Mechanism of GDNF Regulation of Hepatic Steatosis

非酒精性脂肪性肝病（NAFLD）是退伍军人慢性肝病的常见原因， 随着肥胖症的流行，患病率继续上升。目前超过25%的退伍军人 肥胖和超重的人数更多。在这个提议中，我们将研究胶质细胞的多效性作用， 肝细胞上的衍生神经营养因子（GDNF）。我们已经证明了GDNF转基因（GDNF- Tg）小鼠喂养高脂肪饮食，保护从肥胖和发展的肝脂肪变性，尽管类似的 食物摄入和身体活动。GDNF转基因小鼠在GFAP启动子控制下过表达GDNF 在胶质细胞和星状细胞中表达。GDNF及其受体GFRα−1在人类和小鼠中表达 肝细胞因此，存在于肝脏中的GDNF可以局部作用于肝细胞。我们最近展示了 GDNF可以增强自噬，防止肝损伤。我们的初步数据表明， （i）来自脂肪变性和纤维化患者的人肝组织具有减少的GDNF对肝脏的作用， GDNF表达（ii）西方饮食（WD）喂养的小鼠具有降低的Sirt 3水平，并且这在GDNF中得到改善。 Tg小鼠喂食WD;（iii）GDNF处理的人肝细胞线粒体自噬增加。(iv)GDNF阻止 原代人肝细胞凋亡。这些潜在的有益作用的信号通路 GDNF还有待探索。我们推测GDNF预防肝损伤的机制是 通过促进肝细胞Sirt 3信号传导，随后改善线粒体功能， 肝细胞存活率增加。使用遗传学和药理学方法，我们将定义 GDNF诱导线粒体自噬导致线粒体功能改善、脂肪减少和肝细胞减少， 损伤为了验证这一假设，并进一步研究GDNF调节细胞凋亡的潜在机制， 针对肝脂肪变性，我们提出了以下相互关联但独立可实现的目标：具体目标1： 为了确定GDNF调节Sirt 3的机制，初步数据表明GDNF是一种有效的调节Sirt 3的机制。 Sirt 3的体内和体外诱导剂。我们已经证明GDNF可以激活ERK信号通路， 在肝细胞中通过其受体GFRα1。我们将确定GDNF调控Sirt 3的机制是否是 通过GFRα1-ERK-CREB-PGC-1α通路。WT和GDNF-Tg小鼠将一起喂食西方饮食 在饮用水中加入果糖和葡萄糖（WD/FG）16周， 在肝细胞中评估的途径。体外GDNF中ERK-CREB-PGC-1α的必要性和充分性 Sirt 3表达的调节将使用基因敲低和过表达策略来确定。 具体目的2：研究线粒体功能和线粒体自噬在GDNF介导的细胞凋亡中的作用。 肝细胞存活率。我们的初步数据表明，GDNF增加线粒体功能， 肝细胞线粒体自噬。我们将确定GDNF在调节线粒体功能中的作用， 使用GDNF-Tg小鼠的线粒体自噬和涉及用GDNF处理肝细胞的体外实验。 我们将研究GDNF调节线粒体健康的机制，使用基因敲除和过度敲除。 表达策略。我们将研究GDNF诱导的线粒体自噬在调节肝细胞存活中的作用。 具体目的3：确定载GDNF生姜脂质纳米粒对肝脏脂质的保护作用 积累并促进肝细胞存活。我们的初步数据表明我们有能力 肝脏特异性天然生姜纳米颗粒。我们将研究载有GDNF的生姜纳米颗粒对 肝脂肪变性和肝细胞死亡和损伤。载有GDNF的生姜纳米颗粒将注射一次 WT、Sirt 3 KO小鼠和PINK 1 KO小鼠一周，以进一步建立GDNF的作用机制，涉及 Sirt 3和PINK 1。总之，我们从这项提议中获得的数据可能为治疗或治疗提供新的靶点。 预防肝脏脂肪变性和损伤。

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease in veterans and its prevalence continues to increase, with the growing obesity epidemic. Currently more than 25% of our veterans are obese and a larger number are overweight. In this proposal, we will examine the pleiotropic effects of Glial Derived Neurotrophic Factor (GDNF) on hepatocytes. We have demonstrated that GDNF transgenic (GDNF- Tg) mice fed a high fat diet are protected from obesity and the development of hepatic steatosis despite similar food intake and physical activity. GDNF-Tg mice overexpress GDNF under the control of the GFAP promoter expressed in glia and stellate cells. GDNF and its receptor GFRα−1 are expressed in human and murine hepatocytes. Thus, GDNF present in the liver can act locally on the hepatocytes. We recently demonstrated that GDNF can enhance autophagy and prevent liver injury. Our preliminary data demonstrate novel effects of GDNF on the liver that include: (i) Human liver tissue from patients with steatosis and fibrosis have reduced GDNF expression (ii) Western diet (WD)-fed mice have reduced level of Sirt3 and this is ameliorated in GDNF- Tg mice fed a WD; (iii) Human hepatocytes treated with GDNF have increased mitophagy. (iv) GDNF prevents apoptosis in primary human hepatocytes. The signaling pathway for these potentially beneficial effects of GDNF have yet to be explored. We hypothesize that the mechanism of GDNF prevention of hepatic injury is through promoting hepatocyte Sirt3 signaling, subsequent improved mitochondrial function leading to increased hepatocyte survival. Using both genetic and pharmacological approaches we will define the role of GDNF in inducing mitophagy to lead to improved mitochondrial function, fat reduction and reduced hepatic injury. To test the hypothesis and further investigate the underlying mechanism(s) of GDNF regulation of hepatic steatosis, we propose the following interrelated, but independently achievable aims: Specific Aim 1: To determine the mechanism of GDNF regulation of Sirt3 Preliminary data indicate that GDNF is a potent inducer of Sirt3 in vivo and in vitro. We have demonstrated that GDNF can activate the ERK signaling pathway in hepatocytes through its receptor GFRα1. We will establish if the mechanism of GDNF regulation of Sirt3 is through the GFRα1-ERK-CREB-PGC-1α pathway. WT and GDNF-Tg mice will be fed a Western diet together with fructose and glucose added in drinking water (WD/FG) for 16 weeks and ERK-CREB-PGC-1α-Sirt3 pathway assessed in hepatocytes. In vitro the necessity and sufficiency of ERK-CREB-PGC-1α in the GDNF regulation of Sirt3 expression will be determined using gene knock down and overexpression strategies. Specific Aim 2: To examine the role of mitochondrial function and mitophagy in GDNF-mediated hepatocyte survival. Our preliminary data demonstrate that GDNF increases mitochondrial function and mitophagy in hepatocytes. We will establish the role of GDNF in regulating mitochondrial function and mitophagy using the GDNF-Tg mice and in vitro experiments involving treatment of hepatocytes with GDNF. We will examine the mechanism of GDNF regulation of mitochondrial health using gene knock down and over- expression strategies. We will examine the role of GDNF-induced mitophagy in regulating hepatocyte survival. Specific Aim 3: To establish that GDNF-loaded ginger lipid nanoparticles can prevent hepatic lipid accumulation and promote hepatocyte survival. Our preliminary data demonstrate our ability to generate liver-specific natural ginger nanoparticles. We will examine the effect of GDNF-loaded ginger nanoparticles on hepatic steatosis and hepatocyte cell death and injury. GDNF-loaded ginger nanoparticles will be injected once a week into WT, Sirt3 KO mice and PINK1 KO to further establish the mechanism of action of GDNF involving Sirt3 and PINK1. Taken together our data from this proposal may provide novel targets for the treatment or prevention of hepatic steatosis and injury.