The role of FIT2 in VLDL assembly, hepatic triglyceride homeostasis, and lipoprotein atherogenicity

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This R01 application focuses on the mechanisms that control the generation of very low density lipoproteins (VLDLs) in liver cells. Prior work from the MPIs, which has resulted in 15 publications, established that a major mechanism controlling the secretion and circulation of VLDL is the regulated degradation of apolipoprotein (apoB) in the endoplasmic reticulum (ER). This metabolically orchestrated event requires the ER-associated degradation (ERAD) pathway, which was named and first elucidated by MPI Brodsky. In contrast, when neutral lipids (primarily triacylglycerols; TGs) are sufficient, apoB is co-translationally lipidated by MTP, and the nascent VLDL particles are expanded by lipids sourced from ER-resident lipid droplets (LDs). The re-modelled VLDL particles are next packaged into COPII vesicles for delivery to the Golgi and then secreted into the medium from hepatic cells (in vitro) or from liver hepatocytes into the circulation (in vivo). In contrast to these pathways that control the levels of VLDL, the factors that regulate the concentration and composition of lipids assembled onto apoB in the ER are poorly characterized. However, recent results generated by the MPIs and colleagues indicate that an ER-resident membrane protein, FIT2, plays a significant role in controlling TG assembly onto apoB in vitro and in vivo. Specifically, the preliminary data outlined in this application—which were made possible by the generation of novel rodent models and engineered VLDL secreting cell lines—strongly suggest that FIT2 regulates both the concentration and composition of apoB-associated lipids, as well as the atherogenicity of VLDL. Based on these and other new data, the following hypotheses will be tested: 1) FIT2 deficiency will increase ER membrane lipid content along with the generation of TG-depleted VLDL; 2) FIT2 delivers LDs into the ER, which are then integrated into VLDL particles in either an MTP-dependent or independent manner; and, 3) the level of FIT2 activity is a previously unappreciated determinant for the severity of fatty liver disease (NAFLD), steatohepatitis (NASH), and atherosclerosis. Because the efficiency of FIT2-mediated loading of TG onto VLDL also impacts hepatic lipid levels in the ER, FIT2 deficiency may also lead to lipid dysregulation in the ER and toxic stress responses. Moreover, the delivery of lipid-rich VLDLs could be limited by another ER-associated factor, KLHL12, that helps form specialized VLDL- resident COPII vesicles. Thus, another hypothesis is that native KLHL12 levels constrain the capacity of FIT2- supported delivery of VLDL from hepatic cells, thus exacerbating ER stress. Overall, besides dissecting how FIT2 regulates NASH, NAFLD, VLDL biogenesis, hepatic TG homeostasis, and lipoprotein atherogenicity, the clinical relevance of this project also includes the integration of transcriptomic data from the recently generated models with analogous databases obtained from human livers and atherosclerotic plaques. Toward all of these goals, the project benefits from deep and complementary expertise of the MPIs along with the experience of long-term collaborators with a substantial track record in studying lipoprotein metabolism.