Location: Immunity and Disease Prevention Research2011 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. 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. 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.
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).
3. Progress Report
Objective 1: This year we completed enrollment and follow-up of the final subjects (for a total of ten) for this cross-over study involving eight weeks of supplementation with each of the placebo and limonoid-containing drinks. Laboratory work and data analysis began prior to the present year and that work continues. The progress of the study has been frequently discussed by phone and e-mails with the sponsor and ARS collaborators. Objective 2: We analyzed liver and adipose tissue samples from our previous mouse study to examine potential mechanisms by which docosahexaenoic acid prevented conjugated linoleic acid-induced nonalcoholic fatty liver disease. We found that dietary conjugated linoleic acid increased the expression of the hepatic transcription factor PPAR-gamma and of the enzymes stearoyl-CoA desaturase and fatty acid synthase. These changes suggest fatty acid synthesis would be increased and, when we performed fatty acid analysis on these livers, this expectation was confirmed. In contrast, dietary docosahexaenoic acid decreased expression of these enzymes and the levels of their fatty acid product. Docosahexaenoic acid also increased hepatic fatty acid oxidation, which would also decrease liver fatty acid levels, consistent with an increased expression of the enzyme acyl-CoA oxidase in this group. Docosahexaenoic acid also decreased expression of important markers of inflammation in the liver but not in adipose tissue. We continue to analyze the changes in the expression of additional genes which play key roles in lipid and carbohydrate metabolism. Objective 3: Transgenic mice constitutively expressing an activated version of toll-like receptor 4 in adipose tissue were previously prepared by our group. These mice have chronic inflammation mimicking that seen in humans with metabolic syndrome, who also have decreased insulin sensitivity. Contrary to expectation, these mice gained less weight and had better insulin sensitivity than did non-transgenic control mice. Transgenic mice also had higher expression of several negative regulators of the toll-like receptor pathways. One possible explanation for these seemingly paradoxical results is that the transgenic mice increase the expression of not only pro-inflammatory toll-like receptor target genes but also of anti-inflammatory target genes, possibly as a compensatory response to the expression of toll-like receptor 4. Using this model, we had proposed to study anti-inflammatory effects of plant polyphenols contingent upon securing extramural funds. However, this year we obtained a grant from the U.S. Highbush Blueberry Council for a study to determine the efficacy of blueberry powder (containing the polyphenols of interest) in attenuating high fat-diet induced postprandial inflammation in healthy human subjects. Therefore, we are pursuing the anti-inflammatory effects of polyphenols using a whole food source in humans as an alternative to studying the effects of purified compounds in mice.
1. Saturated fatty acids induce inflammasome-mediated inflammation. The pro-inflammatory effect of a diet high in saturated fat is well-known but the mechanism of such effects is poorly understood. An ARS scientist from the Western Human Nutrition Research Center in Davis, CA found that saturated fat activates an immune cell receptor complex (the inflammasome) normally activated by microorganisms to trigger inflammation, and also found that omega-3 polyunsaturated fat blocked this activation. This novel finding helps to explain how saturated fat in food increases the risk of chronic inflammatory disease and it also suggests that increasing the intake of omega-3 fatty acids will decrease this risk. This finding will thus have impact on formulating dietary recommendations as it indicates that two dietary strategies to diminish chronic disease risk would be to (1) decrease intake of saturated fat and (2) increase intake of omega-3 polyunsaturated fat.
2. Diet-induced inflammation and insulin resistance. Poor diets can induce insulin resistance and thus cause the development of type 2 diabetes. ARS scientist from the Western Human Nutrition Research Center in Davis, California, working in collaboration with a scientist at the University of Tennessee, found that the expression of a particular type of proinflammatory receptor (the nucleotide oligomerization binding domain receptor) was increased during the differentiation of a fibroblast-like cell to a fat cell (i.e., an adipocyte) in cell culture. They also found that activation of this receptor impaired insulin signaling in these adipocytes. Different dietary factors can trigger activation of this receptor (e.g., saturated fat) or block its activation (e.g., some phytochemicals found in fruits and vegetables). This work suggests that balancing the intake of such dietary factors will be a useful approach to decreasing the risk of type 2 diabetes.
Dawson, K., Zhao, L., Adkins, Y.C., Rodriguez, R.L., Kelley, D.S., Hwang, D.H. 2011. Modulation of blood cell gene expression by DHA supplementation in hypertriglyceridemic men. Journal of Nutritional Biochemistry. Available: http://www.sciencedirect.com/science/article/pii/S0955286311000933