In this proposal we aim to define the mechanistic basis for neutral lipid translocation into the hepatocyteendoplasmic reticulum (ER), a process that is essential and likely rate limiting for lipoprotein-mediated lipidefflux. In addition, we shall explore whether apoB-mediated lipid transport can be modulated via regulatedparticle expansion, thereby increasing the lipid transport capacity of the hepatocyte without increasingatherogenic particle production.
Three Specific Aims are proposed: 1. Establish whether the phospholipid(PU and neutral lipid (TG) transfer activities of the microsomal triqlvceride transfer protein (MTP) playdistinct and separable roles in hepatic lipid transport. We hypothesize that PL transfer is the primordialfunction of MTP and is essential for the formation of lipoprotein precursors. In contrast, TG transfer is avertebrate adaptation required for TG translocation into the ER, thereby providing lipid for second steplipoprotein expansion. To test this hypothesis, transgenic mice will be generated that overexpress DrpsophilaMTP (PL only) or human MTP (PL and TG transfer) in the liver of both wild type and MTP null mice. Thephenotype of transgenic mice in terms of plasma lipids, apoB particle size and composition, hepatic apoBand TG production rates, and liver lipid content and histology will reveal whether selective inhibition of MTP'sPL transfer activity can reduce production of lipoproteins without causing hepatic lipid accumulation. 2^Determine whether MTP is necessary and sufficient for lipid translocation into the ER. To address thisquestion, human MTP will be expressed in CHO and other nonhepatic cells, in the absence of apoB, and theresulting qualitative and quantitative alterations in subcellular lipid distribution analyzed in cell-based and cellfree lipid translocation assays. In a second approach, human MTP and an epitope tagged form of humanapoBSO (apoBSOF) will be co-expressed ectopically in mouse adipocytes. Plasma apoB50F-TG productionrates and apoBSOF-containing lipoprotein particle characteristics will reveal the extent to which MTP is alonenecessary, sufficient, and rate limiting in the creation of secretion-coupled lipids. 3. Establish the mechanismbv which apoAIV facilitates lipid transport via apoB-containinq lipoprotein particle expansion. Mice thatoverexpress truncated SREBP-1a in liver overproduce cholesterol and fatty acids, giving rise to hepaticsteatosis. Under these lipogenic conditions, the liver produces large, chylomicron-sized lipoprotein particlesconcomitant with a 5.3-fold upregulation of apoAIV mRNA. As our previous and preliminary studies stronglysuggest that apoAIV may play a direct role in lipoprotein expansion, lipoprotein assembly and secretion willbe compared in SREBPIa transgenic mice versus SREBP1a;apoAIV'~ mice. The impact of apoAIVdeficiency on steatosis, steatosis-related pathologies, TG production rates, apoB particle size andcomposition, and apoB secretion kinetics, will be assessed. These studies may provide the first indicationthat the lipid efflux capacity of the hepatocyte can be enhanced without increasing production of atherogeniclipoproteins and, along with Aims 1 and 2, provide strategies for the prevention and treatment ofdyslipidemias associated with cardiovascular and other chronic diseases.
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