In this proposal we aim to define the mechanistic basis for neutral lipid translocation into the hepatocyte endoplasmic reticulum (ER), a process that is essential and likely rate limiting for lipoprotein-mediated lipid efflux. In addition, we shall explore whether apoB-mediated lipid transport can be modulated via regulated particle expansion, thereby increasing the lipid transport capacity of the hepatocyte without increasing atherogenic 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) play distinct and separable roles in hepatic lipid transport. We hypothesize that PL transfer is the primordial function of MTP and is essential for the formation of lipoprotein precursors. In contrast, TG transfer is a vertebrate adaptation required for TG translocation into the ER, thereby providing lipid for second step lipoprotein expansion. To test this hypothesis, transgenic mice will be generated that overexpress Drpsophila MTP (PL only) or human MTP (PL and TG transfer) in the liver of both wild type and MTP null mice. The phenotype of transgenic mice in terms of plasma lipids, apoB particle size and composition, hepatic apoB and TG production rates, and liver lipid content and histology will reveal whether selective inhibition of MTP's PL 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 this question, human MTP will be expressed in CHO and other nonhepatic cells, in the absence of apoB, and the resulting qualitative and quantitative alterations in subcellular lipid distribution analyzed in cell-based and cell free lipid translocation assays. In a second approach, human MTP and an epitope tagged form of human apoBSO (apoBSOF) will be co-expressed ectopically in mouse adipocytes. Plasma apoB50F-TG production rates and apoBSOF-containing lipoprotein particle characteristics will reveal the extent to which MTP is alone necessary, sufficient, and rate limiting in the creation of secretion-coupled lipids. 3. Establish the mechanism bv which apoAIV facilitates lipid transport via apoB-containinq lipoprotein particle expansion. Mice that overexpress truncated SREBP-1a in liver overproduce cholesterol and fatty acids, giving rise to hepatic steatosis. Under these lipogenic conditions, the liver produces large, chylomicron-sized lipoprotein particles concomitant with a 5.3-fold upregulation of apoAIV mRNA. As our previous and preliminary studies strongly suggest that apoAIV may play a direct role in lipoprotein expansion, lipoprotein assembly and secretion will be compared in SREBPIa transgenic mice versus SREBP1a;apoAIV'~ mice. The impact of apoAIV deficiency on steatosis, steatosis-related pathologies, TG production rates, apoB particle size and composition, and apoB secretion kinetics, will be assessed. These studies may provide the first indication that the lipid efflux capacity of the hepatocyte can be enhanced without increasing production of atherogenic lipoproteins and, along with Aims 1 and 2, provide strategies for the prevention and treatment of dyslipidemias associated with cardiovascular and other chronic diseases.
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