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...