Project Summary The number of individuals with metabolic diseases like obesity and type 2 diabetes is growing at overwhelming rates globally, increasingly affecting younger populations, and disproportionally affect under-served communities. Emerging clinical, epidemiological, and laboratory research has demonstrated an essential role for gut bacteria in the regulation of metabolism. Another key contributor to the pathogenesis of these metabolic diseases are the sphingolipids ceramides. Ceramides are produced in response to increased fat intake and mediate many of the molecular pathways that cause increased lipid uptake, insulin resistance, and fatty liver disease. A few studies have begun to investigate the relationship between the gut microbiota, ceramides, and metabolic diseases, and have shown that the microbiota can regulate ceramide production. Despite these studies, it remains unclear which bacteria and how these bacteria impact ceramide production and ultimately host metabolism. Our lab has identified a community of Clostridia Class bacteria that provide metabolic protection from a high-fat diet in the form of decreased weight gain, improved leanness, and lower fasting glucose. Furthermore, we have found that Clostridia reduce the rate of ceramide production. The objective of this proposal is to understand which bacteria are essential to providing metabolic protection and determine how these bacteria are impacting ceramide synthesis and overall host metabolism. We hypothesize that select Clostridia bacteria can protect from features of metabolic disease through decreasing ceramides and reducing lipid absorption in the gut epithelium. We will test this hypothesis in two aims: 1) defining the community of Clostridia that protect from features of metabolic disease induced by a high-fat diet, and 2) determining if Clostridia decrease ceramides and lipid absorption in the gut epithelium. Aim 1 will continue to culture, genetically characterize, and define the most limited community of Clostridia that provides metabolic protection. Aim 2 will use in vitro and in vivo approaches with germ-free (GF) and gnotobiotic mouse models to assess the relationship between Clostridia and ceramides in the context of lipid absorption. This proposed work will have a significant impact on the study of the metabolic disease and the microbiota by providing novel insight into microbiota-driven mechanisms that can be leveraged for therapeutic benefit.