Location: Immunity and Disease Prevention Research2012 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.
3. Progress Report:
Limonoid glucoside (LG) and cherry supplementation trials: Ten subjects completed the trial and laboratory work is in progress. Markers of inflammation, lipid metabolism and organ function have been evaluated, including targeted proteomics and gene arrays. Markers of oxidative stress are being evaluated and results of gene array need to be confirmed by a second method. Most of the response variables did not change with LG treatment. We have also expanded analysis of the plasma samples from our previous cherry study. We found consumption of sweet Bing cherries reduced the plasma concentrations of several markers of inflammation, diabetes, blood pressure, and cardiovascular disease. Prevention of fatty liver in mice: We examined the effects of dietary conjugated linolenic acid (CLA) and docosahexaenoic acid (DHA) on the fatty acid composition of liver, adipose tissue and gastrocnemius muscle. Adipose tissue had the lowest concentration and muscle the highest concentration of long chain polyunsaturated acids (arachidonic acid [AA], eicosapentaenoic acid [EPA], and DHA). CLA increased monounsaturated fatty acids and decreased polyunsaturated fatty acids in the liver; it had only minor effects on the fatty acid composition of muscle and adipose tissue. Concomitant supplementation of DHA with CLA increased EPA and DHA concentrations in all three tissues and reversed CLA-induced increase in monounsaturated fatty acids in liver. Neither CLA nor DHA altered the concentration of saturated fatty acids in all three tissues. Genes whose expression was altered by dietary DHA and CLA also varied in different tissues. Our results show that various tissues respond differently to changes in dietary fatty acid intake. Toll-like receptor 4 (TLR4) transgenic mice: We prepared transgenic mice constitutively expressing an activated version of TLR4 in adipose tissue. 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 TLR 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 TLR4. The manuscript for these results is being prepared for publication. Because of the contradictory phenotypes, we have decided not to pursue this aim. Blueberries and inflammation: We obtained a grant from the US 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. A total of thirty healthy subjects will be studied with placebo, two or four servings of blueberry powder to determine whether the blueberry intake attenuates monocyte activation induced by a moderately high fat single meal.
1. Saturated fat and inflammation – confirming the link. Excessive intake of saturated fats from meat and other foods is associated with chronic diseases, such as heart disease, that are mediated by inflammation triggered by white blood cells of our immune system. Many studies in the scientific literature have shown that saturated fatty acids, when added to cultured white blood cells, trigger early events in inflammation, suggesting a direct cause-effect relationship. Drawing conclusions from this scientific literature has been muddied by an ongoing controversy among scientists about whether contaminants associated with these fatty acids, rather than the fatty acids themselves, cause these inflammatory events. ARS scientists working at the Western Human Nutrition Research Center in Davis, CA, conducted definitive experiments with isolated cells demonstrating that the saturated fatty acids, and not other contaminating molecules, do indeed trigger inflammation. This finding helps clarify the emerging consensus in the scientific literature that saturated fatty acids themselves trigger inflammation that is associated with chronic disease. Further studies will help determine the levels of intake of saturated fat that are optimal to prevent such adverse health consequences.
2. Insulin sensitivity and dietary omega-3 fatty acids. Consumption of fish or supplements that contain long-chain omega-3 fatty acids such as docosahexaenoic acid (or DHA) can decrease the risk of heart disease. Heart disease often occurs together with diabetes because the underlying risk factors (such as elevated blood lipids) are similar. This raises the question of whether increasing DHA intake could decrease the risk of diabetes. Diabetes is caused by insensitivity to insulin, the hormone that regulates blood sugar levels in response to dietary sugar. ARS scientists working at the Western Human Nutrition Research Center in Davis, CA, discovered that supplementing the diets of men with high blood lipids (triglycerides) with DHA improved several markers of insulin sensitivity related to abnormal fat metabolism, including blood levels of free fatty acids, blood levels of lipoprotein particles associated with risk of heart disease (small dense LDL and remnant-like particles), and the ratio between blood triglycerides and HDL cholesterol. These results suggest that an appropriate level of DHA intake may improve insulin sensitivity and thus decrease the risk of diabetes by normalizing fat metabolism.
Kelley, D.S., Adkins, Y.C., Sharma, S., Fedor, D.M. 2011. Modulation of atherosclerosis by N-3 polyunsaturated fatty acids. In: Bidlack, W., Rodriguez, R., editors. The Impact of Dietary Regulation of Gene Function on Human Disease. New York, NY: CRC Press. p. 139-160.
Kelley, D.S., Adkins, Y.C. 2012. Similarities and differences between the effects of EPA and DHA on markers of atherosclerosis in human subjects. Proceedings of the Nutrition Society. 10.1017/S0029665112000080.
Fedor, D.M., Adkins, Y.C., Mackey, B.E., Kelley, D.S. 2012. Docosahexaenoic acid prevents trans-10, cis-12 conjugated linoleic acid-induced non-alcoholic fatty liver disease in mice by altering expression of hepatic genes regulating fatty acid synthesis and oxidation. Metabolic Syndrome and Disorders. 10(3):175-180. DOI: 10.1089/met.2011.0113.
Huang, S., Rutkowsky, J.M., Snodgrass, R.G., Ono-Moore, K., Schneider, D.A., Newman, J.W., Adams, S.H., Hwang, D.H. 2012. Saturated fatty acids activate TLR-mediated pro-inflammatory signaling pathways. Journal of Lipid Research. Epublished. DOI: 10.1194/jlr.D029546.