Project Summary/Abstract Hepatic steatosis (fatty liver) increases the risk for liver disease and type 2 diabetes, major health issues for Veterans. Reduced hepatic mitochondrial function (MitoFX), defined as reduced mitochondrial -respiration and - fat oxidation, increased H202 emission, and uncoupled mitochondria, contribute to hepatic steatosis and liver injury. In contrast, exercise protects against steatosis/liver injury via improving or enhancing MitoFX. Female mice and women are protected against steatosis compared to males. Still, their susceptibility to steatosis and poor metabolic health dramatically increases after the loss of ovarian function, suggesting estrogen is a potent mediator of liver health and metabolism. In the last funding cycle, we showed that female mice display enhanced MitoFX compared to males, which likely underlies their protection against steatosis. In female mice, we have demonstrated that ovariectomy (OVX-eliminates estrogen and ovarian function) dramatically reduces MitoFX, while exercising (partially) and estradiol (fully) restores MitoFX. This data points to estrogen's role in regulating hepatic MitoFX in female mice. Also, OVX in female mice provides a pre-clinical model system to investigate mechanisms and develop therapies that can be used for female Veterans. Estrogen putatively mediates MitoFX via estrogen receptor-a (ERa) signaling, a signaling node that is likely critical for estrogen replacement therapy. A significant knowledge gap exists on how to positively modulate hepatic MitoFX and lower the risk for steatosis after the loss of ovarian function, which is the focus of this proposal. We posit that a primary tool by which exercise drives enhanced MitoFX is through the activation of mitochondrial quality control (MitoQC) mechanisms after each bout, including an induction of mitochondrial dynamics (fission/fusion) followed by mitophagy. These processes remodel or eliminate mitochondria with high oxidative stress or impaired coupling of oxidative phosphorylation, resulting in curated pools of healthy hepatic mitochondria (coupled respiration, low oxidative stress, and enhanced oxidative capacity). However, our preliminary data suggests that OVX impairs the ability of exercise to activate hepatic MitoQC, which we believe will limit the power of exercise to improve MitoFX and treat steatosis. This aligns with clinical data suggesting that exercise is less effective at treating steatosis in post- menopausal women. In this proposal, we will test if exercise and estrogen signaling through ERa are critical for exercise-induced activation of MitoQC and treatment of steatosis and liver injury after OVX. We will also test whether estrogen and exercise require the activation of dynamin-related protein 1 (Drp1). Drp1 is a primary regulator of mitochondrial fission and the initiation of MitoQC. We have recently shown that it is highly upregulated in the liver after acute exercise in mice with normal ovarian function...