PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a common but poorly understood disease with a critical need for improved diagnosis and treatment. It is the most common form of chronic liver disease manifested as a spectrum of hepatic abnormalities ranging from simple steatosis, steatohepatitis (NASH) to fibrosis and cirrhosis in the absence of excessive alcohol consumption. During the present grant cycle, we have used an approach termed “systems genetics” to identify genetic and environmental interactions in NAFLD. Using omics technologies, we directly examined the effects of genetic variation on molecular phenotypes, such as global transcript, protein, or metabolite levels. This enables the identification of causal genes and pathways and the modeling of higher order interactions in the disease process. We validated two key driver genes, matrix gla protein (Mgp) and insulin-like growth factor binding protein 4 (Igfbp4) as causal genes in vivo by showing that experimental modulation of their expression led to significant reduction in liver fibrosis in mice. In Aim 1a, we will examine the role of Mgp in hepatic stellate cells (HSC) physiology by using HSC-specific knockout mice. In Aim 1b, we will use the Four Core Genotypes mouse model to dissect the respective role of gonadal hormones and sex-chromosomes in conferring sex-specific protection of NASH in the Mgp knockout mice. In Aim 1c, we will investigate the mechanisms by which Igfbp4 alters NASH using inducible overexpression and knockout mice. Additionally, its role in TGF-β and SMAD pathways will be examined. Our single-cell transcriptome analysis revealed potential cell-cell interactions important for liver fibrosis. We observed significant information flow from various liver cell types to HSC during the onset of liver fibrosis. In Aim 2a, we will examine the role of these interactions in HSC function using primary HSC. In Aim 2b, we will examine intercellular communication between HSC and other liver cell types by 3D liver spheroid culture. Plasma metabolomic analysis showed that circulating indole-3- propionic acid (IPA) levels were lower in NASH patients with liver fibrosis and that IPA levels were significantly correlated with liver transcripts enriched in HSC activation and hepatic fibrosis signaling. The links among IPA, cellular NAD+ levels and gut microbiota will be the focus for investigation in Aim 3. IPA will be supplemented in mice to test the hypothesis that IPA protects the liver from NASH by increasing NAD+ levels in hepatocytes (Aim 3a). We will use knockout mice of two NAD+ catabolizing enzymes, namely nicotinamide N-methyl transferase (NNMT) and CD38, to examine the link between NAD+ levels and NASH (Aim 3b). To test the causal links between gut bacterial IPA production and host phenotypes, we will perform gnotobiotic studies with germ-free mice. The hypothesis that perturbating IPA levels through transplantation of genetically engineered bacteria into germ-fre...