Project Summary/Abstract Fructose bisphosphate phosphatase (FBP1) is the rate-limiting enzyme in gluconeogenesis (GNG), whereas nuclear factor erythroid-related factor 2 encoded by the NFE2L2 gene (NRF2) is a transcription factor, previously identified as the master activator of the antioxidant defense response. NRF2 also activates the transcription of many metabolic genes, especially in liver. We found that Nrf2Act-HEP mice, in which NRF2 was selectively activated in hepatocytes, and fasted Fbp1ΔHEP mice, in which FBP1 was conditionally deleted in hepatocytes, exhibit similar metabolic phenotypes, including hypoglycemia, hepatomegaly, hepatosteatosis and hypertriglyceridemia, which are also manifested by insulin overdosed individuals and glucose- or carbohydrate-deprived FBP1-deficient children. Given these similarities, we asked whether NRF2 and FBP1 engage in biochemical crosstalk. Surprisingly, we found that hepatic activation of NRF2 induces FBP1 degradation, mediated by NRF2 induced EGF and PDGF expression, which through an autocrine signaling mechanism led to activation of ERK1/2 MAP kinases that phosphorylated FBP1 at serine 271 and triggered its ubiquitination and proteasomal degradation. Even more surprising was the finding that FBP1 expression led to inhibition of AKT, thereby relieving inhibitory phosphorylation of GSK3 isozymes, which phosphorylate a degron embedded within the NRF2 molecule and thereby induce its ubiquitin-dependent proteolysis. These findings led us to hypothesize that the NRF2-FBP1 crossregulatory loop is a key regulator of liver metabolism and homeostasis, whose aberrant function can promote liver damage and cancer. We plan to test this hypothesis through three specific aims: 1). Investigate the hypothesis that FBP1 induces NRF2 degradation in periportal hepatocytes by activating GSK3 or enhancing its recruitment to NRF2; 2). Determine whether NRF2 activation alters liver zonation and contributes to the metabolic defects caused by FBP1 ablation; 3). Investigate whether NRF2-induced FBP1 degradation or NRF2 upregulation control the progression from chronic metabolic stress to hepatocellular carcinoma. Pursuing these aims via new mouse models, cell biological studies and highly innovative Seq-Scope technology, which we had developed for high-content spatial transcriptomic and proteomic profiling of single liver cells, will answer several critical questions of general importance: 1). What are the non-enzymatic mechanisms through which FBP1 has a broad effect on liver metabolism beyond its well-studied involvement in GNG? 2). What is the role of NRF2 in the metabolic alterations caused by FBP1 deficiency? 3). What is the role of a previously described FBP1- aldolase B interaction in AKT inhibition and GSK3-induced NRF2 degradation? and 4). What is the role of AKT activation in the metabolic defects and increased susceptibility to oncogenic transformation exhibited by the FBP1-deficient liver?