2008 Annual Report 1a.Objectives (from AD-416) We previously found that docosahexaenoic acid (DHA) reduced the production of several markers for inflammation and serum triglycerides, and increased serum HDL in healthy men. Whether DHA will provide similar health benefits to men who are at increased risk for cardiovascular disease (CVD) because of elevated serum triglycerides is not known. Overall goal of our studies is to determine the effects of dietary fatty acids on immune and inflammatory responses and on risk factors of chronic diseases. Specific objectives are: 1. Determine effects of DHA supplementation on risk factors for CVD in hypertriglyceridemic men. 2. Determine effects of DHA and arachidonic acid on proliferation and differentiation of granulocytic precursor cells, and elucidate the mechanisms involved. 3. Determine whether different dietary fatty acids modulate the activation of Toll-like receptors, downstream signaling pathways, target gene expression and consequent cellular responses. 1b.Approach (from AD-416) Specific aim 1 will involve supplementing diets of hypertriglyceridemic men with DHA, and monitor its effects on a number on serum markers of inflammation (CRP, inflammatory cytokines, adhesion molecules, granulocyte numbers and maturation, scavenger receptor expression on monocytes), in vitro production of inflammatory markers (cytokines and eicosnoids), white blood cell genes whose expression will be up or down regulated by DHA, serum lipids and markers for diabetes. Specific aims 2 & 3 will involve feeding diets with different fatty acid compositions to animal models, and adding fatty acids to cultured cells. A number of molecular biological approaches including DNA microarrays, ectopic expression of genes, and transgenic animals will be used. Results from this study can validate novel markers for CVD and elucidate mechanisms by which dietary fatty acids modify risks of various chronic diseases. 3.Progress Report Specific aim 1: Sample and data analysis for the DHA study continued. A paper dealing with effects of DHA on the fatty acid composition of plasma and red blood cells, plasma concentration of remnant like cholesterol particles was published in the Journal of Nutrition. We completed analysis of plasma samples for the concentration of inflammatory markers and adhesion molecules and a manuscript is under preparation. We completed data analysis regarding the effects of DHA on gene expression for the human study, and a manuscript is under preparation. We have also completed part of the analysis for oxy derivatives for cholesterol and fatty acids in plasma and red blood cells (collaboration with Dr. Yoshida, Japan. The goals of this research are consistent with the goals of National Program 107 and address Component 5, Health Promoting Properties of Plant and Animal Foods. Specific aim 2: We made substantial progress in our experiments with HL-60 cells; however, because of technical difficulties and critical vacancy, we were unable to fully meet the milestone for this specific aim. Instead of continuing this project, we redirected our research effort to determine the effects of plant and marine omega-3 fatty acids on insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). Our earlier publications have shown that conjugated linoleic acid (CLA) induced both these metabolic conditions in mice. In a study conducted with mice last year we found that flaxseed oil which is rich in alpha linolenic acid (ALA) completely prevented the CLA-induced IR and partly prevented the NAFLD. These findings were presented at the 2008 ISSFAL meeting and a manuscript is in press in the British Journal of Nutrition. Thus, the milestones for the revised specific aim were fully met. The goals of this research are consistent with the goals of National Program 107 and address Component 5, Health Promoting Properties of Plant and Animal Foods. Specific aim 3: We have substantially met our milestones for this sub-objective. We determined which downstream signaling pathways derived from TLR activation are modulated by fatty acids, and in turn which target genes are differentially modulated. Identification of the differentially regulated genes and consequent cellular responses will eventually lead us to understand why types of dietary fatty acids differentially affect the risk of development of chronic diseases. Microarray data are currently being analyzed in collaboration with Dr. Kevin Dawson. We have investigated the mechanism by which fatty acids differentially modulate TLR4 activation. The results showed that saturated fatty acid, lauric acid stimulated TLR4 dimerization and the recruitment of TLR4 to lipid raft fractions, whereas, n-3 PUFA docosahexaenoic (DHA) inhibited the dimerization and recruitment. The goals of this research are consistent with the goals of National Program 107 and address Component 5, Health Promoting Properties of Plant and Animal Foods. 4.Accomplishments 1. Flaxseed oil prevented insulin resistance induced by trans fatty acids According to a 2002 survey, 35% of the US adults have insulin resistance (IR) which leads to the development of type 2 diabetes. ARS scientists in the Immunity and Disease Prevention Research Unit in Davis, CA believe the major reason for this increase in IR is the high intake of trans fatty acids and inadequate intake of omega-3 fatty acids. To test this hypothesis a study was conducted in mice where the diet was supplemented either with 0.5% CLA (a trans fatty acid found in processed vegetable oils) or CLA along with 0.5% flaxseed oil (0.3% alpha linolenic acid). The CLA containing diets caused a 7 fold increase in the concentration of circulating insulin, which was restored to the normal levels by the concomitant feeding of flaxseed oil. If CLA and flaxseed oil have similar effects in human beings, then these findings may have enormous implications for the prevention of diabetes. This addresses NP 107, Component 5. 2. Nuclear factor-kappa B (NFkB) inhibition due to plant polyphenols. Certain anti-inflammatory plant polyphenols (e.g.,resveratrol, curcumin) are known to inhibit NFkB transcription factor. However, the direct molecular targets of these polyphenols are not identified. Since NFkB is a common downstream signaling molecule of Toll-like receptors (TLRs), ARS scientists in the Immunity and Disease Prevention Research Unit in Davis, CA determined whether these polyphenols inhibit TLR signaling pathways. The results showed that curcumin inhibits the activation of TLR4 by interfering the dimerization of TLR4 which is the initial step of receptor activation, whereas resveratrol inhibits the downstream kinase enzyme of TLR4 signaling pathways. These results suggest that signaling molecules in TLR signaling pathways may be attractive targets for dietary preventive strategy for many inflammatory chronic diseases. This relates to NP 107, Component 5. 5.Significant Activities that Support Special Target Populations Results from the DHA and CLA studies discussed above improve the health of obese subjects and those with abnormal blood lipids. 6.Technology Transfer None Review Publications Youn, H., Lim, H., Lee, H., Hwang, D.H., Yang, M., Jeon, R., Ryu, J. 2008. Garlic (Allium sativum) Extracts Inhibits Lipopolysaccharide-Induced Toll-Like Receptor 4 Dimerization. Bioscience Biotechnology and Biochemistry 72(2):368-375, 2008. Youn, H.S., Lee, J.K., Choi, Y.J., Saitoh, S.I., Miyake, K., Hwang, D.H., Lee, J.Y. 2008. Cinnamaldehyde suppresses Toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization. 2007. Biochemical Pharmacology. Vol.75(2), p.494-502, 2008. Zhao, L., Lee, J.Y., Hwang, D.H. The Phosphatidylinositol 3-Kinase/Akt Pathway Negatively Regulates Nod2-Mediated NF-kB Pathway. 2008. Biochemical Pharmacology. 75:1515-1525, 2008. Lee, J., Lee, J., Lee, M., Hwang, D.H., Youn, H. 2007. Acrolein with an alpha, beta-unsaturated Carbonyl Group Inhibits LPS-induced Homodimerization of Toll-like Receptor 4. Molecules and Cells. Vol.25, No2, 253-257. Kelley, D.S., Siegel, D., Vemuri, M., Chung, G.H., Mackey, B.E. 2007. Docosahexaenoic acid supplementation decreases remnant-like particle-cholesterol and improves omega-3 index in hypertriglyceridemic men1-3.. Journal of Nutrition. 2007. Journal Nutrition 138:30-35, 2008. Vermuri, M., Kelley, D.S., Erickson, K. 2008. Health Effects of Foods Rich in Polyphenols. Book Chapter, "Wild-Type Food in Health Promotion and Disease Prevention, The Columbus Concept. De Meester, Fabien; Watson, Ronald R. (Eds.) Humana Press, Chapter 27;pp.393-412. Vemuri, M., Kelley, D.S. 2007. The Effects of Dietary Fatty Acids on Lipid Metabolism. Book Chapter in, "Fatty Acids in Foods and their Health Implications, third edition". CRC Press Taylor & Francis Group pp.591-630.