This is a request for continued funding for a long term program to learn how diet derived cholesterol is metabolized and affects overall cholesterol homeostasis. For a number of years, the program has had as a major focus, investigation of the mechanism of hepatic removal of chylomicron remnants. During the last grant period, studies with liver cell membranes, hepatocytes, and intact mice has led to the hypothesis that there are three pathways for remnant removal. The major determinant of remnant uptake in the normal animal is the LDL receptor. The newly discovered LRP has a lower affinity for these particles and serves as a backup mechanism for the LDL receptor, but is quantitatively less important in the normal animal. In addition, the intact liver may sequester remnants in the space of Disse, a process we have termed, sieving. This would constitute a third pathway and could function to clear even apoE deficient particles. Hepatic lipase, apoE, and glycosaminoglycans, may mediate the sieving, and seem to be paracrine factors that accelerate the uptake of LDL. During the next period, we propose studies to continue to test these pathways and assess their quantitative significance in various states. Pulse chase liver perfusion followed by cell isolation studies are proposed to precisely measure the contribution of sieving. Competitive inhibitors of the LDL receptor and the LRP have, and will be prepared, and their effect on remnant removal in the isolated liver and in vivo will be studied. Lipoprotein uptake by cells that secrete apoE, hepatic lipase, and/or express an anchored form of hepatic lipase, have been prepared. Lipoprotein uptake, both in cell culture, and in vivo will be compared to that of otherwise identical cells that do not secrete these proteins. Once the in vitro systems provide a general understanding of the various contributors to the remnant removal pathways, their relative importance in vivo will be assessed. This will be done using intact mice expressing a variety of genetically engineered defects in the various components of the system. The mice to be studied include apoE deficient and hepatic lipase deficient, as well as mice that over-express LDL receptors in an extra-hepatic site, and mice that express hepatic lipase localized to the liver. From these studies, we will obtain a complete understanding of the components of the remnant uptake mechanism and how alterations and deficiencies affect the entire system.
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