Functional Char. of Pro-Inflammatory Pathways Influencing Insulin Influencing Ins

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Insulin resistance is a common metabolic condition which underlies Syndrome X(Metabolic Syndrome, In- sulin Resistance Syndrome). Syndrome Xis strongly associated with obesity and Type 2 diabetes and the ongoing obesity epidemicin the United States makes this constellation ofabnormalities even more prevalent. An overarchingtheme in this project is that chronic inflammationplays a central role in the etiology of hu- man insulin resistant states. Further, wepropose that the macrophage canbe an initiator ofthis inflamma- tion-induced insulin resistance. Powerful data to support this concept has already come from recent studies of macrophageknockout animals in whichwe showed that disabling the inflammatorypathwaywithin macrophages leads to a state ofglobal insulin sensitivity in mice,whereas, hyperactivating (or derepressing) the inflammatory pathway within macrophages leads to a state ofglucose intolerance, hyperinsulinemia, and insulin resistance. In this proposal, we plan to conduct an extensive series ofin vitro and in vivo studies aimed at identifying the full role ofthe macrophagein inflammation-induced insulin resistance and the un- derlying mechanisms. We will use a gene array approachto elucidate the gene network regulated by PPARy and the two co-repressors N-CoRand SMRT in sTs-Li adipocytes. Wehave developed in vitro assays which allow us to dissect out the various steps ofthe macrophageitinerary, including endothelial transmigration, chemotaxis, and direct binding of macrophages to adipocytes. In addition, we have established a co-culture system in which addition ofmacrophages causes cellular insulin resistance in sTs-Li adipocytes, and a num- ber of experiments are proposed to identify the basic mechanisms ofthis effect. Finally, we willvigorously pursue our mouse studies by using a number ofnewknockout models as well as new strategies. In this ap- proach, we will make heavyuse ofbone marrowtransplantation from a given KOanimal into irradiated C57bl/6 hosts to create functional macrophage KO models on a relativelyhigh throughput scale. Wewill also utilize our lentisiRNAmethod oftreating normal bone marrow with lentisiRNAvectors targeted against spe- cific inflammatory pathway components within macrophages. This creates a bone marrowknockdownof the desired target, and these cells can then be transplanted and engraftedinto an irradiated host mouse. This further enhances the throughput of our approach and also allows the possibility of multiplexing knockouts to assess combinatorial effects. Assuch, 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.