2011 Annual Report
1a.Objectives (from AD-416)
Objective: Evaluate leukocyte patterns, gene expression profiles, and inflammatory mediators in adipose tissue under influence of dietary manipulation that leads to obesity.
1b.Approach (from AD-416)
The research approach to accomplish this objective employs an established animal model of diet-induced obesity, the C57BL/6J mouse strain, fed diets rich in milk fat or corn oil, compared with low-fat control diets matched for all nutrients except the level of milk fat or corn oil. At specified times of feeding, animals will be analyzed for metabolic changes induced by the diets (e.g., insulin sensitivity, RER), systemic inflammatory changes, and local inflammatory changes in intra-abdominal and subcutaneous adipose tissue depots. The inflammatory changes analyzed include gene (qPCR and/or expression arrays) and protein (ELISA and/or flow cytometry) expression of cytokines, chemokines and adhesion molecules, and leukocyte subsets phenotypically characterized (flow cytometry); and mechanistic studies will be carried out using targeted deletions of proteins potentially key to inflammatory cascades (e.g., CD11c knockouts, gamma delta T cell knockouts, TLR4 knockouts, etc., backcrossed to the C67BL/6J strain). The experimental approach analyzing anti-inflammatory factors (e.g, candidate genes such as IL-1ra or expression array analysis) influenced by changes in diet will utilize this animal model with focus on the early times following initiation of the high fat feeding and on times following reversal of the high-fat diet to a low-fat diet, a time we have found involves rapid resolution of inflammation that precede reductions in fat mass.
Five-week-old mice were given a high fat or a low fat diet for varying periods of time. Blood, liver, and adipose (fat) tissues (AT) were collected from these animals for gene expression analysis. A protein called CD11c produced by white blood cells significantly increased in mice on the high fat diet that became obese. To see if CD11c was important to the inflammation that occurs in the AT of obese mice, we studied mice with a genetically engineered CD11c deficiency. They became obese, but failed to develop inflammation as seen in normal mice. CD11c deficiency also prevented the abnormal elevations of blood glucose and insulin resistance that occurs in obesity. We showed that the CD11c increases in diet-induced obesity are important for the development of adipose tissue inflammation and insulin resistance associated with obesity. The earliest evidence of inflammation in AT occurs in mice at 3 days after feeding the high fat diet. We have analyzed the mesenteric AT for proteins that attract white blood cells and for increases in white blood cells in the fat tissue. We found a significant increase in a protein called CCL2, a factor known to attract white blood cells. Since white blood cells can be pro-inflammatory or anti-inflammatory, we sought to determine their characteristics in the mice on high fat diet. Our results indicate a pro-inflammatory activation involving another important protein called TLR4, a factor that can recognize products released from bacteria as well as saturated fatty acids. In mice that are deficient in TLR4, the increased number and activation of white blood cells still occurs, but the activation is toward anti-inflammatory functions of the cells. To see if saturated fatty acids in the diet could influence the activation, we exposed white blood cells in tissue culture to palmitate, an abundant fatty acid in the milk fat diet fed the mice. Future studies will address if the anti-inflammatory activation seen in the TLR4-deficient mice is linked to an effect of the oleic acid in the diet. To extend our studies of a role for TLR2, another member of the family of cell surface proteins that recognize products released by bacteria as well as saturated fatty acids, we studied the connection between intestinal microorganisms and TLR2. We sought to determine the consequences of TLR2 deficiency, since there is now evidence that the organisms in the gastrointestinal tract can markedly influence inflammation. The mouse colon was analyzed for gene expression, as well as bacterial RNA, to identify the diversity of bacteria in the colonic mucosa. We show that the expression of genes involved in inflammation was modified by the absence of TLR2. Several bacterial species were significantly different in abundance between WT and TLR2-deficient animals. Thus, understanding diet-induced obesity must consider changes in gene expression in the colonic mucosa and the balance of microorganisms within the intestines.
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