Hepatic TGFbeta1 in Control of Type 2 Diabetes and NASH via FoxO1 Signaling

项目摘要 营养过剩会导致胰岛素抵抗，这是T2D和NASH的高危因素。这个 胰岛素抵抗诱导肝脏脂肪生成、纤维化、细胞凋亡和炎症的机制 对于纳什来说，这一点还不清楚。血液和组织中高水平的转化生长因子-β-1(转化生长因子-β1) 在患有T2D和NASH的人和小鼠身上观察到。转化生长因子-β1在一系列不同的 细胞反应，包括细胞外基质(ECM)合成和凋亡，这是 组织动态平衡，但转化生长因子β1调节糖代谢和肝脏的分子机制 人们对功能的理解还不完全。 叉头转录因子Foxo1是胰岛素→PI3K→蛋白的关键下游靶点 蛋白激酶B(Akt)信号通路和胰升糖素→-cAMP-→蛋白激酶A(PKA)信号通路。AKT 磷酸化Foxo1-Ser253，触发Foxo1核输出和细胞质隔离 泛素化。相比之下，PKA磷酸化Foxo1-Ser273以促进Foxo1核转位和 肝细胞中蛋白质的稳定性。在代谢压力下，如营养过剩，Foxo1过度激活 促进肝脏葡萄糖生成(HGP)和高血糖。在这项提案中，PI假设 肝转化生长因子β1通过S273(Foxo1-pS273)的磷酸化，促进 Foxo1核活性，并诱导高血糖、肝纤维化和炎症，促进T2D和 纳什。在目标1中，Pi和他的团队将使用遗传方法1)删除肝脏中的转化生长因子β1基因 带有或不带有活性Foxo1-S273D/D突变的小鼠(L-转化生长因子β1KO)，或2)产生肝脏特异性 Foxo1-S273A/A失活突变前后转化生长因子β1过表达小鼠(L-转化生长因子β10)的研究 肝转化生长因子β1是否是Foxo1-pS273促进人血清白蛋白、细胞凋亡、肝纤维化和肝纤维化的关键调控因子 纳什饮食喂养后的炎症。AIM2是用小鼠原代肝细胞、骨髓- 来源的巨噬细胞和肝星状细胞，以确定转化生长因子β1的机制 通过Foxo1-pS273促进肝细胞巨噬细胞去极化和HSC活化。具体的- Smad3和Foxo1的结构域相互作用及相关靶基因在肝细胞中的表达将进一步 调查过了。目的3是研究全身或肝脏转化生长因子β1信号的抑制是否 足以预防小鼠的T2D和NASH，以Foxo1依赖的方式。总体而言，使用 遗传、基因组、生物信息学和药理学方法，PI和他的团队已经完全准备好 肝转化生长因子-β_1-→_foxo_1-pS_(273)→转化生长因子β_1环路系统在大鼠肝损伤中的作用 T2D和NASH的控制，这将为糖尿病和NASH的发病机制提供新的见解。

Project Summary Overnutrition induces insulin resistance, which is a high-risk factor for T2D and NASH. The mechanism underlying insulin resistance-induced hepatic lipogenesis, fibrosis, apoptosis, and inflammation for NASH is unclear. High levels of transforming growth factor-β1 (TGFβ1) in blood and tissues were observed in both human and mice with T2D and NASH. TGF-β1 plays a pivotal role in a diverse range of cellular responses, including extracellular matrix (ECM) synthesis and apoptosis which are essential for tissue homeostasis, but the molecular mechanism by which TGFβ1 regulates glucose metabolism and liver function is incompletely understood. The forkhead transcription factor Foxo1 is a key downstream target of the insulin→ PI3K→protein kinase B (Akt) signaling pathway and the glucagon→cAMP→protein kinase A (PKA) signaling pathway. Akt phosphorylates Foxo1-Ser253, triggering Foxo1 nuclear export and cytoplasmic sequestration for ubiquitination. By contrast, PKA phosphorylates Foxo1-Ser273 to promote Foxo1 nuclear translocation and protein stability in hepatocytes. Upon metabolic stress such as overnutrition, Foxo1 hyperactivation promotes hepatic glucose production (HGP) and hyperglycemia. In this proposal, the PI hypothesizes that hepatic TGFβ1 plays key role in control of Foxo1 via phosphorylation at S273 (Foxo1-pS273), enhancing Foxo1 nuclear activity, and inducing hyperglycemia, liver fibrosis and inflammation, and promoting T2D and NASH. In Aim 1, PI and his team will use genetic approaches to 1) delete the TGFβ1 gene in the liver of mice (L-TGFβ1KO) with or without active Foxo1-S273D/D mutation, or 2) generate liver-specific overexpression TGFβ1 mice (L-TGFβ1OE) with or without inactive Foxo1-S273A/A mutation, investigating whether hepatic TGFβ1 is a key controller for Foxo1-pS273 in promoting HGP, apoptosis, liver fibrosis and inflammation following the NASH diet feeding. Aim2 is to use mouse primary hepatocytes, bone marrow- derived macrophages, and hepatic stellate cells (HSC) to determine the mechanisms by which TGFβ1 promotes macrophage depolarization and HSC activation via Foxo1-pS273 in hepatocytes. The specific- domain interactions of Smad3 and Foxo1 and related target genes expression in hepatocytes will be further investigated. Aim 3 is to investigate whether suppression of systematic or hepatic TGFβ1 signaling is sufficient for the prevention of T2D and NASH in mice, in a Foxo1 dependent manner. Overall, using the genetic, genomic, bioinformatic, and pharmacological approaches, the PI and his team are fully equipped to investigate the new pathophysiological role of hepatic TGF-β1→Foxo1-pS273→TGFβ1 looping system in control of T2D and NASH, which will provide novel insights on mechanism of diabetes and NASH.