Proprotein Convertase Subtilisin/kexin Type 9 Concentrations Research Articles

135 RIP2 LINKS IMMUNE SIGNALING AND SUBENDOTHELIAL LIPID ACCUMULATION BY REGULATING TLR4-DEPENDENT LIPID UPTAKE IN MACROPHAGES

Gain-of-function mutations within proprotein convertase subtilisin kexin type 9 (PCSK9) are linked to familial autosomal dominant hypercholesterolaemia, a disease characterized by elevated plasma concentrations of cholesterol associated with low-density lipoproteins (LDLs). Conversely, PCSK9 loss-of-function mutations result in low levels of LDL cholesterol (LDLC) and protect against coronary heart disease. Although compelling evidence indicates that PCSK9 impairs the LDLR pathway, its role in cholesterol metabolism remains incompletely defined. In the past two years, several new biochemical findings, including the PCSK9 crystal structure and the identification of several transcriptional repressors, were reported. Moreover, new clinical and epidemiological data have revealed the correlation between plasma PCSK9 concentrations and LDLC levels.

Comparison of effects of bezafibrate and fenofibrate on circulating proprotein convertase subtilisin/kexin type 9 and adipocytokine levels in dyslipidemic subjects with impaired glucose tolerance or type 2 diabetes mellitus: Results from a crossover study

BackgroundBezafibrate and fenofibrate show different binding properties against peroxisome proliferator-activated receptor subtypes, which could cause different clinical effects on circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and on various metabolic markers. MethodsAn open, randomized, four-phased crossover study using 400mg of bezafibrate or 200mg of fenofibrate was performed. Study subjects were 14 dyslipidemia with impaired glucose tolerance or type 2 diabetes mellitus (61±16 years, body mass index (BMI) 26±3kg/m2, total cholesterol (TC) 219±53mg/dL, triglyceride (TG) 183±83mg/dL, high-density lipoprotein-cholesterol (HDL-C) 46±8mg/dL, fasting plasma glucose 133±31mg/dL and HbA1c 6.2±0.8%). Subjects were given either bezafibrate or fenofibrate for 8 weeks, discontinued for 4 weeks and then switched to the other fibrate for 8 weeks. Circulating PCSK9 levels and other metabolic parameters, including adiponectin, leptin and urine 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured at 0, 8, 12 and 20 weeks. ResultsPlasma PCSK9 concentrations were significantly increased (+39.7% for bezafibrate and +66.8% for fenofibrate, p<0.001) in all patients except for one subject when treated with bezafibrate. Both bezafibrate and fenofibrate caused reductions in TG (−38.3%, p<0.001 vs. −32.9%, p<0.01) and increases in HDL-C (+18.0%, p<0.001 vs. +11.7%, p<0.001). Fenofibrate significantly reduced serum cholesterol levels (TC, −11.2%, p<0.01; non-HDL-C, −17.3%, p<0.01; apolipoprotein B, −15.1%, p<0.01), whereas bezafibrate significantly improved glucose tolerance (insulin, −17.0%, p<0.05) and metabolic markers (γ-GTP, −38.9%, p<0.01; adiponectin, +15.4%, p<0.05; urine 8-OHdG/Cre, −9.5%, p<0.05). ConclusionBoth bezafibrate and fenofibrate increased plasma PCSK9 concentrations. The addition of a PCSK9 inhibitor to each fibrate therapy may achieve beneficial cholesterol lowering along with desirable effects of respective fibrates.

The Correlation between Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Insulin Resistance and the Components of Atherogenic Lipoprotein Phenotype in Males with Central Obesity

BACKGROUND: Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) promotes the degradation of LDL receptor in hepatocytes. in vitro studies have proven that insulin increases the expression of PCSK9. Insulin resistance, a common condition in central obesity is characterized by dyslipidemia called Atherogenic Lipoprotein Phenotype (ALP). This study aimed to investigate the correlation between insulin resistance (HOMA-IR) and PCSK9, and to analyze whether this condition is related to the development of ALP in central obesity.METHODS: This is an observational study with crosssectional design. The subjects consisted of 62 male adults with central obesity, aged 30-60 years old. ELISA was used to measure plasma PSCK9.RESULTS: The mean plasma PCSK9 concentration of the samples was 283.7 ng/mL. PCSK9 had positive linear correlation with HOMA-IR (r=0.225, p=0.045) and Apo B (r=0.245, p=0.055). After controlling of HOMA-IR, PCSK9 had positive linear correlation with triglycerides (r=0.352, p=0.045). In population with HOMA-IR &gt;2, crosstabs analysis showed that PCSK9 had significant correlation with triglycerides and Apo B with an odd ratio of 6,125 (r=0.376, p=0.037). Triglyceride showed significant negative correlation with HDL cholesterol and ratio of LDL/Apo B, but neither HOMA-IR nor PCSK9 did.CONCLUSION: In males with central obesity, PCSK9 is one of the factors mediating the occurence of ALP in insulin resistance.KEYWORDS: PCSK9, insulin resistance, atherogenic lipoprotein phenotype

Fasting reduces plasma proprotein convertase, subtilisin/kexin type 9 and cholesterol biosynthesis in humans

Proprotein convertase, subtilisin/kexin type 9 (PCSK9), a key regulator of plasma LDL-cholesterol (LDL-c) and cardiovascular risk, is produced in liver and secreted into plasma where it binds hepatic LDL receptors (LDLR), leading to their degradation. PCSK9 is transcriptionally activated by sterol response element-binding protein (SREBP)-2, a transcription factor that also activates all genes for cholesterol synthesis as well as the LDLR. Here we investigated the relationship between plasma PCSK9 levels and the lathosterol-to-cholesterol ratio, a marker of cholesterol biosynthesis, in 18 healthy subjects during a 48 h fast. In all individuals, plasma PCSK9 levels declined steadily during the fasting period, reaching a nadir at 36 h that was ∼58% lower than levels measured in the fed state (P < 0.001). Similarly, the lathosterol-to-cholesterol ratio declined in parallel with plasma PCSK9 concentrations during the fast, reaching a nadir at 36 h that was ∼28% lower than that measured in the fed state (P = 0.024). In summary, fasting has a marked effect on plasma PCSK9 concentrations, which is mirrored by measures of cholesterol synthesis in humans. Inasmuch as cholesterol synthesis and PCSK9 are both regulated by SREBP-2, these results suggest that plasma PCSK9 levels may serve as a surrogate marker of hepatic SREBP-2 activity in humans.

Serum PCSK9 is associated with multiple metabolic factors in a large Han Chinese population

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that regulates cholesterol metabolism through low-density lipoprotein receptor (LDLR) degradation. Gain-of-function and loss-of-function mutations within PCSK9 gene lead to hypercholesterolemia or hypocholesterolemia respectively. Studies in the U.S. and Canada reported a correlation between multiple metabolic factors and circulating PCSK9 concentrations. However, there is no data available on circulating PCSK9 levels in Chinese. A sandwich ELISA assay was applied to measure serum PCSK9 levels in a Chinese population of 2719 adults from Nanjing district, China, which represents a large and uniform ethnic population of Han Chinese. Serum PCSK9 levels ranged from 12.85 to 222.50 ng/ml with a mean concentration of 69.35 ng/ml in this population. Serum PCSK9 levels were slightly higher in women than in men. Compared to premenopausal women, postmenopausal women had significantly higher PCSK9 levels. Serum PCSK9 levels were correlated with multiple metabolic variables including age, BMI, total cholesterol, LDL cholesterol, triglycerides, fasting blood glucose, systolic blood pressure (SP) and diastolic blood pressure (DP) in this population. After stepwise regression analysis, there was a significant positive association between serum PCSK9 levels and total cholesterol, triglycerides and SP in men. In women, there was a positive correlation between PCSK9 levels and total cholesterol, age and DP. Our study indicates that the serum PCSK9 level may be a biomarker of metabolic status and cardiovascular disease.

Fenofibrate concomitantly decreases serum proprotein convertase subtilisin/kexin type 9 and very‐low‐density lipoprotein particle concentrations in statin‐treated type 2 diabetic patients

Diabetic dyslipidaemia, characterized by hypertriglyceridaemia as a result of elevated serum very-low-density lipoprotein (VLDL) concentrations, contributes to the increased risk of cardiovascular disease (CVD) in type 2 diabetes (T2DM). Proprotein convertase subtilisin/kexin type 9 (PCSK9) may play a role in regulating VLDL metabolism. We investigated the effect of fenofibrate on serum PCSK9 and VLDL particle concentrations in T2DM patients already receiving statin therapy. In a double-blind randomized crossover study, 15 statin-treated T2DM patients (63 +/- 8 years, body mass index (BMI) 29 +/- 3 kg/m(2)) were treated with fenofibrate (145 mg/day) or matching placebo for 12 weeks. Serum PCSK9 concentrations were measured by immunoassay. VLDL particle concentration and size were determined by nuclear magnetic resonance spectroscopy. Fenofibrate decreased serum triglycerides (-23%), VLDL-triglycerides (-51%), total cholesterol (-11%), LDL-cholesterol (-16%), apolipoprotein B-100 (-16%), apolipoprotein C-III (-20%) and PCSK9 (-13%) concentrations compared with placebo (p < 0.05). Fenofibrate also decreased serum concentrations of large (-45%), medium (-66%) and small VLDL (-67%) particles (p < 0.05), without altering VLDL particle size. Serum PCSK9 reduction correlated with decreases in total (r = 0.526, p = 0.044) and small (r = 0.629, p = 0.021) VLDL particle concentrations. Fenofibrate concomitantly decreased serum PCSK9 and VLDL particle concentrations in statin-treated T2DM patients. These findings support a mechanistic link between PCSK9 and VLDL metabolism, possibly through an effect of PSK9 on VLDL receptor degradation.

Beyond LDL Cholesterol, a New Role for PCSK9

Elevated low-density lipoprotein cholesterol (LDLC) levels in the plasma is the most important causative factor of atherosclerosis and associated ischemic cardiovascular diseases. The LDL receptor (LDLR) is the preferential pathway through which LDLs are cleared from the circulation. LDLs bound to the LDLR are internalized into clathrin-coated pits and subsequently undergo lysosomal degradation, whereas the LDLR is recycled back to the plasma membrane. See accompanying article on page 1333 Familial hypercholesterolemia (FH) is an autosomal dominant disorder associated with elevated LDL levels and premature coronary heart disease. FH is caused primarily by mutations of the LDLR or of apolipoprotein B100 (apoB100), the protein component of LDL that interacts with the LDLR. In 2003, “gain of function” mutations on a newly identified gene, proprotein convertase subtilisin/kexin type 9 ( PCSK9), were associated with FH. In 2005, a causative association was established between “loss of function” mutations in PCSK9 and low LDLC levels in 2% of the African-American population. The coronary heart disease risk in these individuals was reduced by 88%. As a result of these landmark studies (reviewed in Reference 1), PCSK9 became the subject of intensive research to discover the underlying mechanisms. PCSK9 is a serine protease mainly expressed in the liver and the intestine. It acts by reducing the amount of LDLR in hepatocytes. This was demonstrated in vitro and in mouse models …

Healthy Individuals Carrying the PCSK9 p.R46L Variant and Familial Hypercholesterolemia Patients Carrying PCSK9 p.D374Y Exhibit Lower Plasma Concentrations of PCSK9

We measured plasma PCSK9 concentrations in healthy men with a PCSK9 (proprotein convertase subtilisin/kexin type 9) loss-of-function variant (p.R46L), in statin-treated patients with a clinical diagnosis of familial hypercholesterolemia (FH) and carrying a PCSK9 gain-of-function mutation (p.D374Y), and in statin-treated patients with FH due to different genetic causes. PCSK9 was measured with a previously described ELISA. In 81 healthy middle-aged Caucasian men, the PCSK9 concentration was significantly associated with the concentrations of total cholesterol (r = 0.42; P < 0.0001), LDL cholesterol (r = 0.34; P = 0.01), and triglycerides (r = 0.25; P = 0.02). In p.R46L carriers, mean (SD) concentrations of PCSK9 were 15% lower than in RR individuals [65.5 microg/L (21.6 microg/L) vs 77.5 microg/L (18.2 microg/L); P = 0.03]. In patients with the p.D374Y variant (n = 7), the mean PCSK9 concentration was significantly lower than in the combined group of patients with an LDLR (low density lipoprotein receptor) mutation (n = 25), an APOB [apolipoprotein B (including Ag(x) antigen)] variant encoding p.R3527Q (n = 6), or no detectable mutation (n = 14) [96.4 microg/L (42.5 microg/L) vs 151.6 microg/L (69.6 microg/L); P = 0.02]. Two of the 14 patients with no mutation had PCSK9 concentrations below the mean for p.D374Y carriers; sequencing of the PCSK9 gene and promoter revealed no mutations. Among 409 FH patients, we identified 6 carriers of the promoter variant -287G>A (1.5%), a frequency similar to that (1.0%) previously reported for 2772 healthy men in the UK. In neither group was the -287G>A variant associated with differences in lipid traits. The loss-of-function p.R46L variant is associated with the expected lower concentrations of circulating PCSK9; the gain-of-function p.D374Y mutation is also associated with lower concentrations, presumably because of the higher affinity of this variant for the LDL receptor and its more rapid clearance. In treated FH patients, a low plasma PCSK9 concentration does not appear to be a useful screening tool for identifying novel PCSK9 mutations.

Plasma Proprotein Convertase Subtilisin/Kexin Type 9: A Marker of LDL Apolipoprotein B-100 Catabolism?

Experimental studies suggest that proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of LDL metabolism because of its ability to facilitate degradation of the LDL receptor. We investigated the association between plasma PCSK9 concentration and LDL apolipoprotein B-100 (apo B-100) metabolism in men with a wide range of body mass index values. We used GC-MS to study the kinetics of LDL apo B-100 after intravenous administration of deuterated leucine and analyzed the data by compartmental modeling. The plasma PCSK9 concentration was measured by ELISA. Univariate regression analysis revealed the plasma PCSK9 concentration to be significantly and positively correlated with cholesterol (r = 0.543; P = 0.011), LDL cholesterol (r = 0.543; P = 0.011), apo B-100 (r = 0.548; P = 0.010), and LDL apo B-100 concentrations (r = 0.514; P = 0.023), and inversely correlated with the LDL apo B-100 fractional catabolic rate (FCR) (r = -0.456; P = 0.038). The association between plasma PCSK9 concentration and the LDL apo B-100 FCR remained statistically significant after adjusting for age, obesity, plasma insulin, homeostasis model assessment score, and dietary energy; however, this association had borderline significance after adjusting for plasma lathosterol. In men, variation in plasma PCSK9 concentration influences the catabolism of LDL apo B-100. This finding appears to be independent of obesity, insulin resistance, energy intake, and age.

Plasma PCSK9 Is Associated with Age, Sex, and Multiple Metabolic Markers in a Population-Based Sample of Children and Adolescents

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein convertase that posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLR) in hepatocytes and increases plasma LDL cholesterol (LDL-C). Heterozygote gain-of-function mutations of PCSK9 are associated with the familial hypercholesterolemia phenotype, whereas loss-of-function variants are associated with reduced LDL-C concentrations and lower coronary risk. Plasma PCSK9 correlates with body mass index, triglyceridemia, total cholesterol, and LDL-C in adults, but no data are available in youth. We studied 1739 French Canadian youth ages 9, 13, and 16 years who participated in the Quebec Child and Adolescent Health and Social Survey, a province-wide school-based survey conducted in 1999. An ELISA assay was used to measure plasma PSCK9. The mean (SD) plasma PCSK9 concentration was 84.7 (24.7) microg/L in the sample. In boys, plasma PCSK9 decreased with age, whereas the inverse was true for girls. There were statistically significant positive associations between PCSK9 and fasting glucose, insulin, and HOMA-IR (homeostasis model assessment of insulin resistance). In multivariable analysis, a 10% higher fasting insulin was associated with a 1%-2% higher PCSK9 in both sexes. There were also positive associations between PCSK9 and total cholesterol, LDL-C, and triglycerides, as well as with HDL-C and apolipoproteins A1 and B. PCSK9 is associated with age, sex, and multiple metabolic markers in youth. A novel finding is that PCSK9 is associated with fasting insulinemia, which suggests that PCSK9 could play a role in the development of dyslipidemia associated with the metabolic syndrome. .

Plasma PCSK9 preferentially reduces liver LDL receptors in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-of-function mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 microg) to mice by a single injection reduced hepatic LDLRs by approximately 90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be approximately 5 min. Continuous infusion of PCSK9 (32 microg/h) into wild-type mice caused a approximately 90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo.

Plasma PCSK9 Concentrations Correlate with LDL and Total Cholesterol in Diabetic Patients and Are Decreased by Fenofibrate Treatment

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the LDL receptor (LDLr) in hepatocytes, and its expression in mouse liver has been shown to decrease with fenofibrate treatment. We developed a sandwich ELISA using recombinant human PCSK9 protein and 2 affinity-purified polyclonal antibodies directed against human PCSK9. We measured circulating PCSK9 concentrations in 115 diabetic patients from the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study before and after fenofibrate treatment. We found that plasma PCSK9 concentrations correlate with total (r = 0.45, P = 0.006) and LDL (r = 0.54, P = 0.001) cholesterol but not with triglycerides or HDL cholesterol concentrations in that cohort. After 6 weeks of treatment with comicronized fenofibrate (200 mg/day), plasma PCSK9 concentrations decreased by 8.5% (P = 0.041 vs pretreatment). This decrease correlated with the efficacy of fenofibrate, as judged by a parallel reduction in plasma triglycerides (r = 0.31, P = 0.015) and LDL cholesterol concentrations (r = 0.27, P = 0.048). We conclude that this decrease in PCSK9 explains at least in part the LDL cholesterol-lowering effects of fenofibrate. Fenofibrate might be of interest to further reduce cardiovascular risk in patients already treated with a statin.

GENDER DIFFERENCES IN RISK FACTOR CONTROL OF PATIENTS ON LIPID LOWERING DRUGS, A NATIONAL PRIMARY HEALTH CARE STUDY

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is highly expressed in the kidney, where its function remains unclear. In vitro data suggested that PCSK9 could impair the trafficking of the epithelial Na channel (ENaC). Here, we aimed at determining the consequences of PCSK9-deficiency on blood pressure, sodium balance and ENaC function in vivo in mice.Blood pressure was measured using non-invasive tail-cuff system or radiotelemetry under basal conditions in male and female PCSK9+/+ and PCSK9−/− mice, as well as in models of hypertension: l-NAME (2 mg/kg/day), angiotensin II (1 mg/kg/day) and deoxycorticosterone acetate (DOCA)-salt in male mice only. Plasma and urine electrolytes (Na+, K+, Cl−) were collected under basal conditions, after DOCA-salt and amiloride treatment. Renal expression of ENaC subunits was assessed by western blotting.PCSK9-deficiency did not alter both basal blood pressure and its increase in salt-insensitive (l-NAME) and salt-sensitive (Ang-II and DOCA-salt) hypertension models. Plasma PCSK9 concentrations were increased by 2.8 fold in DOCA-salt-induced hypertension. The relative expression of the cleaved, active, 30-kDa αENaC subunit was significantly increased by 32% in kidneys of PCSK9−/− mice under basal, but not under high-Na+ diet or DOCA-salt conditions. Amiloride increased urinary Na+ excretion to similar level in both genotypes, indicating that ENaC activity was not affected by PCSK9-deficiency.Despite an increase of cleaved αENaC under basal condition, PCSK9−/− mice display normal sodium balance and blood pressure regulation. Altogether, these data are reassuring regarding the development of PCSK9 inhibitors in hypercholesterolemia.

Statins Induce Plasma Levels of Proprotein Convertase Subtilisin/Kexin Type 9

LDL receptor (LDLR) number is regulated transcriptionally by sterol regulatory element‐binding protein (SREBP)‐2. Studies have shown that inhibitors of HMG‐CoA reductase (statins) used to treat hypercholesterolemia trigger a regulatory response in cells leading to elevated levels of active SREBP‐2, thereby increasing LDLR expression. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a SREBP‐2 regulated protein secreted by the liver, binds to the LDLR and mediates its degradation. Thus, induction of PCSK9 may counter‐balance the effect of statins on liver LDLR protein levels. We demonstrate that simvastatin‐fed rats exhibit elevated levels of nuclear SREBP‐2 in liver and increased transcription of SREBP‐2 target genes, including PCSK9. This was associated with a &gt;3‐fold increase in circulating PCSK9 levels compared to control rats. In human studies, 13 subjects with moderate to slightly elevated plasma LDL‐cholesterol levels (127 – 238 mg/dl) were administered a statin for 7 days (Lipitor ‐ 40 mg/day), which resulted in a 25% decrease in plasma cholesterol levels. ELISA measurements of circulating PCSK9 concentrations showed a mean increase of 35% in these subjects. These studies demonstrate that circulating PCSK9 levels are reflective of liver SREBP‐2 activity and the induction of PCSK9 by statins may partially offset their efficacy in lowering plasma LDL‐cholesterol levels. Supported by CIHR.

Serum Proprotein Convertase Subtilisin Kexin Type 9 Is Correlated Directly with Serum LDL Cholesterol

Proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining attention as a key regulator of serum LDL-cholesterol (LDLC). This novel serine protease causes the degradation of hepatic LDL receptors by an unknown mechanism. In humans, gain-of-function mutations in the PCSK9 gene cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDLC and decreased cardiovascular risk. Relatively little is known about PCSK9 in human serum. We used recombinant human PCSK9 protein and 2 different anti-PCSK9 monoclonal antibodies to build a sandwich ELISA. We measured PCSK9 and lipids in 55 human serum samples and correlated the results. We used the anti-PCSK9 antibodies to assay lipoprotein particle fractions separated by sequential flotation ultracentrifugation. Serum concentrations of PCSK9 ranged from 11 to 115 microg/L and were directly correlated with serum concentrations of LDLC (r = 0.45, P = 0.001) and total cholesterol (r = 0.50, P = 0.0003), but not with triglycerides (r = 0.15, P = 0.28) or HDL cholesterol concentrations (r = 0.13, P = 0.36). PCSK9 was not detectable in any lipoprotein particle fraction, including LDL. PCSK9 is present in human serum, likely not associated with specific lipoprotein particles. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations.