The overall long term goal of this project is to understand the mechanisms that generate fatty liver and hepatic insulin resistance in obesity, and the molecular relationships between these two phenomena. We seek to unravel the paradox whereby hepatic steatosis is tightly correlated with insulin resistance in humans, whereas these pathways can be clearly dissociated in experimental models. We hypothesize that this paradox may be solved by investigating Kupffer cells as mediators of insulin resistance in neighboring hepatocytes through their release of inflammatory cytokines in response to innate immune receptors and lipid sensors CD36 and Msr1 scavenger receptor. New evidence that these lipid sensors activate inflammasome and NFkB pathways in Kupffer cells provides a plausible mechanism whereby over-nutrition in obesity can lead to activation of liver inflammation. In order to attack this problem, we developed a unique glucan encapsulated siRNA (GeRP) delivery method to silence genes selectively in macrophages and Kupffer cells that reside specifically in liver, while leaving hepatocytes and macrophages in other tissues unaffected. With this method we can selectively probe Kupffer cell contributions to hepatocyte function in vivo in various mouse models of obesity. Using this approach we shall selectively silence Kupffer cell NFkB p65, Nlrp3, TLR4 co-receptor CD14, CD36 and Msr1 and other targets, alone and in combinations, to disrupt release of inflammatory factors in vivo. Metabolic profiling will be performed on these mice (glucose tolerance, insulin tolerance, systemic insulin sensitivity, hepatic glucose output and other metabolic parameters) to assess systemic effects of silencing Kupffer cell inflammation. We will then test the hypothesis that Kupffer cell factors that modulate whole body metabolism do so by paracrine signaling to neighboring hepatocytes. We also seek to discover novel Kupffer cell factors that disrupt hepatic insulin sensitivity using deep sequencing, proteomics and lipidomics approaches. Finally, we shall identify which intracellular pathways within hepatocytes cause impaired hepatocyte insulin signaling in response to Kupffer cell factors. Candidates such as DAG, ceramides, JNK as well as novel signaling components found in unbiased screens will be evaluated following GeRP-mediated silencing of inflammatory pathways in Kupffer cells in vivo.