2009 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. Formerly 5306-51530-011-00D (4/04). 3.Progress Report A DHA study with hypertriglyceridemic men was successfully conducted. Results from this study showed that DHA supplementation decreased both the fasting and postprandial plasma triglyceride concentrations, number of total and small dense LDL particles, and of remnant chylomicron particles; it increased the number of large LDL and HDL particles. DHA supplementation also decreased the number of circulating granulocytes and plasma concentrations of a number of inflammatory markers including CRP, IL-6, and GM-CSF. Thus, DHA decreased the risk for cardiovascular disease by improving the blood lipid profile and by decreasing inflammation. Three papers based on the results from this study have been published, one has been submitted and one is under preparation. The milestones for this specific aim were completely met. The original specific aim 2 had to be replaced because of technical difficulties and because the support scientist working on this project moved to a different location. This specific aim was replaced with investigating the role of individual n-3 polyunsaturated fatty acids (ALA, EPA, and DHA) in the prevention of insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD) in mice model. Results of our studies showed that DHA prevented both CLA induced IR and NAFLD, while EPA prevented only NAFLD. ALA form flaxseed oil completely prevented IR and partially prevented NAFLD. Results show that individual n-3 PUFA differ in their efficacy and specific effects. We also examined the mechanisms by which CLA caused these metabolic disorders and the n-3 PUFA prevented them. This specific aim was completely met. We have found that saturated fatty acids can activate TLR2 in addition to TLR4. However, n-3 polyunsaturated fatty acids (PUFAs) inhibit all TLRs tested. Therefore, saturated fatty acids can activate TLR2 and TLR4, but n-3 PUFAs are pan inhibitors for all TLRs. N-3 PUFAs such as eicosapentaenoic acid and docosahexsaenoic acid are more potent in inhibiting TLR4 activation than n-6 PUFAs such as linoleic acid and arachidonic acid. These results reveal the relative anti-inflammatory (TLR-mediated) potency of different PUFAs. We have found that saturated fatty acids and n-3 PUFAs reciprocally modulate multiple downstream TLR4-siganling pathways involving MyD88, TRIF and PI3-Kinase/Akt, and the expression of TLR target genes. As a functional consequence of this modulation, saturated fatty acids and n-3 PUFAs also reciprocally modulate both innate and adaptive immune responses. We have found that saturated fatty acid, lauric acid stimulated TLR4 dimerization and the recruitment of TLR4 to lipid raft fractions, whereas, n-3 PUFA docosahexaenoic (DHA) inhibits these processes in a reactive oxygen species dependent manner. These results reveal the molecular mechanism by which fatty acids differentially modulate TLR-mediated inflammatory responses which are implicated to be linked to the risk of many chronic diseases. 4.Accomplishments 1. Reciprocal modulation of immune cell function by saturated and polyunsaturated fatty acids (PUFA). Polyunsaturated fatty intake is associated with decreased risk of chronic inflammatory disease, such as cardiovascular disease, while intake of saturated fatty acids is associated with greater disease risk. How PUFA decrease disease risk is not completely understood and recent findings suggest that PUFA may be anti-inflammatory while saturated fats may be pro-inflammatory. ARS scientists at the Western Human Nutrition Research Center in Davis, CA conducted studies with isolated immune cells that normally promote inflammation (dendritic cells) to determine the effects of these fatty acids on the expression Toll-like receptors on these cells that regulate inflammation. These experiments demonstrated that saturated fatty acids increased the activity of this receptor while PUFA decreased the activity. These results suggests that PUFA may decrease the risk of chronic inflammatory disease by decreasing inflammation mediated by dendritic cells, while saturated fatty acids have the opposite effect. The impact of this research is to strengthen the scientific basis for recommending increased PUFA and decreased saturated fatty acid intake in order to promote health. 2. Biomarkers of cardiovascular disease risk decreased by consumption of docosahexaenoic acid supplements. Cardiovascular disease (CVD) is a major cause of death in the United States that can be reduced with appropriate dietary interventions such as the consumption of fish or supplements containing fish oils such as docosahexaenoic acid (DHA). The precise role of DHA as compared to other fatty acids in fish oil is unclear. ARS Researchers in the Immunity and Disease Prevention Management Unit at the Western Human Nutrition Research Center in Davis, CA conducted a study with 34 hypertriglyceridemic men (45-65 y) who were randomly divided into 2 equal groups receiving DHA (3 g/d) or a placebo. DHA supplementation significantly reduced both fasting and postprandial circulating triglycerides, the blood concentration of CVD-promoting total and small dense low density lipoprotein particles and remnant chylomicron particles, blood levels of a number of inflammatory markers including C-reactive protein (CRP), and also decreased heart rate and blood pressure. These results suggest that the risk for CVD can be significantly reduced by taking DHA supplements. 3. Dietary trans fatty acids interfere with the metabolism of health-promoting omega-3 fatty acids: Trans fatty acids, such as conjugated linoleic acid (CLA), are known to increase the risk for cardiovascular disease, but the mechanisms involved are poorly understood. ARS scientists at the Western Human Nutrition Research Center in Davis, CA examined the effect of supplementing mice diets with a particular, common dietary form of CLA (trans-10, cis-12 CLA) on the fatty acid composition of tissues. Feeding CLA caused adverse health consequences, including the development of a fatty liver, but also caused unexpected, dramatic changes in the fatty acid composition of other tissues. In particular, it reduced the amount of an important polyunsaturated fatty acid (docosahexaenoic acid; DHA) in heart tissue and increased the DHA content of the spleen by 700%. These dramatic alterations in tissue distribution of the essential fatty acid DHA may help explain the adverse health risks associated with CLA intake from certain processed foods. The impact of this research is to provide a stronger scientific basis for dietary recommendations for Americans. 4. Consumption of Bing sweet cherries reduces the plasma concentration of markers of inflammatory disease progression. Fruits and vegetables contain compounds which can decrease inflammation and thus decrease the risk of development of chronic inflammatory diseases. However, the content and potency of such compounds varies between fruits and within different varieties of specific fruits and while studies of specific fruits on the development of disease (e.g., cardiovascular disease) would take years to complete, scientists often examine the effect of short-term dietary interventions on markers of disease risk in blood. The effect of cherry consumption on the plasma concentration of inflammatory markers is not known and was investigated in this study. ARS scientists at the Western Human Nutrition Research Center in Davis, CA studied eighteen healthy men and women who consumed Bing Sweet cherries for one month. Cherry consumption reduced the plasma concentration of several markers of inflammation by 18-25%. These results suggest cherry consumption may reduce the risk for inflammatory diseases including arthritis and cardiovascular disease. The impact of this research is to provide a stronger scientific basis for dietary recommendations for Americans. 5. Inhibition of Inflammatory Responses by Plant Polyphenols. Chronic inflammatory diseases, such as arthritis and autoimmune disease, are important health problems for Americans. The risk and severity of these diseases may be reduced with appropriate diets. Many polyphenolic chemicals normally found in plant foods (resveratrol, curcumin, epigallocatechin) have anti-inflammatory properties that are thought to decrease the risk of such chronic inflammatory diseases, but their mechanism of action at the cellular and molecular level is not known. ARS scientists at the Western Human Nutrition Research Center in Davis, CA investigated whether certain plant polyphenols inhibit inflammation mediated by two immune cell types, macrophages and B lymphocytes, by decreasing their responsiveness to normal inflammatory stimuli via a cell-surface receptor known as “Toll-like receptor”. The results of these experiments indicate that these polyphenols do not alter cell-surface expression of this receptor but do decrease its ability to transmit a signal to the cell nucleus, which is necessary to trigger greater inflammation. This work demonstrates how specific dietary components act to reduce inflammation. The impact of this research is to provide a stronger scientific basis for dietary recommendations for Americans. 6. Some but not all polyunsaturated fatty acids (PUFA) counteract the damaging effects of trans fat on glucose tolerance: Trans fat consumption increases the risk of many chronic inflammatory diseases, including diabetes, among Americans consuming a typical high-fat diet. However, PUFA intake can be beneficial and it may counteract some of the detrimental effects of high intake of trans fat. ARS scientists at the Western Human Nutrition Research Center in Davis, CA conducted a study in mice to determine if PUFA intake could counteract the expected increase in insulin resistance, which is a hallmark of type 2 diabetes, that would be seen with high trans fat intake. Increased trans fat intake caused a 10-fold increase in circulating insulin, indicating the development of insulin resistance, but concomitant supplementation with two different PUFAs (eicosapentaenoic acid, or EPA; and docosahexaenoic acid, or DHA) prevented the development of some adverse effects of trans fat intake, while only DHA prevented the development of insulin resistance. These results suggest that increasing intake of DHA from fish or supplements by Americans may decrease the risk of insulin resistance caused by diets containing processed foods rich in trans fat. The impact of this research is to provide a stronger scientific basis for dietary recommendations for Americans. Review Publications Adams, S.H., Hoppel, C.L., Lok, K.H., Zhao, L., Wong, S.W., Minkler, P.E., Hwang, D.H., Newman, J.W., Garvey, W.T. 2009. Plasma Acylcarnitine Profiles Suggest Incomplete Fatty Acid ß-Oxidation and Altered Tricarboxylic Cycle Activity in Type 2 Diabetic African-American Women. Journal of Nutrition, 139:1073-1081.