The incidence of obesity in the US has increased dramatically over the last few decades. Obesity develops due to an imbalance between food intake and energy expenditure and is significantly associated with several complications, including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Unfortunately, current treatments are limited in their ability to mitigate obesity and its complications. Therefore, there is an urgent need to better understand the mechanisms governing energy balance and metabolism in order to develop novel strategies to treat obesity and its complications. Mammalian 5′-AMP-activated protein kinase (AMPK) is an essential sensor of cellular nutrition and energy homeostasis. Given its key role in controlling energy homeostasis, AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases, including obesity, T2D, and NAFLD. AMPK is expressed in several tissues including the intestine, which is considered critical for metabolic regulation and mediating the therapeutic effects of drugs such as metformin. However, in contrast to the relatively well-established functions of AMPK in other tissues, the role of intestinal AMPK in regulating energy and metabolism remains largely unexplored. In order to fill the knowledge gap in understanding intestinal AMPK action, we generated an intestinal epithelium-specific AMPK knockout (AMPK- IKO) mouse strain. We observed a surprising finding of impaired BAT thermogenesis and exacerbated insulin resistance in the diet-induced obese (DIO) AMPK-IKO mice. Interestingly, we found that the gut microbiota profile of AMPK-IKO mice was shifted compared to that of AMPKfl/fl control mice, and fecal microbiota transplantation (FMT) from AMPK-IKO mice recapitulated the AMPK-IKO phenotype in wild-type mice. More interestingly, we found that expression of intestinal antimicrobial peptides (AMPs) was significantly lower in the intestines of AMPK-IKO mice compared to AMPKfl/fl mice, and metformin treatment induced AMP expression in AMPKfl/fl mice but not AMPK-IKO mice. Based on these and other preliminary results, we hypothesize that intestinal AMPK cross-talk with BAT regulates thermogenesis and metabolism through modulating gut microbiota composition. We propose three Specific Aims to test our hypothesis: 1) Determine the role of intestinal AMPK in regulating energy and metabolism; 2) Determine the role of gut microbiota and their metabolites in mediating the regulatory effects of intestinal AMPK on energy balance and metabolism; and 3) Investigate the mechanism by which intestinal AMPK modifies the gut microbiota profile. These studies will identify and characterize an important new mechanism of intestine-BAT communication, which may fill the gap of knowledge of our current understanding how intestine is a key site of AMPK action and metformin in metabolic regulation. We also expect that this project will identify novel targets and potential novel therapies ...