Location: Immunity and Disease Prevention Research2013 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: Limonoids are unique, naturally occurring, highly oxygenated triterpenoid compounds that have significant biological activity. We completed a human intervention study with citrus limonoid glucoside (LG) which was supported by the Beverage Research Institute. A provisional patent regarding health benefits of LG was filed. LG caused a greater than 2-fold increase in the expression of 277 genes from white blood cells of study volunteers and a decrease in the expression of 142 genes when compared to placebo group (p< 0.05), indicating potent biological effects of LG. We categorized the stimulated and suppressed genes into functionally related groups, for biochemical-pathways, and associations with diseases. In another human study we reported in 2006 that cherry consumption decreased some markers of inflammation. Using the targeted proteomic arrays, we re-analyzed the plasma samples from that study and found that cherry consumption selectively reduced several additional biomarkers associated with inflammatory diseases. Objective 2: We previously reported that a trans fatty acid found in processed foods called conjugated linoleic acid or CLA caused insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD) in a mouse model. IR is a pre-diabetic condition and can progress to diabetes, while NAFLD can advance to liver failure. Our recent results showed that CLA not only caused NAFLD, but also nonalcoholic steatohepatitis (NASH) which is an advanced liver disease. This mouse model has many features of human NAFLD and IR (concurrent occurrence of IR and NAFLD, progression to NASH, decreased adiponectin and leptin) and is therefore relevant to study those metabolic disorders in humans. Objective 3: Transgenic (Tg) mice expressing an activated version of toll-like receptor 4 (TLR4) in adipose tissue were previously prepared by our group as a mouse model for chronic inflammation. These mice have chronic inflammation mimicking that seen in humans with metabolic syndrome. Contrary to expectation, Tg mice gained less weight and had better insulin sensitivity than did wild-type mice. Tg mice also had higher expression of several negative regulators of TLR4 pathways. One possible explanation for these seemingly paradoxical results is that Tg mice increase the expression of not only pro-inflammatory TLR4 target genes but also of anti-inflammatory target genes, possibly as a compensatory response to the expression of TLR4. Using this model, we had proposed to study anti-inflammatory effects of plant polyphenols. Because of the unexpected findings with Tg mice, we redirected our research focus from mice to a more translational approach. We studied whether endogenous saturated fatty acids derived from high fat meal can activate TLR2 and induce inflammasome-mediated blood monocyte activation, which is the triggering event to induce enhanced inflammation in peripheral tissues, using human primary monocytes. Further, we studied whether high fat diet-induced monocyte activation can be alleviated by fruit (blueberries) or docosahexaenoic acid in a study with human volunteers. These data are currently being analyzed.
1. Health benefits of citrus limonoid glucosides. Studies conducted in animal models suggested that LG have several health benefits including prevention of cancer, however, such information for humans is missing. An ARS scientist at Davis, California, discovered that supplementing the diets of overweight and obese subjects with citrus limonin glucoside (LG) significantly decreased several plasma biomarkers for inflammatory diseases. In addition, gene array analysis performed using the RNA from white blood cells of the same study volunteers showed that LG supplementation caused a greater than 2-fold increase in the expression of 277 genes and a decrease in the expression of 142 genes when compared to placebo treatment (p< 0.05). By using Systems Biology Analysis, the same Davis group found that the genes whose expression was altered by LG mediated several disease pathways including inflammation, insulin signaling, and cancer. These findings suggest that increased LG consumption may prevent several inflammatory diseases in humans.
2. A new mouse model to study liver disease. Relevance of currently available animal models of fatty liver disease to human liver disease is questionable because they lack several aspects of human liver disease. In collaboration with a scientist from the Universtiy of California, Davis (UCD), Medical Center, an ARS scientist in Davis, California, discovered that supplementing mice diets with a trans fatty acid called conjugated linoleic acid or CLA not only caused insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD) but also nonalcoholic steatohepatitis (NASH). Occurrence of NASH was confirmed by increased liver inflammation, oxidative stress, lipotoxicity, and fibrosis. This mouse model has many features of human NAFLD and IR (concurrent occurrence of IR and NAFLD, progression to NASH, decreased adiponectin and leptin) and is therefore relevant to those human metabolic disorders.
3. Anti-inflammatory effects of sweet Bing cherries. Consumption of cherries reduced circulating concentrations of a few biomarkers of inflammation as previously reported by us and other investigators. Previous studies did not monitor global changes in gene expression. By using a proteomic array analysis of plasma samples collected after the consumption of Bing sweet cherries, an ARS scientist in Davis, California, discovered that cherry consumption for 28 days significantly decreased circulating concentrations of biomarkers associated with several chronic inflammatory diseases including arthritis, diabetes, hypertension, cardiovascular disease and cancer. Thus, cherry consumption may be helpful in the prevention and reversal of these diseases.
4. Saturated fatty acid (SFA) palmitic acid induces activation of blood monocytes. Endogenous SFAs derived from triglyceride rich lipoproteins isolated from postprandial blood samples from subjects consuming a high fat breakfast, also induced inflammasome-mediated IL-1ß release in primary blood monocytes. ARS scientists from Davis, California, determined that postprandial monocyte activation is dynamically modulated by the types of dietary fat we consume. These results are significant in view of the fact that blood monocytes are sentinel immune cells that are in constant surveillance for invading pathogens, tissue injury and metabolic abnormality. Activated blood monocytes can transmigrate and become macrophages that stimulate proinflammatory pathways in tissues. Therefore, the activation of blood monocytes is a key triggering step toward enhanced inflammation in peripheral tissues. Our results signify that postprandial monocyte activation after a single meal can be dynamically modulated by the types of dietary fat we consume, and it can serve as an important platform for evaluating pro- or anti-inflammatory effects of different dietary regimens.
Fedor, D.M., Kelley, D.S. 2012. Polyunsaturated fatty acids and insulin resistance. In: Watson, R.R., Preedy, V.R., editors. Bioactive Food as Dietary Interventions for Diabetes. San Diego, CA: Academic Press, Elsevier. pp 183-194.