Novel regulatory mechanisms controlling hepatic apoB-Lp lipid loading and secretion

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The global hypothesis underlying this PPG application is that crosstalk between adipose, liver, and intravascular lipolysis regulates the biogenesis of atherogenic apoB-containing lipoproteins, most notably VLDLs. Project 2 (P2) focuses on mechanisms that control the generation of apoB/VLDL in liver cells. Prior work from the PI and Co-I, which has resulted in 14 publications, established that a major mechanism controlling circulating apoB/VLDL is the regulated degradation of apoB in the endoplasmic reticulum (ER). This metabolically orchestrated event requires the ER-associated degradation (ERAD) pathway, which was named and first elucidated by the Co-I. Under lipid-poor conditions, the ERAD of apoB begins with its selection by molecular chaperones, which is followed by apoB ubiquitinylation and delivery to the cytoplasmic proteasome. In contrast, when neutral lipids (primarily triacylglycerols) are in excess, apoB is co-translationally lipidated by MTP, and the nascent apoB/VLDL particles are expanded by lipids sourced from ER resident lipid droplets (LDs), and the re- modelled apoB/VLDL particle is packaged into COPII vesicles for delivery to the Golgi and then into circulation. In contrast, the events that regulate the number and composition of lipids assembled with apoB—as well as the packaging of engorged, lipid-rich apoB/VLDL particles into COPII vesicles—are poorly characterized. To these ends, results generated by the PI and his colleagues indicate that two factors, ER-resident proteins KLHL12 and FIT2, play a significant role in controlling lipid assembly onto apoB and the encapsulation of apoB/VLDL into COPII vesicles in vitro and in vivo. These recently acquired data support several new hypotheses: First, that KLHL12 resides at apoB exit sites on the ER that include COPII components, and second that the inefficient integration of apoB into COPII vesicles selects the protein for the recently characterized ER-phagy pathway, which was also elucidated by the Co-I. A characterization of a new liver-specific KLHL12 knockout mouse will be used to define the role of KLHL12 in non-alcoholic fatty liver disease and steatohepatitis. In turn, other recently acquired preliminary data indicate that FIT2 deficiency increases ER membrane lipid content along with the generation of lipid-depleted apoB/VLDLs. Based on the function of FIT2 in forming cytosolic LDs, these data underscore another novel hypothesis, that FIT2 delivers LDs into the ER, where they are then integrated into pre-apoB/VLDL particles in either an MTP-dependent or independent manner. A new liver-specific FIT2 knockout mouse will concomitantly allow for a study of the links between FIT2 and fatty liver disease and steatohepatitis, and also atherosclerosis (because of FIT2 regulation of apoB/VLDL lipid content and composition). Broadly, the Specific Aims of this application are: (1) To define the mechanisms underlying KLHL12-dependent regulation of lipid-rich apoB/VLDL encapsulation into hepatic ER-derived, Golgi-directed transport vesicles, and (2) To define the impact of FIT2 on lipid loading of apoB/VLDL particles and their atherogenicity, both of which require critical collaborations with P1, P3, and Core facilities.