Identification of metabolic regulators of hepatic phosphatidylcholine synthesis - Abstract Hepatic phosphatidylcholine (PC) metabolism is essential for maintaining lipid homeostasis, as PCs are required for very-low-density lipoprotein (VLDL) secretion and protection against hepatic fat accumulation. Disruptions in PC synthesis are linked to metabolic dysfunction-associated fatty liver disease (MAFLD), a condition affecting over 25% of the U.S. population. However, the genetic regulation of PC metabolism in response to metabolic stress, and its relevance to MAFLD prevention and progression, remain poorly understood. Our preliminary data reveal that the transmembrane oxidoreductase VKORC1L1 regulates hepatic PC levels through a function independent of its enzymatic activity. Furthermore, loss of hepatic Vkorc1l1 in mice decreases PC levels, reduces VLDL secretion, and promotes hepatic fat accumulation. Lastly, human genetic data indicate a potential link between VKORC1L1 variants and liver steatosis, suggesting a role in MAFLD progression. Hepatic PC levels are regulated through three primary pathways: de novo synthesis from choline via the Kennedy pathway, methylation of phosphatidylethanolamine (PE), and the salvage of exogenous lipids. We aim to define the role of VKORC1L1 in hepatic PC metabolism and MAFLD by addressing three key questions. First, we will determine whether VKORC1L1 is necessary and sufficient to regulate PC levels and mitigate MAFLD progression in mouse models under varying dietary conditions, focusing on two MAFLD-relevant nutrients: fat and choline—a precursor for PC synthesis. Using liver-specific Vkorc1l1 knockout and overexpression mice, we will assess its impact on VLDL secretion, hepatic lipid accumulation, and whole-body metabolic health. Second, we will delineate the broader impact of VKORC1L1 on hepatic PC homeostasis by assessing its influence on the three major pathways that maintain PC levels—the Kennedy pathway, PE methylation, and lipid salvage—using isotope-labeled metabolic precursors and phospholipid analytical approaches. Finally, we will investigate the molecular mechanism by which VKORC1L1 influences de novo PC synthesis, building on our preliminary data showing that VKORC1L1 binds to and activates CCTα, the rate-limiting enzyme of the Kennedy pathway. By combining biochemical and structural approaches, including cryo-electron microscopy, we will test whether this interaction is influenced by membrane PC content in cells and mouse liver. By uncovering how VKORC1L1 regulates PC metabolism and VLDL secretion, our study will reveal key molecular mechanisms linking choline and PC deficiency to MAFLD. This is particularly relevant, as low-choline and high-fat diets are prevalent in segments of the U.S. population. Our findings could inform therapeutic strategies targeting VKORC1L1 to restore PC homeostasis and mitigate MAFLD progression—an urgent priority given the widespread prevalence of MAFLD and its considerable variability in severity and treatment response.
