Hepatic proprotein convertases modulate HDL metabolism

Abstract

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.

Figure 1. Profurin and Plasma Lipid Levels after Inhibition of Hepatic Proprotein Convertases in Wild-Type Mice

A single dose of Ad-null or Ad-profurin was injected in wild-type mice. (A) Western blotting of profurin protein expression in vivo after transduction. Similar amounts of proteins from liver lysates (10 μg/lane) were loaded. (B and C) Blood was sampled at the indicated time points, and plasma lipid profile for high-density lipoprotein cholesterol (HDL-C) (B) and phospholipids (C) was determined as described in Experimental Procedures. n ≥ 4 mice per group; *p < 0.05, **p < 0.01. Each experiment was repeated 12 times. In this and all other figures, error bars represent ± SD.

Figure 2. Inhibition of Hepatic Proprotein Convertases Affects HDL Levels through Endothelial Lipase in Mice

(A) Effects of inhibition of hepatic proprotein convertases (PCs) on the cleavage of endothelial lipase (EL) in mice. Ten microliters of plasma was separated by two-dimensional electrophoresis, and EL protein was detected by western blotting. F: full-length protein (dotted box); N: cleaved N-terminal fragment (oval). (B) Effects of inhibition of hepatic PCs on postheparin plasma phospholipase activity. n = 4; *p < 0.05. (C) Effects of inhibition of hepatic PCs on HDL-phospholipid catabolism in wild-type (WT) mice. n = 4; p < 0.01. (D) Effects of inhibition of hepatic PCs on HDL levels in EL knockout mice (top) and western blotting of profurin in liver lysates after transduction (bottom). (E) Effects of inhibition of hepatic PCs on HDL-phospholipid catabolism in EL knockout mice. n = 4; p < 0.01. (F) Effects of inhibition of hepatic PCs on HDL levels in HL knockout mice (top) and western blotting of profurin in liver lysates after transduction (bottom). (G and H) Effects of PCSK5A on HDL levels in wild-type (G) and EL knockout mice (H). n ≥ 4 mice per group; *p < 0.05.

Figure 3. Effects of Inhibition of Hepatic PCs on ANGPTL3

(A) ANGPTL3 inhibited EL activity in vitro. (B) Profurin prevented the cleavage of ANGPTL3 in vitro. (C) The N terminus of ANGPTL3 (R221X) was a more potent inhibitor of EL than uncleavable ANGPTL3 (R224A). Data are expressed as percent of the control. n = 3; *p < 0.05. (D) Effects of inhibition of hepatic PCs on the cleavage of ANGPTL3 in mice. Upper panel: plasma (0.1 μl/lane); lower panel: FPLC fractions 39-41, which elute later than HDL. F: full-length protein; C: cleaved C-terminal fragment.

Figure 4. Effects of Inhibition of Hepatic PCs on Reverse Cholesterol Transport

(A) The measurement of ABCA1-specific efflux to mouse plasma in J774 cells. Data in (A) and (B) are expressed as percent of the control. n = 3; *p < 0.05. (B) The measurement of SR-BI-specific efflux to mouse plasma in Fu5AH cells. (C-F) Mice were injected intraperitoneally with ac-LDL-loaded J774 macrophages labeled with [³H]cholesterol. The percent of macrophage-derived [³H]cholesterol appearing in plasma (C), liver at 48 hr (D), bile at 48 hr (E), and feces over 48 hr (F) after macrophage injection is shown. Data are expressed as percent of the initial amount of [³H]cholesterol injected intraperitoneally. n = 6; *p < 0.05.