PROJECT SUMMARYAmong the many effects that the gut microbiome elicits on its host, the regulation of metabolism is arguably themost 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 practicalsolutions must be found. Microbiome-based interventions hold great promise as a way to correct energybalance in a more natural, physiological manner than pharmacological agents that often have off target sideeffects. In order to accomplish this, we must find novel solutions to unravel the complex and dynamicrelationships between the gut microbiome and host metabolic systems in order to advance the field beyonddescription and association. To move the needle, our group proposes a deep dive into studies that will revealtargets 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 lensof hepatic circadian and metabolic networks (the liver being the main metabolic organ). These studies buildupon a paradigm-shifting discovery our group made showing that the gut microbiome undergoes diurnalvariation, which is essential and intricately intertwined with host circadian rhythms (CRs) that influence hostmetabolism. We will test the hypothesis that HF diet reprograms the gut microbiota to promote loss ofcritical microbial inducers (Aim 1) and gain of disruptors (Aim 2) that impact downstream host hepaticcircadian and metabolic networks, leading to DIO. To gain mechanistic clarity, each will be studiedseparately. Additionally, we will test the hypothesis that microbiota-derived inducers and disruptorsdifferentially interact with the core or auxiliary hepatic circadian components, which functionally affectthe phase and/or amplitude of the pacemaker. For these studies, we have selected two indigenous gutmicrobial 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 circadiansystem i.e. conditional liver-specific Bmal1 knock-out mice, gnotobiotic mouse technology, and hepaticorganoid systems which have all been validated. In Aim 3, we will explore if the culmination of this knowledgecan be leveraged into microbiome-based interventions for DIO by testing the hypothesis that butyrate andpotentially 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 thoroughunderstanding of cellular, tissue, and systems complexity involved in dietary and microbial regulation ofmetabolism mediated by host circadian networks.