An Extrahepatic Receptor-associated Protein-sensitive Mechanism Is Involved in the Metabolism of Triglyceride-rich Lipoproteins

Abstract

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)-independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor-associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre ؉ LRP flox/flox LDLR ؊/؊ ) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDLsized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre ؉ LRP flox/flox LDLR ؊/؊ mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K d ‫؍‬ 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation.Hypertriglyceridemia, combined with the accumulation of remnant lipoproteins in the circulation, is a major risk factor for atherosclerosis and coronary artery disease. The genetic bases of this clinically important syndrome are complex and incompletely understood. Two endocytotic receptor systems are known to remove the lipolyzed remnants of chylomicrons and very low density lipoproteins (VLDL) 1 from the circulation. They are the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) (1, 2). Following lipolysis in the peripheral capillaries of muscle, heart, and adipose tissue, where chylomicrons deliver most of the triglyceride load they carry, the remnants have shrunk to a size at which they can permeate the fenestrated endothelium separating the hepatocyte surface and the space of Disse from the circulation (for review see Ref.3). LRP and LDL receptors at the surface of hepatocytes bind and clear remnant lipoproteins from an intermediate binding site. This intermediate compartment is created by interactions of heparan sulfate proteoglycans (4), hepatic lipase (5, 6), lipoprotein lipase (7), and apoE (8) with the remnants.Gene knockout and gene transfer experiments in mice have defined the roles of the receptors, apoproteins, and lipases in the remnant clearance process. Although the LDL receptor efficiently removes apoB100-containing LDL, as well as apoB48-containing remnants thr...

Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor–related protein deficiency

“…We previously demonstrated that the cre/loxP recombination system effectively achieves inducible disruption of LRP in adult mice, thereby allowing in vivo studies on the role of LRP for at least 6 weeks after pI:pC injection. 26,27 To investigate the role of LRP in regulating plasma FVIII in vivo, both plasma FVIII and its carrier protein VWF were measured in MX1cre ϩ LRP flox/flox and control LRP flox/flox mice.…”

“…Expression of RAP in mouse plasma was quantified by enzyme-linked immunosorbent assay, with the use of polyclonal antibodies against RAP for capture and detection. 27 …”

“…27 Briefly, mouse liver membrane extracts (50 g per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on 4% to 15% polyacrylamide gels under nonreduced conditions. Nitrocellulose membranes were blocked for 30 minutes at room temperature in 0.5% (vol/vol) Tween 20, 2% (wt/vol) BSA, 5% (wt/vol) milk powder, and PBS (pH 7.4), followed by a 60-minute incubation of a peroxidase-labeled rabbit polyclonal antibody directed against the 85-kDa subunit of LRP in the same buffer.…”

“…Nitrocellulose membranes were blocked for 30 minutes at room temperature in 0.5% (vol/vol) Tween 20, 2% (wt/vol) BSA, 5% (wt/vol) milk powder, and PBS (pH 7.4), followed by a 60-minute incubation of a peroxidase-labeled rabbit polyclonal antibody directed against the 85-kDa subunit of LRP in the same buffer. 27 Bound immunoglobulin G (IgG) was detected by means of the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Uppsala, Sweden).…”

“…Previously, this LRPdeficient mouse model proved to be successful for studying the role of LRP in regulating plasma lipoprotein levels in vivo. 26,27 In addition, other RAP-sensitive mechanisms involved in regulating plasma FVIII in vivo were addressed with the use of adenovirusmediated gene transfer of RAP in LRP-deficient mice.…”