2009 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: Six subjects have already completed the study; one more is currently enrolled and another qualified subject will start the study within the next couple of weeks. We have drinks for three more subjects and hope to recruit two more needed within the next few weeks. We have done partial analysis of the samples from the samples from the first 6 subjects. Those subjects who have completed the study tolerated the limonoid containing and placebo drinks well and there were no serious adverse effects. 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 the director of Bioanalytical laboratory.
In addition to the limonoid study we have continued sample and data analysis from our previous DHA study with hyperlipidemic men. A manuscript based on the effects of DHA supplementation on markers of lipid peroxidation has been submitted to the Journal of Nutrition; another manuscript based on the gene array data from this study is under preparation.
Specific Aim 2: A mice study testing the dose of DHA needed to prevent adverse effects of conjugated linolenic acid has been conducted, and samples have been collected. Analysis of these samples will suggest the dose of DHA to be used for future studies.
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.
Our recent results revealed that saturated fatty acids induce dimerization and recruitment of TLR4 into lipid rafts, whereas n-3 polyunsaturated fatty acids (PUFA) inhibit the processes. The dimerized TLR4 is found only in lipid rafts, but not in non-lipid rafts. These results suggest that n-3 PUFAs inhibit the activation of TLRs by interfering dimerization and recruitment of TLR4 to lipid rafts where the receptor interact with other downstream signaling molecules.
Celery and green pepper compounds may decrease risks for cardiovascular disease. Cardiovascular disease (CVD) is a major cause of death in the United States that can be reduced with appropriate dietary interventions that have anti-inflammatory activity, including increased consumption of fruits and vegetables containing polyphenolic compounds. ARS researchers in the Immunity and Disease Prevention Management Unit at the Western Human Nutrition Research Center in Davis, CA conducted experiments in collaboration with a scientist at Gwangju University in South Korea using a cell culture system to examine the ability of the polyphenol luteolin (abundant in celery and green pepper) to inhibit inflammation stimulated by activation of toll-like receptor 4 (TLR4), which is a key regulator of inflammation. Luteolin was found to inhibit the activation of TLR4 by inhibiting the downstream kinase (TBK1) of TLR4. This information clarifies the precise anti-inflammatory role of this compound and will allow greater specificity in predicting the benefits of dietary interventions and may allow development of more potent health-promoting food products.
Fish oil supplements do not cause oxidative stress. Foods or supplements rich in polyunsaturated fatty acids (PUFA), including fish oil supplements containing docosahexaenoic acid (DHA), can decrease the risk of cardiovascular disease but may increase lipid peroxidation thus negating some of the anti-inflammatory, protective benefits of fish oil and other foods rich in polyunsaturated fatty acids. ARS researchers in the Immunity and Disease Prevention Management Unit at the Western Human Nutrition Research Center in Davis, CA conducted a study in hypertriglyceridemic men receiving fish oil supplements. The study measured PUFA peroxidation products in lipid samples collected from hypertriglyceridemic men before and after DHA supplementation. DHA supplementation did not increase markers of lipid peroxidation in red blood cell and plasma lipids. Since DHA supplementation provides a number of health benefits, it can be taken without the concern of increasing oxidative stress. These findings alleviate the concern that DHA supplementation may increase lipid peroxidation and increase the risk of chronic diseases.
Huang, S., Zhao, L., Kim, K., Lee, D., Hwang, D.H. 2008. Inhibition of Nod2 signaling and target gene expression by curcumin. Molecular Pharmacology 74(1):274-281, 2008.