Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

Abstract

Objective-In humans, apolipoprotein (apo) E4 is associated with elevated plasma cholesterol levels and a high risk of developing atherosclerosis, whereas apoE2 is protective. Here we investigate the mechanism by which mice expressing human apoE isoforms recapitulate this association when they also express high levels of human low-density lipoprotein receptor (LDLR). Methods and Results-Primary hepatocytes from apoE4 mice secreted less apoE into the medium than hepatocytes from apoE2 mice. Increased LDLR expression decreased this secretion and increased degradation of apoE4. An apoE4-GFP fusion protein expressed in the liver of apoE-deficient mice accumulated on the hepatocyte surface bordering the space of Disse in an LDLR-dependent manner. Fluorescence-labeled very low-density lipoprotein (VLDL) remnants accumulated on the hepatocyte surface in apoE4 mice with high LDLR, but they were internalized poorly. In contrast, apoE2-GFP did not accumulate on the hepatocyte surface even when the LDLR expression was high, but apoE2 mice with high LDLR internalized the remnants avidly without sequestering them on the hepatocyte surface. Conclusions-The high affinity of apoE4 to the LDLR enhances VLDL sequestration on the hepatocyte surface but delays their internalization. This delay likely increases VLDL conversion to cholesterol-enriched remnants in apoE4 mice with high LDLR, and probably to LDL in humans with apoE4. A polipoprotein E (apoE) and the low-density lipoprotein receptor (LDLR) play a pivotal role in the clearance of lipoproteins by the liver, thereby reducing plasma cholesterol, a leading determinant of atherosclerosis susceptibility. 1,2 Uptake of triglyceride-rich lipoproteins (TRL) occurs via multiple receptors and perhaps in several steps. 3 The first, most important step appears to be the sequestration of TRL on the microvilli of hepatocytes, where LDLR, heparan sulfate proteoglycans (HSPG), and LDLR related proteins (LRP) are located. These molecules bind apoE proteins secreted by the liver thereby serving as reservoirs for enriching remnant particles with apoE. 4 They also serve as the receptors for internalization of apoE-enriched remnants. 3 In humans, the Apoe gene is polymorphic, resulting in production of 3 common isoforms, apoE2, E3, and E4. They differ in primary structure at 2 positions, E2 having Cys at both positions 112 and 158, E3 having a Cys at 112 and an Arg at 158, and E4 having Arg at both positions. They also differ in their LDLR binding affinity; apoE4 binds LDLR with a slightly higher affinity than apoE3, whereas apoE2 has much reduced binding compared to the other 2 isoforms. [5][6][7][8][9] Despite the low receptor binding of apoE2, the majority of individuals carrying apoE2 have lower plasma LDL cholesterol and reduced atherosclerosis risk, although 5% to 10% of apoE2 homozygotes develop type III hyperlipoproteinemia characterized by markedly elevated plasma lipid levels. 2,10 Equally paradoxical is the association of the apoE4 isoform with high LDL-cholesterol, low plasma tr...

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