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，其 随着肥胖症的流行，患病率继续上升。肝脏脂肪变性是 非酒精性脂肪肝，其中甘油三酯在肝脏中积聚。最近，我们发表了GDNF 尽管转基因小鼠喂食了高脂肪食物，但仍可防止肥胖和肝脏脂肪变性的发生 相似的食物摄入量和体力活动。胶质细胞源性神经营养因子转基因小鼠在胶质细胞中的高效表达 胶质细胞表达的GDNFTG启动子及其受体CRET和GfR-1在人中的表达 和小鼠肝细胞。因此，肝脏中存在的GDNF可以局部作用于肝细胞。GDNF-TG小鼠 展示通过间接量热法评估的增强能源利用率。在确立了 在预防肝脏脂肪变性中我们最近研究了GDNF在预防肝损伤中的作用。我们的 初步数据表明，GDNF对肝脏有新的作用，包括：(I)GDNF预防HFD诱导的 肝脏损伤(Ii)喂食HFD的GDNF-TG小鼠肝脏自噬水平增加；(Iii)肝细胞 GDNF治疗改善了线粒体的功能，表现为基础和线粒体的增加 呼吸作用GDNF的这些潜在有益影响的机制还有待探索。我们 GDNF预防肝损伤的机制可能是通过促进肝细胞 自噬和改善线粒体功能从而增加肝细胞存活率。GDNF可以 增加肝细胞脂滴周转、脂肪氧化和氧化磷酸化 自噬途径。在这项建议中，我们将建立GDNF对肝细胞的调节机制 自噬和改善线粒体功能。目前还没有FDA批准的治疗这种疾病的药物 肝脏脂肪变性。我们已经确定了一种新的神经营养因子GDNF，它可以降低肝脏和肝脏 脂肪变性和损伤，通过增强自噬和线粒体功能。同时使用遗传和 药理学方法我们将确定GDNF在诱导自噬导致氧化脂质中的作用 新陈代谢，降脂，减轻肝脏损伤。我们提出以下相互关联但又相互独立的建议 可实现的目标：具体目标1：确定GDNF是否通过降低肝细胞脂质的机制 积聚是通过增强的自噬介导的脂解，通过抑制mTOR信号介导的。 初步数据表明GDNF是一种有效的自噬诱导剂。我们将确定自噬是否 通过研究GDNF的过度表达和基因敲除来介导GDNF诱导的细胞脂质储备的减少 自噬相关基因。初步数据表明，GDNF抑制pmTOR信号，而pmTOR信号是 自噬的主要抑制途径。我们将确定GDNF上调自噬的机制 关注mTOR途径。WT和GDNF-TG小鼠将以高脂饲料喂养8周，并对基因 将对肝细胞自噬的调节进行评估。体外实验研究了棕榈酸酯对GDNF的调节机制 诱导的自噬将集中在mTOR/TFEB/Pgc1-途径上确定。具体目标2： GDNF通过促进线粒体呼吸促进肝细胞存活的机制研究 增强的β-肾上腺素能信号和有丝分裂。我们的初步数据表明，GDNF增加 肝细胞线粒体功能：基础呼吸和最大呼吸增加 并改善在棕榈酸酯存在下培养的肝细胞的线粒体呼吸。GDNF 也通过激活-肾上腺素能信号促进脂解。我们将确定GDNF在以下方面的作用 利用GDNF-TG小鼠调节线粒体呼吸。我们将探讨GDNF的作用机制 线粒体健康的调节，侧重于线粒体吞噬作用及其对改善线粒体呼吸的作用。 综上所述，我们的数据可能为肝病的治疗或预防提供新的靶点。 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. Hepatic steatosis is a component of NAFLD, wherein there is accumulation of triglycerides in the liver. Recently we published that GDNF transgenic mice fed a high fat diet are protected from obesity and the development of hepatic steatosis despite similar food intake and physical activity. GDNF transgenic mice over express GDNF in glia under the GFAP promoter (GDNF-tg) GDNF (expressed in glia) and its receptors, cRET and GFR-1 are expressed in human and murine hepatocytes. Thus, GDNF present in the liver can act locally on the hepatocytes. GDNF-tg mice demonstrate enhanced energy utilization assessed by indirect calorimetry. Having established a role for GDNF in preventing hepatic steatosis we recently examined the role of GDNF in preventing liver injury. Our preliminary data demonstrate novel effects of GDNF on the liver that include: (i) GDNF prevents HFD-induced liver injury (ii) GDNF-tg mice fed a HFD have increased hepatic levels of autophagy; and (iii) Hepatocytes treated with GDNF have improved mitochondrial function as seen by increased basal and mitochondrial respiration The mechanisms 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 autophagy and by improving mitochondrial function leading to increased hepatocyte survival. GDNF can increase lipid droplet turnover, fat oxidation and oxidative phosphorylation in hepatic cells through the autophagic pathway. In this proposal we will establish the mechanism of GDNF regulation of hepatocyte autophagy and improved mitochondrial function. Currently there are no FDA approved drugs for the treatment of hepatic steatosis. We have identified a novel neurotrophic factor GDNF that decreases both hepatic steatosis and injury through enhancing autophagy and mitochondrial function. Using both genetic and pharmacological approaches we will define the role of GDNF in inducing autophagy to lead to oxidative lipid metabolism, fat reduction and reduced hepatic injury. We propose the following interrelated, but independently achievable aims: Specific Aim 1: To determine if the mechanism by which GDNF decreases hepatocyte lipid accumulation is through enhanced autophagy mediated lipolysis mediated by suppression of mTOR signaling. Preliminary data indicate that GDNF is a potent inducer of autophagy. We will establish whether autophagy mediates the GDNF-induced reduction in cellular lipid stores by studies of overexpression and knockdown of autophagy related genes. Preliminary data indicate that GDNF suppresses pmTOR signaling which is the prime inhibitory pathway of autophagy. We will determine the mechanism of GDNF up regulation of autophagy focusing on the mTOR pathway. WT and GDNF-tg mice will be fed a high fat diet for 8 weeks and genes regulating autophagy in hepatocytes will be assessed. In vitro the mechanism GDNF regulation of palmitate- induced autophagy will be determined focusing on the mTOR/TFEB/PGC1- pathway. Specific Aim 2: To establish that GDNF promotes hepatocyte survival by enhancing mitochondrial respiration by the mechanism of enhanced beta-adrenergic signaling and mitophagy. Our preliminary data demonstrate that GDNF increases mitochondrial function in hepatocytes as seen by increasing basal and maximal mitochondrial respiration as well as improving the mitochondrial respiration in hepatocytes cultured in the presence of palmitate. GDNF also promotes lipolysis through activation of -adrenergic signaling. We will establish the role of GDNF in regulating mitochondrial respiration using the GDNF-tg mice. We will examine the mechanism of GDNF regulation of mitochondrial health focusing on mitophagy and its effects on improving mitochondrial respiration. Taken together our data from this proposal may provide novel targets for the treatment or prevention of hepatic steatosis and injury.