Project Summary/Abstract Hepatic steatosis (fatty liver) is a risk factor for type 2 diabetes, cardiovascular disease, and further liver injury, all major health issues for Veterans. Currently there are 1.6 million women Veterans, a number predicted to grow steadily making women's health issues a major concern going forward. Prior to menopause, women are protected against steatosis, but risk dramatically increases after loss of ovarian function and accumulating evidence shows that differences in estrogen signaling are a primary mediator. Physical inactivity and low fitness also drive increased risk for hepatic steatosis and associated pathologies. In contrast, increased physical activity and exercise protects and treats steatosis, even in obese patients. Abnormalities in hepatic mitochondrial function strongly contribute to the pathology of steatosis and are likely a primary target for the effects of physical activity and exercise to mitigate the condition, but mechanisms remain largely unknown. Estrogen is likely the cause of protection against hepatic steatosis in female rodents but the direct effects of estrogen signaling on hepatic mitochondria function have received little attention. Our recent findings show that female mice display increased mitochondrial respiration, lower reactive oxygen species (H2O2) emission and protection against steatosis in a sedentary condition compared to males. Female hepatic mitochondria respiratory capacity was also more responsive to diet- and exercise-induced metabolic stress, but these adaptive traits were partially diminished in mice with genetic ablation of mitochondrial turnover (biogenesis and mitophagy). These data form our hypothesis that enhanced mitochondrial function in females is critical for their inherent protection against steatosis and adaptive responses to metabolic stress. We will test the hypothesis that estrogen signaling through estrogen receptor α (ERα) is obligatory for elevated hepatic mitochondrial function and adaptability in females by driving enhanced mitochondrial biogenesis and mitophagy. A second objective of this proposal will test if differences in bile acid (BA) metabolism provide protection against steatosis in females. Female rodents display chronically higher serum and fecal BA levels, paired with higher expression of hepatic genes controlling cholesterol/BA synthesis. Increasing rates of BA synthesis and fecal excretion via BA sequestrant drugs and chronic CYP7a1 overexpression also prevent and treat hepatic steatosis, suggesting a similar affect to what we see in female livers. Our preliminary data suggest that estrogen and exercise synergize to increase BA synthesis and fecal excretion only in females. We will test the hypothesis that trafficking of excess acetyl CoA away from de novo lipogenesis (synthesis of new fatty acids) and towards BA synthesis and fecal loss during postprandial conditions is an additional mechanism that protects females against hepatic steatosis. Overall, this proposal will examine if hepatic ERα signaling is obligatory for sex differences in hepatic mitochondrial function and BA metabolism and if these factors independently impact risk for hepatic steatosis in female mice. We will test these questions by utilizing liver- specific ERα knockout mice (LERKO), exercise, surgical (ovariectomy), pharmacological (estradiol), and molecular (AAV for shRNA CYP7a1) approaches combined with novel in vivo metabolic tracing techniques, and direct measures of mitochondrial quality control and function. The overall objective of this proposal is to determine mechanistic interactions between estrogen, exercise, and mitochondrial function that drive risk for hepatic steatosis with a goal of determining therapeutic targets for female Veterans.

Project Narrative Hepatic steatosis (fatty liver) increases risk for liver complications including steatohepatitis, cirrhosis, and liver cancer. Hepatic steatosis also conveys significant metabolic and cardiovascular risk. Rising rates of hepatic steatosis are a major health problem in Veteran patients as it has increased by 2.8-fold from 2003 to 2011. Currently, women represent 9% of the Veteran population, a number predicted to increase to ~15% by 2040. Pre-menopausal women are protected against steatosis but display increased risk compared to men after they lose ovarian function and have reduced estrogen. Thus, hepatic steatosis will become a major health burden for women and the VA as more women lose ovarian function due to normal aging. The mechanisms underlying how estrogen signaling mediates risk for hepatic steatosis in women is the focus of this proposal. We will examine mechanistic interactions between liver estrogen signaling, exercise, and mitochondrial function that drive risk for hepatic steatosis with an underlying goal of discovering therapeutic targets.