Insulin resistance is a common metabolic condition which underlies Syndrome X (Metabolic Syndrome, Insulin Resistance Syndrome). Syndrome X is strongly associated with obesity and Type 2 diabetes and the ongoing obesity epidemic in the United States makes this constellation of abnormalities even more prevalent. An overarching theme in this project is that chronic inflammation plays a central role in the etiology of human insulin resistant states. Further, we propose that the macrophage can be an initiator of this inflammation- induced insulin resistance. Powerful data to support this concept has already come from recent studies of macrophage knockout animals in which we showed that disabling the inflammatory pathway within macrophages leads to a state of global insulin sensitivity in mice, whereas, hyperactivating (or derepressing) the inflammatory pathway within macrophages leads to a state of glucose intolerance, hyperinsulinemia, and insulin resistance. In this proposal, we plan to conduct an extensive series of in vitro and in vivo studies aimed at identifying the full role of the macrophage in inflammation-induced insulin resistance and the underlying mechanisms. We will use a gene array approach to elucidate the gene network regulated by PPARy and the two co-repressors N-CoR and SMRT in sTs-Li adipocytes. We have developed in vitro assays which allow us to dissect out the various steps of the macrophage itinerary, including endothelial transmigration, chemotaxis, and direct binding of macrophages to adipocytes. In addition, we have established a co-culture system in which addition of macrophages causes cellular insulin resistance in sTs-Li adipocytes, and a number of experiments are proposed to identify the basic mechanisms of this effect. Finally, we will vigorously pursue our mouse studies by using a number of new knockout models as well as new strategies. In this approach, we will make heavy use of bone marrow transplantation from a given KO animal into irradiated C57bl/6 hosts to create functional macrophage KO models on a relatively high throughput scale. We will also utilize our lentisiRNA method of treating normal bone marrow with lentisiRNA vectors targeted against specific inflammatory pathway components within macrophages. This creates a bone marrow knockdown of the desired target, and these cells can then be transplanted and engrafted into an irradiated host mouse. This further enhances the throughput of our approach and also allows the possibility of multiplexing knockouts to assess combinatorial effects. As such, these results should identify relevant mechanisms whereby activation of macrophages causes global insulin resistance in insulin target tissues and should also better elucidate the mechanisms of action, and tissue sites of action of currently available TZDs.
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