The NRF2-FBP1 crossregulatory loop and the control of healthy and diseased liver metabolism

项目概要/摘要 果糖二磷酸磷酸酶（FBP 1）是植物异生（GNG）的限速酶， 由NFE 2L 2基因编码的核因子红细胞相关因子2（NRF 2）是一种转录因子，以前 被认为是抗氧化防御反应的主要激活剂。NRF 2还激活了 许多代谢基因，特别是在肝脏中。我们发现NRF 2选择性激活的Nrf 2Act-HEP小鼠， 在肝细胞中，和禁食的Fbp 1 ΔHEP小鼠，其中FBP 1在肝细胞中被条件性删除，表现出类似的 代谢表型，包括低血糖、肝肿大、脂肪肝和高脂血症， 也表现为胰岛素过量的个体和葡萄糖或碳水化合物缺乏的FBP 1缺乏的个体。 孩子鉴于这些相似性，我们询问NRF 2和FBP 1是否参与生化串扰。 令人惊讶的是，我们发现NRF 2的肝激活诱导FBP 1降解，由NRF 2诱导的FBP 1降解介导。 EGF和PDGF的表达，通过自分泌信号机制导致ERK 1/2 MAP的激活 在丝氨酸271处磷酸化FBP 1并触发其泛素化和蛋白酶体降解的激酶。 更令人惊讶的是，发现FBP 1表达导致AKT抑制，从而缓解了抑制性AKT。 GSK 3同工酶的磷酸化，其磷酸化嵌入NRF 2分子内的降解决定子， 从而诱导其泛素依赖性蛋白水解。这些发现使我们假设NRF 2-FBP 1 交叉调节环是肝脏代谢和稳态的关键调节器，其异常功能可 促进肝损伤和癌症。我们计划通过三个具体目标来验证这一假设：1）。探讨 假设FBP 1通过激活GSK 3或增强NRF 2在门脉周围肝细胞中的降解， 2.加入NRF; 2）。确定NRF 2激活是否改变肝脏分区并有助于 FBP 1消融引起的代谢缺陷; 3）.研究NRF 2诱导的FBP 1降解或NRF 2 上调控制从慢性代谢应激到肝细胞癌的进展。追求这些 通过新的小鼠模型，细胞生物学研究和高度创新的Seq-Scope技术， 开发用于单个肝细胞的高含量空间转录组学和蛋白质组学分析，将回答几个 具有普遍重要性的关键问题：1）。FBP 1通过哪些非酶机制 对肝脏代谢的广泛影响超出了对GNG的充分研究？2）。NRF 2的作用是什么 FBP 1缺乏引起的代谢改变3）。先前描述的FBP 1的作用是什么？ 醛缩酶B在AKT抑制和GSK 3诱导的NRF 2降解中的相互作用？和4）。AKT的作用是什么 活化的代谢缺陷和增加的易感性致癌转化所表现出的 FBP 1缺乏的肝脏？

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?