PROJECT SUMMARY Aberrant hepatic mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activity is associated with obesity and insulin resistance. Insights into the physiological inputs and mechanisms that regulate mTORC1 signaling in the liver are needed in order to understand how regulation of hepatic mTORC1 becomes disrupted and how this contributes to insulin resistance and metabolic disease. mTORC1 is a central regulator of cellular growth and proliferation that promotes anabolic processes in response to nutrients and growth factors. In the liver, mTORC1 activity is sensitive to physiologic fluctuations during fasting and feeding, with reduced mTORC1 signaling during fasting and acute induction of mTORC1 upon feeding. This dynamic regulation of mTORC1 in the liver is abolished in obese rodents, highlighting the importance of flexible mTORC1 signaling for systemic metabolic health. The goal of this study is to define mechanisms of dynamic mTORC1 regulation in the liver, with a focus on two hormonal cues. mTORC1 activity is induced by insulin, and both mTORC1 signaling and insulin have been shown to regulate glucose and lipid metabolism in the liver. The mechanism by which insulin induces mTORC1 activity is well-established in cell culture models. However, it is not known how insulin regulation contributes to the induction of mTORC1 activity in the liver upon feeding. Conversely, published reports and our preliminary data demonstrate that mTORC1 signaling is suppressed by glucagon in primary hepatocytes. Glucagon signaling orchestrates an adaptive response to fasting in the liver, and hepatic mTORC1 activity blocks multiple glucagon-mediated metabolic changes. However, the mechanism of mTORC1 repression by glucagon and whether glucagon contributes to the repression of mTORC1 during fasting are unknown. I hypothesize that the glucoregulatory hormones insulin and glucagon contribute to the dynamic regulation of mTORC1 in the liver with feeding and fasting, respectively. I will test this hypothesis through two aims: 1) Use of a new precision genetic mouse model to define the role of liver insulin signaling to mTORC1, in which I will generate and characterize mice with mTORC1 signaling that is disconnected from insulin regulation specifically in the liver, and 2) Determine the physiological significance and mechanism of glucagon-mediated mTORC1 suppression, in which I will determine if glucagon is necessary and sufficient for hepatic mTORC1 suppression during fasting, as well as evaluate potential downstream mediators connecting glucagon and mTORC1 signaling in primary hepatocytes. The proposed experiments have the potential to identify targetable insights into chronic mTORC1 activation in metabolic disease. The study will be conducted in the lab of Dr. Brendan Manning at the Harvard T.H. Chan School of Public Health. Dr. Manning is an expert in the mTORC1 field and has extensive knowledge of insulin-PI3K-Akt signaling. Additionally, many of t...