Project Abstract Obesity is the predominant risk factor for Non-Alcoholic Fatty Liver Disease (NAFLD), the leading cause for chronic liver disease with prevalence approaching 30% in certain populations. NAFLD, however, may in fact be considered a “pre-disease” state for Non-Alcoholic Steatohepatitis (NASH), which is defined by liver injury and associated inflammation and fibrosis. NASH has no approved pharmacotherapy, and is thus the fastest-growing reason for liver transplantation. As the prevalence of obesity-related NASH continues to rise, and available livers for transplantation remain limiting, this unmet need grows more urgent. In obese mice and patients undergoing liver biopsy for suspected NASH, we observed an unexpected and aberrant activation of hepatocyte Notch, a morphogenic pathway critical for cell fate decision-making in normal liver development. Validation studies revealed that Notch activity was positively associated with markers of liver inflammation and fibrosis and tracked with disease severity in patients with pre- and post-intervention biopsies in randomized clinical trials. Our mechanistic work in the first cycle of this grant showed that Notch is causal to disease, as hepatocyte-specific Notch loss-of-function – by genetic or pharmacologic ablation of either the Notch ligand Jagged1, or by blocking Notch transcriptional activity – protected mice from diet- induced steatohepatitis and fibrosis, whereas forced Jagged1 or Notch activity exacerbated inflammation and fibrosis phenotypes. To determine downstream factors responsible for Notch activity, we performed a series of RNAseq and scRNAseq screens that revealed LTBP3 as a novel Notch target. In Aim 1, we study with novel genetic and pharmacologic tools whether increased hepatocyte LTBP3 secretion mediates Notch-induced hepatic inflammation and fibrosis, and potential mechanisms of these effects. Beyond endocrine communication, hepatocytes can directly interact with non-parenchymal cells, including hepatic stellate cells (HSC), to determine obesity-induced liver pathology. Intriguingly, we observed increased Notch activity in HSC isolated from NASH diet-fed mice; other preliminary data suggest that the same proximal signal (Jagged1) may mediate HSC activation and lead to liver fibrosis, which we test in Aim 2. Finally, we leverage a novel mouse model of reversible hepatocyte Notch activity to discover potential fibrosis regression pathways in Aim 3. Achieving the goals of this application will identify mechanistic determinants of Notch-induced liver pathology and potentially lead to application of novel Jagged-Notch or LTBP3 inhibitors for NASH and fibrosis.