Proprotein Convertase Subtilisin Kexin Type 9 Null Mice Are Protected From Postprandial Triglyceridemia

Abstract

Objectives-Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the low-density lipoprotein receptor, and its deficiency in humans results in low plasma LDL-cholesterol and protection against cardiovascular disease. We explored whether PCSK9 expression impacts postprandial triglyceridemia, another important cardiovascular risk factor. Methods and Results-Real-time PCR and confocal microscopy were used to show that PCSK9 is expressed throughout the entire small intestine and in human enterocytes. On olive oil gavage, PCSK9-deficient mice showed a dramatically decreased postprandial triglyceridemia compared with their wild-type littermates. Lymph analysis revealed that intestinal TG output is not quantitatively modified by PCSK9 deletion. However, PCSK9 Ϫ/Ϫ mice present with a significant reduction of lymphatic apoB secretion compared to PCSK9 ϩ/ϩ mice. Modulating PCSK9 expression in polarized CaCo-2 cells confirmed the relationship between PCSK9 and apoB secretion; PCSK9 Ϫ/Ϫ mice consistently secrete larger TG-rich lipoprotein than wild-type littermates. Finally, kinetic studies showed that PCSK9-deficient mice have an increased ability to clear chylomicrons compared to wild-type littermates. G ain-of-function mutations affecting proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with autosomal dominant hypercholesterolemia and premature atherosclerosis. 1 It is now established that PCSK9 is a natural inhibitor of the low-density lipoprotein receptor (LDLr), acting posttranscriptionally. 2 Circulating PCSK9 binds to the EGF-A extracellular domain of the hepatic LDLr and prevents its recycling to the cell surface. 3 A breakthrough study reported that blacks harboring PCSK9 loss-of-function mutations had an 88% risk reduction for coronary heart disease. 4 Although the lower concentrations of LDL cholesterol over one's lifetime is suggested to be the main reason for this very high level of protection, we hypothesized that PCSK9 deficiency might also affect other risk factors. Low levels of HDL cholesterol (HDL-c) and elevated nonfasted triglycerides (TG) levels are associated with increased risk for cardiovascular disease. No association between PCSK9 loss-of-function mutations and plasma HDL-c levels has been noticed. 4,5 However, to our knowledge, nonfasting plasma TG were not measured in PCSK9-deficient individuals. They are an important contributor to the risk for cardiovascular disease. 6,7 Rashid et al reported the phenotype of PCSK9-deficient mice (PCSK9 Ϫ/Ϫ ) mice and observed that they exhibit lower plasma cholesterol levels (Ϫ50%) and are hypersensitive to statins. 8 The potential role of PCSK9 in the intestine in mice and humans remains unexplored. Conclusion-TheseDuring the postprandial period, dietary lipids are actively absorbed into the enterocyte. After intracellular reesterification, long chain fatty acids and cholesterol esters are associated with phospholipids and apolipoproteins, particularly apoB48, to produce TG-rich lipoproteins, mainly chy...

Response to Letter Regarding Article, “Proprotein Convertase Subtilisin Kexin Type 9 Promotes Intestinal Overproduction of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both Low-Density Lipoprotein Receptor–Dependent and –Independent Mechanisms”

“…We presented data from PCSK9 added to the apical side but obtained similar results after adding it to the basolateral side. This is in line with previous work in which Levy et al 4 show that CaCo-2 cells secrete PCSK9 mostly into the basolateral medium, whereas Le May et al 2 state that enterocytes show “strong PCSK9 immunostaining mainly in the apical compartment” and that “PCSK9…accumulates at the subapical and basolateral compartments of the enterocyte.”…”

“…In their letter, Drs Le May and Cariou point out the omission of 2 references. The first, published by Le May et al 2 in 2009, reports that PCSK9 -/- mice display reduced postprandial hypertriglyceridemia and apolipoprotein B levels after olive oil gavage and that adenoviral PCSK9 overexpression or targeted shRNA silencing of PCSK9 increased and reduced apolipoprotein B secretion, respectively. In that article, Le May et al 2 acknowledge an earlier observation, published by 1 of us (S.R.)…”

Letter From Le May and Cariou Regarding Article, “Proprotein Convertase Subtilisin Kexin Type 9 Promotes Intestinal Overproduction of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both Low-Density Lipoprotein Receptor–Dependent and –Independent Mechanisms”

“…2 Finally, we confirmed this relationship between intestinal PCSK9 and apolipoprotein B secretion in vitro in differentiated human Caco-2 cells by showing that adenoviral PCSK9 overexpression or silencing of PCSK9 with targeted SiRNA led to a significant increase or reduction of apolipoprotein B secretion, respectively. 2 In 2013, Levy and collaborators 3 further confirmed the role of PCSK9 in lipid metabolism in Caco-2 cells. In their study, they revealed a role for PCSK9 in cholesterol absorption.…”

“…2 Using the lymph duct cannulation method, we also showed that PCSK9-deficient mice have reduced intestinal apolipoprotein B production compared with wild-type mice. 2 Finally, we confirmed this relationship between intestinal PCSK9 and apolipoprotein B secretion in vitro in differentiated human Caco-2 cells by showing that adenoviral PCSK9 overexpression or silencing of PCSK9 with targeted SiRNA led to a significant increase or reduction of apolipoprotein B secretion, respectively. 2 In 2013, Levy and collaborators 3 further confirmed the role of PCSK9 in lipid metabolism in Caco-2 cells.…”

Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans

“…Overexpression of PCSK9 in either the liver or the intestine increases secretion of apoB-lipoproteins, 29,30 whereas knockdown of PCSK9 has the opposite effect. 30,31 Additionally, individuals with gain-of-function mutations in PCSK9 were found to have increased secretion of VLDL-, IDL-, and LDL-apoB. 32 Our finding that alirocumab did not affect either VLDL PR or PP lipemia is, therefore, in contrast to these reports, but it is possible that these differences also reflect the absence of underlying abnormalities in either lipolysis- or receptor-mediated removal of apoB48 particles in the individuals we studied.…”