Project Summary The worldwide obesity epidemic presents a significant public health crisis that is progressively worsening. This has led to intensive efforts to identify the host and environmental factors that regulate human metabolism and energy homeostasis. The gut microbiota has been identified as an environmental factor that regulates lipid metabolism and absorption in the intestine, and thus promotes high fat diet-induced obesity. However, a major knowledge gap remains about the specific bacterial and host factors that regulate intestinal lipid absorption and metabolism. The overall goal of this proposal is therefore to identify the intestinal bacteria and host immune recognition pathways that regulate intestinal lipid metabolism. Prior work by our group identified the circadian transcription factor NFIL3 as essential for the gut microbiota’s role in driving obesity in mice fed a high-fat, high sugar Western-style diet. NFIL3 expression is regulated by the microbiota, and promotes the transcription of intestinal epithelial genes that regulate lipid absorption and metabolism. Preliminary studies of monocolonized mice revealed that flagellated Gram-negative bacteria induced NFIL3 expression. Therefore, my central hypothesis is that Gram-negative flagellated bacterial species, such as Escherichia coli, selectively promote lipid absorption in intestinal epithelium. I will test this hypothesis by using gnotobiotic mice and genetic manipulation of both mice and bacteria. My first aim is to identify intestinal bacteria that regulate intestinal lipid uptake and metabolism through NFIL3. Monocolonized mice will be fed a Western style diet and analyzed for lipid content, gene expression changes in the small intestine, and metabolic syndrome. I will then use genetically-altered bacteria to identify specific bacterial factors that are required to promote intestinal lipid absorption. My second aim is to identify the host immune pathways that are required for bacterial activation of NFIL3-regulated metabolic pathways. Genetically altered mice with deletions of specific pattern recognition receptors will be used to identify host factors required for lipid uptake and metabolism. These studies will provide new insight into how the microbiota regulates lipid metabolism of the host and should identify new avenues for therapeutic interventions into obesity.