2010 Annual Report
1a.Objectives (from AD-416)
Objective 1: Define the specific classes of resident and emigrated leukocytes in adipose tissue, their phenotypic changes during development of obesity, and inflammatory mediators involved.
Subobjective 1A – Detect inflammatory changes after 7 days, 5 weeks, and 6 months on a high fat diet. These studies will analyze cytokines, chemokines, adhesion molecules and leukocyte subtypes in adipose tissue and blood.
Subobjective 1B – Analyze monocyte/macrophages in adipose tissue. It is well known that macrophages are present in adipose tissue, but the specific subsets and differentiation characteristics remain to be defined.
Subobjective 1C – Define patterns of leukocyte activation induced by serum from obese mice. These studies will analyze the ability of serum from mice at different stages of obesity and systemic inflammation to activate normal blood leukocytes.
Objective 2: Characterize leukocyte patterns and gene expression in adipose tissue of animals fed normal and high fat diets. Determine if there is expression of anti-inflammatory factors or the accumulation of regulatory leukocytes.
Subobjective 2A – Detect anti-inflammatory factors modulated or induced by obesigenic diets. We have detected expression of potentially anti-inflammatory factors early in the development of diet-induced obesity. We will expand these analyses to later stages of obesity, include in our analysis a broader array of potentially anti-inflammatory factors, and focus on the resolution of inflammation following removal of the obesigenic diet.
Subobjective 2B – Analyze yo T cells in adipose tissue. We have recently recognized this category of T cells in adipose tissue is important to the inflammatory aspects of diet-induced obesity and will define which subsets are present.
1b.Approach (from AD-416)
The research approach to accomplish these objectives 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.
Male mice of a particular strain (C57BL/6) at 5 weeks of age were given a high fat or a low fat diet for varying periods of time. The food was fully available, and the weight of the animals was recorded periodically. At defined times animals were sacrificed and blood, liver, and adipose tissues were collected for analysis of inflammatory changes. The earliest detected inflammatory changes were found in the fat tissues of the intestine after 3 days on the high fat diet (HFD). By 5 weeks on the HFD, other fat development occurred in the abdomen. The livers exhibited fatty liver changes at 5 weeks, but no evidence of inflammation. Investigation of proinflammatory receptors of possible importance in the activation of these genes revealed that TLR2 and TLR4, members of a family of cell surface receptors on cells in adipose tissue that are known to recognize products released from bacteria, are necessary. Mice deficient in these receptors failed to show this proinflammatory gene expression after 3 days on the HFD and had significantly reduced expression at 5 weeks. Analysis of the mice fat tissues on the HFD for 5 weeks revealed a significant increase in macrophages that express a cell surface receptor (CD11c) that indicates a state of cell activation. These cells were isolated and analyzed further for gene expression. In addition, we identified two distinct populations of lymphocytes in the fat tissues. The function of the CD11c macrophage surface protein was analyzed by studying mice deficient in CD11c. We also performed studies of diet reversal to analyze the changes in inflammatory gene expression that occur in obese mice put on a low fat diet. The HFD was extended to 12 weeks of feeding to produce maximum expression of inflammatory genes in adipose tissue. Since white blood cells migrate into fat tissues during the development of obesity, basic studies of the molecular mechanisms of white blood cell migration were undertaken to define possible targets for intervention. We have begun collaborating on a study on inflammatory bowel disease utilizing our mouse strains with genetically engineered deletions of proteins important in obesity-related inflammation. The initial studies in normal mice revealed developmental changes in susceptibility to inflammatory bowel disease associated with developmental changes in DNA methylation. Older mice had more severe disease. Studies begun in the TLR2 deficient mice reveal these mice are less susceptible to inflammatory bowel disease that normal mice.
The ADODR monitors activities for the project by routine site visits, and review of major purchases of supplies/equipment, use of SCA funds for foreign travel, and submission of grant applications by investigators funded through the SCA.
Studies on fat as a danger signal. Most of the body's cells have proteins on their surface that allow them to activate inflammation when they recognize different danger signals such as foreign biochemicals. Scientists at the Children's Nutrition Research Center, Houston, Texas, are studying one such protein, called TLR2, because this protein may recognize certain types of fat as a danger signal. Mice genetically engineered to be deficient in this protein when placed on a high fat diet developed very little signs of the inflammation that causes obesity-related symptoms, in contrast to normal mice that readily developed obesity and symptoms of disease. These findings show for the first time that TLR2 is involved in high fat diet-induced inflammation, and they indicate that future in-depth study of TLR2 will provide new insights into how the body responds to excess fat as a danger signal.
The importance of a white blood cell protein. During the development of obesity in children and adults, certain types of white blood cells migrate into fat tissue and cause some of the disease symptoms, such as diabetes, common in obese individuals. Scientists at the Children's Nutrition Research Center, Houston, Texas, are studying a protein called CD11c found on the surface of white blood cells in obese fat tissues. To determine the importance of this protein in obesity-related disease, they studied mice that were genetically engineered to be unable to make this protein. Animals that lacked CD11c became obese when put on a high fat diet, but they showed very little signs of diabetes in contrast to normal mice that developed signs of diabetes within a few weeks of eating a high fat diet. These results show for the first time that the white blood cell protein CD11c contributes to obesity-related disease, and they raise the possibility that a drug blocking CD11c could be used to reduce obesity-related disease.