Hepatic sortilin regulates both apolipoprotein B secretion and LDL catabolism

Abstract

Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.

Figure 1. Sortilin demonstrates high-affinity binding to APOB.

(A) Preparations of LDL at the indicated concentrations in HBS-EP buffer (pH 7.4) were passed across the sensor chip for 240 seconds to monitor the association of LDL, HDL, and APOE DMPC discs and sortilin, and then the chip was exposed to buffer alone for an additional 240 seconds to monitor the dissociation phase. (B) 100 μg/μl of LDL was bound to immobilized human sortilin in HBS-EP buffer at pH 7.4. At the indicated time point, the buffer was exchanged with HBS-EP at lower pH values.

Figure 2. SORT1 expression reduces APOB secretion in a lysosome-dependent manner.

(A and B) Control and Sort1-expressing mice were fasted for 4 hours and then injected intraperitoneally with the detergent Pluronic to inhibit lipolysis, followed by radiolabeling. Mice were sacrificed 1 hour after radiolabeling, blood was harvested by retroorbital bleed, and VLDL was isolated by ultracentrifugation. Sort1 expression resulted in a 30% reduction in VLDL/APOB secretion on a wild-type background and (B) a 50% reduction in VLDL/APOB secretion on an Ldlr^–/– background. (C) APOB secretion from primary mouse hepatocytes from APOBEC1^–/–;APOB Tg;Ldlr^+/– mice following radiolabeling for 3 hours, with or without exposure to E64d. Labeled APOB was immunoprecipitated and measured from medium and normalized to total labeled secreted protein measurements. Sort1 expression resulted in a 30% reduction in APOB secretion in the absence of E64d; the effect was reversed in the presence of E64d. (D) Control and Sort1-expressing mice were fasted for 4 hours and then injected intraperitoneally with the detergent Pluronic to inhibit lipolysis. Plasma was drawn serially 1, 2, and 4 hours after Pluronic injection to permit assessment of the VLDL-TG secretion rate. Wild-type sortilin reduced the VLDL-TG secretion rate by 30%; neither Sort.Stop nor Sort.LAYA reduced VLDL-TG secretion. (E and F) GFP-expressing (control) and sortilin-expressing HuH7 cells were metabolically labeled for 3 hours, and labeled APOB was immunoprecipitated and measured from medium and standardized to total protein measurements. Wild-type sortilin reduced secreted APOB by 40%, while Sort.Stop and Sort.LAYA did not affect secreted APOB. Albumin secretion was not affected by induction of Sort1.

Figure 3. Sortilin expression promotes LDL uptake and lysosomal catabolism in vitro.

(A and B) HuH7 cells expressing wild-type sortilin, Sort.Stop, Sort.LAYA, or GFP (control) under control of a dox-inducible promoter were assayed for (A) LDL cell association and (B) LDL degradation after incubation with ¹²⁵I-LDL for 3 hours. Both wild-type sortilin and Sort.LAYA increased ¹²⁵I-LDL cell association 5-fold, while only wild-type sortilin increased ¹²⁵I-LDL degradation. Sort.Stop did not increase ¹²⁵I-LDL cell association or degradation (C–G) Cells were incubated with fluorescently labeled LDL for 6 hours and subjected to confocal microscopy to visualize intracellular LDL. Compared with cells containing only endogenous sortilin (C), wild-type sortilin (D) increases intracellular LDL, while Sort.Stop (E) does not increase intracellular LDL. Sort.LAYA (F) resulted in profound accumulation of LDL at the cell surface without increasing intracellular LDL (G) Sortilin and LDL show strong colocalization with the lysosomal marker LAMP1. Scale bars: 15 μm.

Figure 4. Sortilin expression promotes LDL uptake and lysosomal catabolism in vivo.

(A–D) Control mice and mice receiving AAVs encoding wild-type or mutant Sort1 were injected with 2 × 10⁶ counts of ¹²⁵I-LDL via tail vein. Blood was drawn serially 2 minutes and 1, 3, 6, 9, and 24 hours after injection. All counts were normalized to the 2-minute count. Clearance curves (A) and FCRs (B) demonstrated a dose-dependent increase in LDL clearance in wild-type mice expressing Sort1. Clearance curves (C) and FCRs (D) demonstrated that wild-type sortilin and Sort.LAYA increased LDL clearance, while Sort.Stop did not increase plasma LDL clearance (E and F) Clearance and FCRs demonstrating a 40% reduction in FCR in Sort1^–/– mice compared with wild-type mice and a 50% reduction in FCR in Ldlr^–/–;Sort1^–/– mice compared with Ldlr^–/– mice.

Figure 5. Sortilin deficiency is associated with reduced or normal plasma cholesterol and reduced APOB/VLDL secretion.

(A–D) Mice (8- to 10-week-old females) were fasted for 4 hours, and blood was drawn by retroorbital bleed; plasma total cholesterol was measured by autoanalyzer and FPLC. (A and B) Sort1 deficiency on an Ldlr^–/– background was associated with a 20% reduction in plasma cholesterol. (C–E) Sort1 deficiency on an APOBEC1^–/–;APOB transgenic background did not affect plasma cholesterol or APOB levels. (F) Mice (8- to 10-week-old females) were fasted for 4 hours and injected intraperitoneally with the detergent Pluronic to inhibit lipolysis followed by radiolabeling. Mice were sacrificed 1 hour after radiolabeling, plasma was collected, and VLDL was isolated by ultracentrifugation. Sort1 deficiency was associated with a 60% reduction in VLDL/APOB secretion on an APOBEC1^–/–;APOB transgenic background.