2004 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? We are studying the liver enzymes that hydrolyze retinyl esters, the form in which newly absorbed dietary vitamin A is delivered to the liver. These enzymes (retinyl ester hydrolases or REHs) are important in both the uptake of dietary vitamin A by the liver and in the mobilization of stored vitamin A from the liver when dietary input is inadequate to meet the body's need for this essential nutrient. Although a number of liver enzymes can catalyze the hydrolysis of vitamin A esters the molecular identity and physiological role of these enzymes is unclear. Our research attempts to fill these gaps in knowledge by isolating these enzymes in pure form using classical techniques of enzymology, and by using gene "knockout" techniques to study the physiological role of these enzymes in animal models. We are also studying the impact of dietary carotenoids on the oxidation of human plasma lipoproteins, a process thought to be involved in the etiology of atherosclerosis. We approach this by altering the cellular content of specific dietary carotenoids and asking whether this inhibits the oxidation of the lipoprotein by human aortic endothelial cells and macrophages in culture. We use this system as a model of what may be occurring in the aortic wall in the intact human. We are also studying the basic mechanisms involved in the intestinal absorption of vitamin A and carotenoids. Objectives: 1. Mechanisms of intestinal absorption of carotenoids & their incorporation into chylomicrons in CACO2 cells in culture. We will test the hypothesis that other fat-soluble nutrients inhibit the incorporation of ß-carotene into chylomicrons. We will test the hypothesis that SRB1, CD-36, FAT, and other putative lipid transporters mediate the uptake of ß-carotene into the cell. We will test the hypothesis that increasing the expression of ß-carotene 15,15' dioxygenase increases the total flux of ß-carotene through the cell. 2. Modulation of macrophage function by dietary carotenoids. We will use the U937 cell line as well as human peripheral blood monocyte-macrophages to examine the partitioning of carotenoids into blood-borne cells and the effects of carotenoid enrichment on monocyte macrophage function. We will test the hypothesis that plasma carotenoids partition into various blood cells as well as into plasma lipoproteins. We will test the hypothesis that carotenoid enrichment alters monocyte-macrophage phenotype, either enhancing or inhibiting potentially pro-atherosclerotic aspects of cell function including response to chemotactic stimuli, adhesion to endothelial cells, differentiation into macrophages, ability to oxidize LDL, and ability to form foam cells. 3. Cell type distribution of retinyl ester hydrolases (carboxylesterases) in liver. Laser Capture Microdissection and cell separations coupled with real-time PCR, immunodetection, and enzyme assays will be used to define the distribution of carboxylesterases between stellate cells & hepatocytes and to test the hypotheses that carboxylesterase ES-2 is localized exclusively in hepatocytes and that carboxylesterase ES-10 is localized in hepatocytes and stellate cells. 4. Physiologic roles of the carboxylesterases in the metabolism of chylomicron retinyl esters in cultured cells. cDNA transfection and RNA interference will be used to modulate carboxylesterase enzyme levels to test the hypothesis that the various carboxylesterases play a metabolic role in the hepatic uptake and/or metabolism of chylomicron RE. Vitamin A deficiency is the leading cause of blindness in the world. More marginal deficiency is associated with increased risk of infections such as measles. Our work is on the basic biochemical mechanisms involved in the metabolism of vitamin A in the intestine and in the liver, the major site of vitamin A storage in the body. Cardiovascular disease, resulting from the underlying process called atherosclerosis, is a leading cause of death and morbidity in the United States population. There is a good deal of evidence that this disease process is related in part to the oxidation of plasma lipoproteins by cells of the blood vessel wall. There is also strong epidemiological evidence relating the consumption of fruits and vegetables (which contain high amount of antioxidant phytonutrients such as carotenoids) to a decreased incidence of cardiovascular disease. It is important to define the effects of specific antioxidant nutrients on lipoprotein oxidation to be able to make reasonable recommendations for the consumption of these nutrients. It is also important to understand the basic mechanisms of the disease process in order to define potential targets for therapeutic intervention. Finally, it is important to understand the mechanisms of intestinal absorption of carotenoids because the extent of bioavailability needs to be considered when formulating recommended intakes. Our research falls under the National Program on Human Nutrition Requirements, Food Composition, and Intake. As indicated in the Program Statement, "human nutrition science has moved from a focus on the prevention of nutrient deficiencies to an emphasis on health-maintenance and reduced risks of chronic diseases." Our research on carotenoids exemplifies that focus by examining the effects of components of the diet (ie., carotenoids) that are not nutritionally essential but that may have impact on a major chronic disease. As indicated in the Program Statement the annual economic impact of cardiovascular disease in the U.S. exceeds $80 billion. Specific program components are focused also on "definition of marginal deficiencies..." and "bioavailability of nutrients in foods." Our research on vitamin A exemplifies those foci by examining the basic biochemistry of enzymes involved in the storage, release, and bioavailability of vitamin A in the body. 2.List the milestones (indicators of progress) from your Project Plan. Major milestones for Objectives 1 and 4 are the construction of the transfected cell lines and RNAi cells, and we will reach this milestone with 2-3 years. The testing of the cell lines will then be accomplished. Objective 3 is fairly straightforward and will be carried out in the first two years (with the development of the antibodies as an important preliminary milestone). Objective 2 will be worked on throughout as time permits, with the major focus on this objective coming in the last two years of the 5-year cycle. Year 1 Year 2 Year 3 Year 4 Year 5 Objective 1 ---------------------> ----------------------> Construction of transfected cells Characterization of cells Objective 2 --------------------------------> Characterization of carotenoid-enriched cells Objective 3 ---------> -----------------> Ab development Localization studies Objective 4 -----------------------> --------------------------> Construction of Characterization of cells transfected & RNAi cells 3.Milestones: A. List the milestonest that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. For CRIS 1235-51000-049-00D, this is a new CRIS and hence there were no milestones achieved this year. For the previous CRIS 1235-51000-040-00D, milestones were not specified. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? In the next year our efforts will be focused on constructing of transfected cell lines for use in studies on vitamin A and carotenoid metabolism. In year two our efforts in the vitamin A field will be focused on understanding the localization, expression and function carboxylesterases ES-2, ES-3, ES-4 and ES-10 and in particular the role these enzymes play in vitamin A metabolism. We will study the cell and tissue-specific expression of these enzymes using real-time PCR and laser capture microdissection. In year three we will begin the characterization of the cells so as to define the mechanisms involved in the intestinal absorption and hepatic metabolism of vitamin A and carotenoids. 4.What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: Very little is known about the molecular mechanisms involved in the intestinal absorption of vitamin A. Thus, understanding the basic aspects of the bioavailability of this nutrient will aid not only basic scientists, but also nutritionists and policymakers in promulgating appropriate recommendations for intake of this essential nutrient. We sought to apply our recently developed cell culture model to study the mechanisms of vitamin A absorption in the intestine. We used CaCo2 cells in culture to study the kinetics of retinol (vitamin A) absorption. The results showed that there was substantial "absorption" of free (unesterified) retinol and that the transport of retinol across the basolateral membrane was likely to be a facilitated transport. This work should lead to a rethinking of the mechanism by which the essential nutrient, vitamin A, is absorbed in the human intestine. B. Other Significant Accomplishments: None. C. Significant Accomplishments/Activities that Support Special Targeted Populations: None. D. Progress Report: None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project on vitamin A moved to ARS when Dr. Harrison joined DHPL on August 31, 1998, and has been actively pursued by Dr. Harrison for a number of years. Since joining ARS, the major accomplishments of this project were defining the role (or lack of role) of three specific enzymes: namely, carboxylester lipase (CEL), lipoprotein lipase (LPL), and pancreatic triglyceride lipase (PTL) on the metabolism of dietary vitamin A. This work has had major impact in the field of vitamin A metabolism, by defining which of the many enzymes (lipases and carboxylesterases) that can hydrolyse vitamin A esters actually do have a physiological role in the metabolism of this essential nutrient. Our recent work on mechanisms of intestinal absorption of vitamin A is also being recognized as indicated by invitations to prepare invited reviews. The project on carotenoids also moved to ARS when Dr. Harrison joined DHPL, and has been actively pursued. Since joining ARS the major accomplishments have been the demonstration of the specificity of the effects of dietary carotenoids on the oxidation of human low-density lipoprotein (LDL) by endothelial cells in culture. We found that enrichment of LDL with beta-carotene protected LDL from oxidation. However, enrichment with the two other major carotenoids in the diet (and in plasma), namely, lutein and lycopene, actually enhanced cell-mediated oxidation of the LDL. Thus, the specific content of carotenoids in LDL clearly modulates its susceptibility to oxidation, but individual carotenoids may either inhibit or promote LDL oxidation. This underscores the importance of studying individual dietary carotenoids and not just beta-carotene. We also found that enrichment of LDL with beta-carotene by supplementation of the diet protected LDL from oxidation. However, enrichment with another major carotenoid in the diet (and in plasma), namely lycopene (achieved by ingestion of tomato juice), had no effect on cell-mediated oxidation of the LDL. By studying the impact of enriching lipoproteins with carotenoids by dietary supplementation and comparing this method with the in vitro enrichment methods previously developed, we clearly showed that dietary supplementation is more effective in inhibiting LDL oxidation than is in vitro enrichment. These results have shown us and other scientists that in vitro supplementation methods are not appropriate for studying the impact of carotenoid content of lipoproteins on their oxidation. Our work on the molecular mechanisms of intestinal absorption of carotenoids has demonstrated an important in vitro model system for studying bioavailability of these important phytonutrients. We have also reported the cloning of the enzyme that converts beta-carotene to vitamin A. The impact of our work both in vitamin A and in carotenoids is indicated by our continuing (1) publication in high impact journals, (2) invitations to present the work at major scientific meetings, and (3) continued grant support from the National Institutes of Health. 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? Our work in vitamin A has been presented at scientific conferences and published in the peer-reviewed literature. Other scientists are aware of our work and will likely adopt our methodology for isolating and studying the REHs and for studying intestinal absorption of vitamin A. Our work on carotenoids has been presented at scientific conferences and published in the peer-reviewed literature. Other scientists are aware of our work and will likely adopt our methodology for enrichment of lipoproteins with individual carotenoids, our new cell culture medium for oxidation experiments, and our new model system for studying carotenoid intestinal absorption using a cell culture model. Our work on proteomics has been extended to other investigators at the Beltsville Human Nutrition Research Center (BHNRC), and we serve as a resource for Beltsville Agricultural Research Center (BARC) in this field. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. During, A. and E.H. Harrison . Intestinal absorption and metabolism of carotenoids: Insights from cell culture. Presented at the Gordon Research Conference on Carotenoids, Ventura, CA, January, 2004 (by invitation). Linke. T., A. During and E.H. Harrison. Mechanisms involved in the intestinal and hepatic metabolism of retinoids and carotenoids. Presented at the FASEB Summer Research Conference on Retinoids, Pine Mountain, GA, June, 2004 (by invitation).