2010 Annual Report
1a.Objectives (from AD-416)
The overall goal of the proposed studies is to determine whether enhanced inflammation promotes development of insulin resistance, and the mechanisms by which dietary fatty acids and certain plant phytochemicals alleviate insulin resistance Objective 1: Determine the effect of citrus limonoid glucoside (LG) on risk factors for cardiovascular disease including blood lipids and markers of inflammation in hypercholesterolemic humans (D. Kelley with collaboration of D. Hwang). Objective 2: Evaluate the impact of dietary docosahexaenoic acid (DHA) on the development and reversal of fatty liver and insulin resistance induced by conjugated linoleic acid (t10,c12 CLA) in animal (mouse) models. Roles of inflammation, adipokines, and insulin signaling will be investigated to understand the changes in lipid and glucose metabolism, and the mechanisms involved (D. Kelley with collaboration of D. Hwang).Objective 3: Prepare transgenic mice in which Toll-like receptor 4 (TLR4) is over-expressed in adipose tissue. Then, determine whether enhanced inflammation in adipose tissue induced by over-expression of TLR4 promotes the development of insulin resistance, and whether dietary n-3 PUFAs ameliorate these processes (D. Hwang with collaboration of D. Kelley).
1b.Approach (from AD-416)
APPROACH: Proposed experiments will involve studies in human volunteers, and in animal and cell culture models. For specific objective 1, we will determine the safety and metabolism of LG and its effects of on serum concentrations of lipids, lipoproteins and their sub-fractions, markers of inflammation and oxidative stress in hypercholesterolemic human subjects. We will determine the responsiveness of monocytes and T lymphocytes by testing various immunological parameters, such as production of inflammatory cytokines, lymphocyte activation, proliferation, and phenotypic analysis for subtypes before and after limonoid ingestion. We will also determine the pharmacokinetics of the metabolism of limonoids by examining the blood and urine concentrations of different LG metabolites. Experiments for specific objective 2 will be conducted in the mouse model to determine the prevention and reversal of CLA induced insulin resistance and non-alcoholic fatty liver disease. To understand the mechanisms involved we will investigate the effects of these fatty acids on the expression of genes involved in fatty acid and lipid metabolism. Further, we will determine the effects of these fatty acids on insulin secretion and insulin signaling pathways. For specific objective 3, we will determine whether enhanced sterile inflammation promotes development of insulin resistance, and the mechanism by which n-3 fatty acids alleviate insulin resistance using transgenic mice in which inflammation is enhanced in adipose tissue. The first study is to prepare and characterize phenotypes of the transgenic mice that over-express a constitutively active form of TLR4 in adipose tissue, in an organ specific manner. The second study is to determine whether dietary n-3 PUFA diet alleviates insulin resistance in these transgenic mice. The third study is to elucidate the mechanism by which n-3 fatty acids alleviate insulin resistance. The fourth study is to determine the efficacy and mechanism by which plant polyphenols alleviate insulin resistance using the transgenic mice described above. The fourth study will be performed only if extramural funding becomes available. Replaces 5306-51530-015-00D (1/09).
Specific Aim 1: Our study protocol called for 10 subjects to complete the cross over study with placebo and limonoid containing drinks. Six subjects had completed the study in FY 2009. Four subjects were enrolled in the study during FY 2010; 3 of them have completed the study and the fourth one will complete it in August 2010. A total of 60 subjects were screened to find 10 subjects who met the inclusion criteria. All study subjects tolerated the liminoid drinks well without serious adverse effects. We will proceed with sample and data analysis once samples from the last subject have been collected. The progress of the study has been frequently discussed by phone and e-mails with the sponsor of the study (Beverage institute) and collaborators, director of human studies and director of Bioanalytical laboratory.
Specific Aim 2: We analyzed the samples from the 2009 DHA dose response mice study for the prevention of insulin resistance and fatty liver; we also conducted another mice study to determine if DHA can reverse the CLA-induced fatty liver and insulin resistance. Results from the 2009 DHA dose response study indicated that a DHA dose of 1.5 wt% was more effective than a dose of 0.5% in the prevention of CLA-induced insulin resistance and fatty liver. In the 2010 experiment we fed mice CLA containing diets for four weeks and then for the next four weeks we changed the diet to either CLA and DHA free control diet, or CLA and DHA containing diet or CLA free but DHA containing diet. Results showed that removal of CLA or adding DHA with CLA both partially reversed the fatty liver, but the removal of CLA along with the addition of DHA completely reversed the fatty liver. Thus, the removal of trans fatty acids and the addition of DHA both contribute to the reversal of fatty liver and the two treatments together were most effective.
Specific Aim 3: We have obtained two founder mice for adipose specific TLR4 transgenic mice that express transgene protein. These mice showed elevated expression of many proinflammatory genes in adipose tissue suggesting enhanced inflammation as a result of transgene expression. The results showed that female TLR4 transgenic mice fed low fat commercial rodent diet gain less weight and improved insulin sensitivity compared with the wild type mice. These results suggest a lean body phenotype for the TLR4 transgenic mice. Food intake by the transgenic mice was less than that by the wild type mice. These results are somewhat opposite to what we hypothesized. Before we publish these results, we plan to repeat phenotyping the second line of the transgenic mice to see whether such a lean body phenotype is reproduced. Currently, the second line of the transgenic mice are being expanded to conduct the phenotyping study.
Fatty acid metabolism and inflammation: Acylcarnitine, a byproduct of dysfunctional fatty acid metabolism, accumulates in tissues of obese subjects and its chemical structure suggests that it could stimulate inflammation and thus be causally associated with development of cardiovascular disease. ARS researchers at Davis, CA developed and tested this hypothesis using cell-culture experiments and found that acylcarnitine does, in fact, promote inflammation by stimulating toll-like receptors (TLR) to activate the NFkappaB signaling pathway. These results suggest that dietary changes to normalize acylcarnitine levels may decrease the risk of cardiovascular disease and provide a potential biomarker (acylcarnitine) for monitoring the success of such dietary interventions.
Cruciferous vegetables and inflammation: Sulforaphane, which is abundant in cruciferous vegetables including broccoli and cauliflower, has anti-inflammatory and anti-cancer activities but the mechanism of action of this compound is not known. ARS researchers at Davis, CA with colleagues from South Korea, used cell-culture experiments to demonstrate that sulforaphane inhibits inflammation by blocking the activation of toll-like receptor 4 (TLR4), a key mediator of inflammation. These results indicate that inflammation stimulated by this pathway can be specifically blocked by this compound from cruciferous vegetables and this knowledge increases the scientific basis for making dietary recommendations to decrease the risk of chronic inflammatory disease in humans.
Docosahexaenoic acid (DHA) and fatty liver: Diet-induced fatty liver is a component of metabolic syndrome that is associated with obesity and an increased risk of cardiovascular disease. ARS researchers at Davis, CA used a mouse model to test the impact of increased intake of the omega-3 fatty acid DHA on fatty liver induced by a trans fatty acid, conjugated linoleic acid (CLA). The study found that that removal of CLA from the diet or adding DHA to a diet containing CLA partially reversed development of fatty liver while the combination treatment (removal of CLA plus addition of DHA) completely eliminated development of fatty liver. This study suggests that both removal of trans fatty acids and the addition of DHA to human diets may reverse fatty liver and decrease the risk of cardiovascular disease, though intervention trials are needed to confirm this hypothesis.
Zhao, L., Kwon, M., Lee, J.Y., Fukase, K., Inohara, N., Hwang, D.H. 2007. Differential Modulation of Nods Signaling Pathways by Fatty Acids in Human Colonic Epithelial HCT116 cells. Journal of Biological Chemistry. Vol.282, No.16,pp.11618-11628.
Lee, J.K., Hwang, D.H., Lee, J.Y. 2009. Toll-like receptors in the pathogenesis of inflammatory diseases. Journal of Organ Dysfuntion. 5:2,119-128, 2009.
Lee, J., Kim, S., Kim, Y., Lee, W., Hwang, D.H., Lee, J. 2009. Suppression of the TRIF-dependent signaling pathway of Toll-like receptors by luteolin. Biochemical Pharmacology. Vol.77:1391-1400.
Kelley, D.S., Siegel, D., Fedor, D.M., Adkins, Y.C., Mackey, B.E. 2009. Docosahexaenoic Acid Supplementation Decreases Serum C - Reactive Protein and Other Markers of Inflammation in Hypertriglyceridemic Men1-3. Journal of Nutrition. 138: 30-35, 2008.