PROJECT SUMMARY Among the many effects that the gut microbiome elicits on its host, the regulation of metabolism is arguably the most significant because of its potential impact on human health and relevance to diet-induced obesity (DIO). DIO is such an enormous and vexing public health concern that there is unanimity that effective and practical solutions must be found. Microbiome-based interventions hold great promise as a way to correct energy balance in a more natural, physiological manner than pharmacological agents that often have off target side effects. In order to accomplish this, we must find novel solutions to unravel the complex and dynamic relationships between the gut microbiome and host metabolic systems in order to advance the field beyond description and association. To move the needle, our group proposes a deep dive into studies that will reveal targets and mechanisms of action of specific microbial drivers of host metabolism in an organ-specific context. We propose to define how diet-induced changes of the gut microbiota affect host metabolism through the lens of hepatic circadian and metabolic networks (the liver being the main metabolic organ). These studies build upon a paradigm-shifting discovery our group made showing that the gut microbiome undergoes diurnal variation, which is essential and intricately intertwined with host circadian rhythms (CRs) that influence host metabolism. We will test the hypothesis that HF diet reprograms the gut microbiota to promote loss of critical microbial inducers (Aim 1) and gain of disruptors (Aim 2) that impact downstream host hepatic circadian and metabolic networks, leading to DIO. To gain mechanistic clarity, each will be studied separately. Additionally, we will test the hypothesis that microbiota-derived inducers and disruptors differentially interact with the core or auxiliary hepatic circadian components, which functionally affect the phase and/or amplitude of the pacemaker. For these studies, we have selected two indigenous gut microbial strains (the recently identified and cultivated Ilealbaculum butyricum [E14] and Bilophila wadsworthia [Bw]) promoted either by LF or HF diet and their known metabolic products (butyrate and H2S, respectively). To achieve higher mechanistic and temporal resolution, we will use genetic manipulation of the circadian system i.e. conditional liver-specific Bmal1 knock-out mice, gnotobiotic mouse technology, and hepatic organoid systems which have all been validated. In Aim 3, we will explore if the culmination of this knowledge can be leveraged into microbiome-based interventions for DIO by testing the hypothesis that butyrate and potentially other LF diet-induced microbe-derived inducers can override the actions of existing HF diet- induced microbial CR disruptors (H2S). These studies serve as a starting point towards a thorough understanding of cellular, tissue, and systems complexity involved in dietary and microbial regulation of metabolism media...