Project Summary NAFLD is closely associated with obesity and type-2 diabetes. Both improved understanding of the disease pathophysiology and discovery of novel therapeutic targets for NAFLD treatment are urgently needed. Cullin- RING E3 ligases (CRLs) are a unique sub-class of ubiquitin ligases that are activated upon neddylation of the cullin scaffold. Mammalian cells contain functionally distinct CRL complexes that recognize specific protein substrates for ubiquitination and subsequent proteasomal degradation. CRLs are considered promising drug targets due to their regulation of disease relevant cellular pathways, higher substrate selectivity and lack of broad cellular impact upon inhibition. Recently, CRLs have emerged as novel regulators of nutrient signaling and inflammatory response in different organ systems. However, the roles of hepatic CRLs in NAFLD pathophysiology is still largely unknown. The goals of this project are to delineate the functional impact and underlying mechanisms by which CRLs regulate hepatic lipid and glucose metabolism and to establish CRLs as valid therapeutic targets for developing NAFLD and diabetes treatment. This project is developed based on our key findings that hepatic CRLs are abnormally activated in human NASH livers and murine NAFLD, and pharmacological inhibition of hepatic CRL activity by targeting cullin neddylation improved insulin sensitivity, reduced hyperglycemia and liver steatosis by countering the abnormally elevated gluconeogenic and lipogenic activity that drives NAFLD progression. We will perform mechanistic studies to establish a working model whereby pharmacological CRL inhibition prevents abnormal liver insulin receptor substrate degradation, the key underlying cause of hepatic insulin resistance in NAFLD, and attenuates ChREBP-driven de novo lipogenesis, therefore simultaneously achieves glucose and lipid-lowering effects. Specific aims are designed to dissect the specific downstream CRL complex that mediates the beneficial effects of cullin neddylation inhibitors and to identify and functionally validate the CRL substrate receptors that can serve as new therapeutic targets for future drug development. This study employs new tissue-specific genetic knockout mice, AAV8-mediated liver-specific gene delivery, and pharmacological treatment models. These models will be investigated with a combination of molecular and cell biology techniques, metabolic phenotyping, hyperinsulinemic-euglycemic clamp, and unbiased transcriptomics and proteomics approaches. By employing these state-of-the-art models and techniques, our working hypothesis can be rigorously tested and new mechanistic insights will be obtained.