PCSK9 is expressed in pancreatic δ-cells and does not alter insulin secretion

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic δ-cells, with no expression in α- and β-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in β-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9^+/+ and Pcsk9^−/− mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.     

PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ～2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.     

Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.     

Common PCSK1 Haplotypes Are Associated With Obesity in the Chinese Population

Prohormone convertase subtilisin/kexin type 1 (PCSK1) genetic polymorphisms have recently been associated with obesity in European populations. This study aimed to examine whether common PCSK1 genetic variation is associated with obesity and related metabolic phenotypes in the Chinese population. We genotyped nine common tag single‐nucleotide polymorphisms (tagSNP) of the PCSK1 gene in 1,094 subjects of Chinese origin from the Stanford Asia‐Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family study. One SNP in the PCSK1 gene (rs155971) were nominally associated with risk of obesity in the SAPPHIRe cohort (P = 0.01). A common protective haplotype was associated with reduced risk of obesity (23.79% vs. 32.89%, P = 0.01) and smaller waist circumference (81.71 ± 10.22 vs. 84.75 ± 10.48 cm, P = 0.02). Another common haplotype was significantly associated with increased risk of obesity (37.07% vs. 23.84%, P = 0.005). The global P value for haplotype association with obesity was 0.02. We also identified a suggestive association of another PCSK1 SNP (rs3811951) with fasting glucose, fasting insulin, homeostasis model assessment of insulin resistance (HOMA_IR), triglycerides, and high‐density lipoprotein cholesterol (P = 0.05, 0.003, 0.001, 0.04, and 0.04, respectively). These data indicate common PCSK1 genetic variants are associated with obesity in the Chinese population.     

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.     

Targeted deletion of the tub mouse obesity gene reveals that tubby is a loss-of-function mutation

The mouse tubby phenotype is characterized by maturity-onset obesity accompanied by retinal and cochlear degeneration. A positional cloning effort to find the gene responsible for this phenotype led to the identification of tub, a member of a novel gene family of unknown function. A splice defect mutation in the 3' end of the tub gene, predicted to disrupt the C terminus of the Tub protein, has been implicated in the genesis of the tubby phenotype. It is not clear, however, whether the Tub mutant protein retains any biological activity, or perhaps has some dominant function, nor is it established that the tubby mutation is itself responsible for all of the observed tubby phenotypes. To address these questions, we generated tub-deficient mice and compared their phenotype to that of tubby mice. Our results demonstrate that tubby is a loss-of-function mutation of the tub gene and that loss of the tub gene is sufficient to give rise to the full spectrum of tubby phenotypes. We also demonstrate that loss of photoreceptors in the retina of tubby and tub-deficient mice occurs by apoptosis. In addition, we show that Tub protein expression is not significantly altered in the ob, db, or melanocortin 4 receptor-deficient mouse model of obesity.     

PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1

We have recently identified a new form of post-translational regulation of BACE1 (beta-site amyloid precursor protein (APP)-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid beta-peptide (Abeta). Specifically, BACE1 is transiently acetylated on seven lysine residues in the lumen of the endoplasmic reticulum/endoplasmic reticulum-Golgi intermediate compartment (ER/ERGIC). The acetylated intermediates of the nascent protein are able to reach the Golgi apparatus, whereas the non-acetylated ones are retained and degraded in a post-ER compartment. Here, we report that the serine protease PCSK9 (proprotein convertase subtilisin kexin type 9) contributes to the disposal of non-acetylated BACE1. Both overexpression and small interfering RNA-mediated downregulation of PCSK9 affected the levels of BACE1. The downregulation of PCSK9 affected the levels of the loss-of-acetylation mutants (BACE1(Ala) and BACE1(Arg)) but not those of the gain-of-acetylation mutant (BACE1(Gln)). In addition, Pcsk9(-/-) mice showed increased levels of BACE1 and Abeta in the brain. Finally, we found that nascent low-density lipoprotein receptor, a known substrate of PCSK9, is also acetylated.     

Genetics of the First Seven Proprotein Convertase Enzymes in Health and Disease

Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature pro-proteins into their biologically active forms. By cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia. The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently, shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments in genome-wide methodology have generated a large amount of novel information on the genetics of the first seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes.     

The polymorphisms of bovine PCSK1 gene and their associations with growth traits

Proprotein convertase 1 (PCSK1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells and mutations in PCSK1 gene are thought to cause obesity. In the present study, based on PCR-SSCP and DNA sequencing methods, polymorphisms of the PCSK1 gene were detected in 858 individuals from five breeds (Nanyang cattle, Qinchuan cattle, Jiaxian cattle, Luxi cattle and Chinese Holstein). The results showed that only P₈ locus showed polymorphisms and 3 synonymous SNPs of PCSK1 gene were identified. Additionally, significant statistical difference was found in bovine birth weight and diplotype MM were 7.35% higher than diplotype XY.     

Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4‐null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4^75–90), which contains its primary autocatalytic cleavage site. ProPC4^75–90 inhibited recombinant PCSK4's activity with a K_i value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4^75–90 inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co‐efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)‐induced acrosome reaction, since proPC4^75–90‐treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4‐null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm–egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4^75–90‐treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process. J. Cell. Physiol. 226: 2817–2826, 2011. © 2011 Wiley‐Liss, Inc.     

The role of the C-terminal domain of PCSK9 and SEC24 isoforms in PCSK9 secretion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that promotes low-density lipoprotein receptor (LDLR) degradation and thereby regulating plasma levels of LDL cholesterol. Previous studies have revealed the role of the C-terminal domain (CTD) of PCSK9 in its secretion, however, how CTD regulates PCSK9 secretion is not completely understood. Additionally, SEC24A, the cargo adaptor protein of the coat protein complex II, has been implicated in the secretion of mouse PCSK9. Here, we investigated how CTD and SEC24 regulated PCSK9 secretion in humans. We found that mutant PCSK9^1–528, in which amino acids from 529 to the end (amino acid 692) were deleted, was maturated and secreted from cells as effectively as the wild-type protein. On the other hand, lacking amino acids 454 to 692 in mutant PCSK9^1–453 significantly reduced its maturation and secretion, but to a lesser extent when compared to mutants PCSK9^1–446, PCSK9^1–445 and PCSK9^1–444, that all markedly impaired PCSK9 maturation. However, mutant PCSK9^1–444 virtually eliminated PCSK9 secretion while PCSK9^1–446 and PCSK9^1–445 could still be adequately detected in culture medium. Interestingly, mutation of Pro⁴⁴⁵ to other amino acid residues considerably impaired the secretion of mutant PCSK9^1–445 but not the full-length protein. We also found that natural variants in CTD including S462P, S465L, E482G, R495Q and A522T impaired PCSK9 secretion. Further, the knockdown of SEC24A, SEC24B, SEC24C but not SEC24D reduced secretion of the full-length PCSK9 but not mutant PCSK9^1–446. Therefore, SEC24A, SEC24B, and SEC24C facilitate endogenous PCSK9 secretion from cultured human hepatocytes, that are most likely mediated by the CTD of PCSK9. Our studies also indicate that the CTD of PCSK9 may allosterically and independently modulate the stability of the hinge region. Collectively, these data revealed that the CTD of PCSK9 and the hinge region play a critical role in PCSK9 maturation and secretion.     

Inactivation of the MSLNL gene encoding mesothelin-like protein during African great ape evolution

Loss of gene function is implicated in the emergence of novel phenotypes during organism evolution. Here, we report the inactivation of the MSLNL gene encoding mesothelin-like protein in African great ape evolution. Human MSLNL has a nonsense mutation in exon 10 and two polymorphic mutations: a frameshift in exon 3 and a nonsense codon in exon 8. The gorilla gene also shows multiple deleterious mutations, including a premature stop codon, a deletion, and a splice site mutation. Molecular evolutionary analysis indicated relaxed selection pressure on MSLNL in African great ape lineages, which suggested that MSLNL might have become inactivated before the divergence of human, chimpanzee and gorilla. The mouse Mslnl gene is highly expressed in olfactory epithelium and moderately expressed in several other tissues. We propose that the loss of MSLNL may be associated with the evolution of the olfactory system in African great apes including human.     

Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimer’s-like phenotypes in mice

Obesity is a risk factor for Alzheimer’s disease (AD), which is characterized by amyloid β depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD.     

Polymorphisms in an obesity-related gene (PCSK1) are associated with fat deposition and production traits in Italian heavy pigs

The proprotein convertase subtilisin/kexin type 1 (PCSK1) gene encodes the prohormone convertase 1/3 enzyme that processes prohormones into functional hormones that, in turn, regulate central and peripheral energy metabolism. Mutations in the human PCSK1 gene cause severe monogenic obesity or confer risk of obesity. We herein investigated the porcine PCSK1 gene with the aim of identifying polymorphisms associated with fat deposition and production traits in Italian heavy pigs. By re-sequencing about 5.1 kb of this gene in 21 pigs of different breeds, we discovered 14 polymorphisms that were organized in nine haplotypes, clearly distributed in two clades of putative European and Asian origin. Then we re-mapped this gene on porcine chromosome 2 and analysed its expression in several tissues including gastric oxyntic mucosa of weanling pigs in which PCSK1 processes the pre-pro-ghrelin into ghrelin, which in turn is involved in the control of feed intake and energy metabolism. Association analyses between PCSK1 single-nucleotide polymorphisms (SNPs) and production, carcass and several other traits were conducted on five groups of pigs from three different experimental designs, for a total of 1221 animals. Results indicated that the analysed SNPs were associated (P < 0.01 or P < 0.05) with several traits including backfat thickness and visible intermuscular fat in Italian Duroc (ID) and growth performances in Italian Large White (ILW) and in ILW × Italian Landrace pigs. However, the effects estimated in the ILW were opposite to the effects reported in the ID pigs. Suggestive association (P < 0.10) was observed with muscle cathepsin B activity, opening, if confirmed, potential applications to reduce the excessive softness defect of the green hams that is of particular concern for the processing industry. The results obtained supported the need to further investigate the PCSK1 gene to fully exploit the value of its variability and apply this information in pig breeding programmes.