investigated in expression systems. Successful completion of these objectives will lead to the identification of new therapeutic approaches to metabolic disorders. 2.List the milestones (indicators of progress) from your Project Plan. Project 1: Natural products and nuclear receptors: PPARs Year 1 (FY 2005): Define roles of soy compounds as ligands for PPAR isoforms and begin characterization of effects of soy compounds on wild type mice. Year 3 (FY 2007): Characterize effects of soy compounds on other PPAR isoforms and other nuclear receptors. Initial characeterization of nuclear receptor effects of biochanin A, formononetin and additional potential metabolic regulators. Define role of soy compounds as PPAR ligands at the levels of target genes and appropriate mouse knockouts. Year 5 (FY 2009): Define role of nuclear receptors in mediating effects of additional potential metabolic regulators. Characterize role of nuclear receptors in metabolic regulatory effects at the levels of target genes using gene arrays and appropriate mouse knockouts. Project 2: Maltase-glucoamylase, regulator of starch digestion Year 1 (FY 2005): Explore starch partitioning and dietary composition, generate the Mgamso construct and define the effect of weaning on transcription factor binding to the Mgam promoter. Weaning on footprinting and EMSA TF confirmation. Conduct a food starch feeding trial. Year 2 (FY 2006): Determine the fate of 13C-starch in the Mgamme KO, generate the Mgamso KO mice, and define the effect of diet on transcription factor binding to the Mgam promoter. Diet on footprinting and EMSA TF confirmation. Year 3 (FY 2007): Initiate phenotypic analysis of the Mgamso KO mice and characterize the effect of dietary glucose on transcription factor binding to the Mgam promoter. Glucose on footprinting, EMSA TF confirmation, and mlCcl2 promoter expression. Year 4 (FY 2008): Determine the fate of 13C-starch in the Mgamso KO and characterize the effect of microflora on transcription factor binding to the Mgam promoter. Microflora on footprinting, EMSA TF confirmation, and mlCcl2 promoter expression. Year 5 (FY 2009): Test the effect of a Mgam enzyme inhibitor used in management of type II diabetes, acarbose, on phenotype and on Mgamme and Mgamso message levels. Feedback regulation of footprint, EMSA TF confirmation, TF KO on Mgamme and Mgamso. 4a.What was the single most significant accomplishment this past year? Project 1: Natural products and nuclear receptors: PPARs UNDERSTANDING THE MOLECULAR BASIS OF THE HEALTH BENEFITS ASSOCIATED WITH ISOFALVONES The potential health benefits of soy isoflavones have been widely publicized, but the molecular basis for such effects is unclear. Researchers hypothesized that, in addition to their modulation of estrogen receptor activity, they would also adjust the activity of other nuclear receptors. This hypothesis has now been shown to be correct, by our lab as well as simultaneously in the labs of others. Specifically, Children's Nutrition Research Center scientists have found that both genistein and daidzein activate PPAR, the anti-diabetic target receptor PPARgamma. These results directly link nutritional isoflavones to metabolic regulatory pathways and provide a basis for specific tests of their biological effects and the mechanisms that underlie them. 4b.List other significant accomplishments, if any. Project 2: Maltase-glucoamylase, regulator of starch digestion GENE REPLICATION IN MAMMALIAN SPECIES A greater understanding of gene replication in mammalian species is needed. Researchers at the Children's Nutrition Research Center in Houston, TX, have discovered that the gene for Mgam is replicated in all mammalian species. The replicated regions are expressed as a variable region and contain a fixed catalytic site but variable substrate binding domains. Food starches vary greatly between plant species, and we hypothesize that the ability to differentially splice Mgam is a response to the variability of food starch. It is recognized that starch digestion can be just as complex as the spectrum of food starches. Our strategy is to express the spliced messages as recombinant proteins so that the substrate binding can be analyzed and recognizing that Mgam is replicated in all mammalian species is a considerable finding that will enhance research in this area. 4c.List any significant activities that support special target populations. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Project 1: Natural products and nuclear receptors: PPARs The potential health benefits of soy isoflavones have been widely publicized, but the molecular basis for such effects is unclear. Researchers hypothesized that, in addition to their modulation of estrogen receptor activity, they would adjust the activity of other nuclear receptors. This hypothesis has been shown to be correct, by our lab as well as others. CNRC scientists have found that both genistein and daidzein activate PPAR the anti-diabetic target receptor PPARgamma. These results directly link nutritional isoflavones to metabolic regulatory pathways and provide a basis for specific tests of their biological effects and the mechanisms that underlie them. The customers include the very large number of people with metabolic disorders, including syndrome X, as well as women who supplement their diet with soy products. Project 2: Maltase-glucoamylase, regulator of starch digestion While the digestion of sucrose and lactose is accomplished by a single message, the digestion of starch is either redundant or highly variable in the response to complex nature of food starches. The future design of food starches for meeting health objectives will need to recognize the spectrum of Mgam splicing and starch digestion. We have determined that the paralogous regions also exist in the human MGAM gene and that two variable regions between exons 22-44 exist at the message level. The design of food starches will need to consider these findings for considering appropriate consumer digestion. This research addresses ARS Human Nutrition National Program (NP107), Component 2: Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease, Priority Objective B. Mechanism of Action - Identify and fully characterize mechanisms of action for beneficial effects of known nutrients and other potentially beneficial dietary chemicals; measure the size of the effects associated with specific amounts of the chemical or nutrient component in question. It conforms to ARS Strategic Plan Goal 4 Improve the Nation's Nutrition and 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? None. 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 1: Natural products and nuclear receptors: PPARs Moore, D. 2005. Nuclear hormone receptors are targets for new metabolic regulators. Invited presentation, October 15, 2005, Beijing, China. Moore, D. 2005. New targets for old treatments - An interface between traditional and modern medicine. Invited presentation, University of Houston, March 30, 2005. Moore, D. 2004. The coffee diterpene cafestol as an FXR and PXR ligand. FASEB 2004, San Diego, California. Review Publications Delgado, A.F., Leone, C., Okay, T.S., Nichols, B.L., Feferbaum, R., Costa Vaz, F.A. 2005. "Silent" tumor necrosis factor alpha (TNF alpha) and interleukin 6 (il-6) serum elevations in critically ill malnourished children do not correlate with therapeutic intervention scoring system (TISS) at 10 days recovery [abstract]. Pediatric Academic Society Meeting. Pediatric Academic Society 57:Abstract 2428. 2005 CDROM.