Differential effects of PCSK9 loss of function variants on serum lipid and PCSK9 levels in Caucasian and African Canadian populations.

OBJECTIVES: Variants of the secreted glycoprotein, proprotein convertase subtilisin/kexin 9 (PCSK9), associate with both hypo- and hyper-cholesterolemic phenotypes. Herein, we carried out full exonic sequencing of PCSK9 documenting the frequency of single and multiple PCSK9 variations and their effects on serum lipoprotein and PCSK9 levels in Caucasian Canadians. METHODS: The 12 exons of PCSK9 were sequenced in 207 unrelated Caucasian Canadians. Minor allele frequencies of PCSK9 variants were compared amongst LDL cholesterol (LDLC) quintiles. Serum PCSK9 levels were measured by ELISA and lipoproteins by enzymatic methods. Comparisons were made with a Caucasian family cohort (n=51) and first generation African Canadians (n=31). RESULTS: In Caucasians, but not African Canadians, the c.61_63insCTG (denoted L10Ins) and A53V PCSK9 variations were linked and their frequency was significantly higher among Caucasian Canadians with LDLC levels in the <25th percentile. In both the unrelated and family Caucasian cohorts those carrying the L10A53V PCSK9 variant had significantly lower LDLC without reduction in plasma PCSK9. The I474V PCSK9 variant associated with significantly lower serum PCSK9 and LDLC. A novel PCSK9 variant was identified; E206K. We found that the frequency of multiple PCSK9 variations was higher in first generation African Canadians. CONCLUSIONS: We showed that the L10A53V and I474V PCSK9 variants were significantly associated with lower LDLC levels in Caucasian Canadians but differed in their effect on serum PCSK9 concentrations, illuminating differences in their mechanism of inaction and indicating that that PCSK9 measurement alone may not always be a good indicator of PCSK9 function.Full exonic sequencing of PCSK9 pointed to factors that may contribute to L10Ins PCSK9 variant loss of function in Canadians of Caucasian but not those of African descent. These included; (1) its tight linkage with the A53V variant in Caucasians and/or (2) for both the L10 and I474V, the combined (and negating) effect of multiple, differing phenotypic PCSK9 variants within individuals of African ancestry for which combinations of PCSK9 variations and their overall frequency was higher. No population studies, to our knowledge, have addressed or accessed the effect of multiple PCSK9 variants on cholesterol profiles. Our results indicate that this should be considered.

Novel loss-of-function PCSK9 variant is associated with low plasma LDL cholesterol in a French-Canadian family and with impaired processing and secretion in cell culture.

BACKGROUND: PCSK9 (proprotein convertase subtilisin/kexin type 9) is a polymorphic gene whose protein product regulates plasma LDL cholesterol (LDLC) concentrations by shuttling liver LDL receptors (LDLRs) for degradation. PCSK9 variants that cause a gain or loss of PCSK9 function are associated with hyper- or hypocholesterolemia, which increases or reduces the risk of cardiovascular disease, respectively. We studied the clinical and molecular characteristics of a novel PCSK9 loss-of-function sequence variant in a white French-Canadian family. METHODS: In vivo plasma and ex vivo secreted PCSK9 concentrations were measured with a commercial ELISA. We sequenced the PCSK9 exons for 15 members of a family, the proband of which exhibited very low plasma PCSK9 and LDLC concentrations. We then conducted a structure/function analysis of the novel PCSK9 variant in cell culture to identify its phenotypic basis. RESULTS: We identified a PCSK9 sequence variant in the French-Canadian family that produced the PCSK9 Q152H substitution. Family members carrying this variant had mean decreases in circulating PCSK9 and LDLC concentrations of 79% and 48%, respectively, compared with unrelated noncarriers (n=210). In cell culture, the proPCSK9-Q152H variant did not undergo efficient autocatalytic cleavage and was not secreted. Cells transiently transfected with PCSK9-Q152H cDNA had LDLR concentrations that were significantly higher than those of cells overproducing wild-type PCSK9 (PCSK9-WT). Cotransfection of PCSK9-Q152H and PCSK9-WT cDNAs produced a 78% decrease in the secreted PCSK9-WT protein compared with control cells. CONCLUSIONS: Collectively, our results demonstrate that the PCSK9-Q152H variant markedly lowers plasma PCSK9 and LDLC concentrations in heterozygous carriers via decreased autocatalytic processing and secretion, and hence, inactivity on the LDLR.

Plasma PCSK9 levels are significantly modified by statins and fibrates in humans.

BACKGROUND: Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a secreted glycoprotein that is transcriptionally regulated by cholesterol status. It modulates levels of circulating low density lipoprotein cholesterol (LDLC) by negatively regulating low density lipoprotein receptor (LDLR) levels. PCSK9 variants that result in 'gain of function' have been linked to autosomal dominant hypercholesterolemia, while significant protection from coronary artery disease has been documented in individuals who carry 'loss of function' PCSK9 variants. PCSK9 circulates in human plasma, and we previously reported that plasma PCSK9 is positively correlated with total cholesterol and LDLC in men. RESULTS: Herein, we report the effects of two lipid-modulating therapies, namely statins and fibrates, on PCSK9 plasma levels in human subjects. We also document their effects on endogenous PCSK9 and LDLR expression in a human hepatocyte cell line, HepG2, using immunoprecipitation and immunoblot analyses. Changes in plasma PCSK9 following fenofibrate or gemfibrozil treatments (fibric acid derivatives) were inversely correlated with changes in LDLC levels (r = -0.558, p = 0.013). Atorvastatin administration (HMGCoA reductase inhibitor) significantly increased plasma PCSK9 (7.40%, p = 0.033) and these changes were inversely correlated with changes in LDLC levels (r = -0.393, p = 0.012). Immunoblot analyses of endogenous PCSK9 and LDLR expression by HepG2 cells in response to statins and fibrates showed that LDLR is more upregulated than PCSK9 by simvastatin (2.6x vs 1.5x, respectively at 10 muM), while fenofibrate did not induce changes in either. CONCLUSION: These results suggest that in vivo (1) statins directly increase PCSK9 expression while (2) fibrates affect PCSK9 expression indirectly through its modulation of cholesterol levels and (3) that these therapies could be improved by combination with a PCSK9 inhibitor, constituting a novel hypercholesterolemic therapy, since PCSK9 was significantly upregulated by both treatments.

The Effect of PCSK9 Loss-of-Function Variants on the Postprandial Lipid and ApoB-Lipoprotein Response.

Context: Proprotein convertase subtilisin kexin 9 (PCSK9) mediates degradation of the low-density lipoprotein receptor (LDLR), thereby increasing plasma low-density lipoprotein cholesterol (LDL-C). Variations in the PCSK9 gene associated with loss of function (LOF) of PCSK9 result in greater expression of hepatic LDLR, lower concentrations of LDL-C, and protection from cardiovascular disease (CVD). Apolipoprotein-B (apoB) remnants also contribute to CVD risk and are similarly cleared by the LDLR. We hypothesized that PCSK9-LOF carriers would have lower fasting and postprandial remnant lipoproteins on top of lower LDL-C. Objective: To compare fasting and postprandial concentrations of triglycerides (TGs), total apoB, and apoB48 as indicators of remnant lipoprotein metabolism in PCSK9-LOF carriers with those with no PCSK9 variants. Design: Case-control, metabolic study. Setting: Clinical Research Center of The Ottawa Hospital. Participants: Persons with one or more copies of the L10ins/A53V and/or I474V and/or R46L PCSK9 variant and persons with no PCSK9 variants. Intervention: Oral fat tolerance test. Main Outcomes Measures: Fasting and postprandial plasma TG, apoB48, total apoB, total cholesterol, and PCSK9 were measured at 0, 2, 4, and 6 hours after an oral fat load. Results: Participants with PCSK9-LOF variants (n = 22) had reduced fasting LDL-C (-14%) as well as lower fasting TG (-21%) compared with noncarrier controls (n = 23). LOF variants also had reduced postprandial total apoB (-17%), apoB48 (-23%), and TG (-18%). Postprandial PCSK9 declined in both groups (-24% vs -16%, respectively). Conclusions: Participants carrying PCSK9-LOF variants had attenuated levels of fasting and postprandial TG, apoB48, and total apoB. This may confer protection from CVD and further validate the use of PCSK9 inhibitors to lower CVD risk.

The anti-adipogenic effect of peripheral blood mononuclear cells is absent with PCSK9 loss-of-function variants.

OBJECTIVE: To determine the effect of (1) an oral fat load and (2) pro-protein convertase subtilisin/kexin type (PCSK) 9 loss-of-function (LOF) variant status on the ability of peripheral blood mononuclear cells (PBMC) to inhibit human adipogenesis. METHODS: PBMC from subjects with one or more PCSK9 LOF variants versus non-variant controls were compared in the fasting state and after an oral fat load. RESULTS: Fasting triglyceride (TG) levels were lower in the LOF variant versus non-variant group but rose to the same level after the oral fat load. Conditioned medium from PBMC was obtained in fasting (PBMC-CM-F) and 4-h postprandial (PBMC-CM-PP) states. PBMC-CM-PP from non-variant controls inhibited adipogenesis of human preadipocytes more than did PBMC-CM-F. In contrast, PBMC-CM-F or -PP from PCSK9 LOF variant subjects had no effect on adipogenesis. After the oral fat load, PBMC from PCSK9 LOF variant subjects showed significant increases in mRNA levels of interleukin-1ß, tumor necrosis factor-α, sterol regulatory element binding protein-1c, CD36, and monocyte chemoattractant protein-1 (MCP-1), only MCP-1 mRNA levels increased in PBMC from non-variant controls. CONCLUSIONS: The absence of anti-adipogenic action of PBMC from PCSK9 LOF variant subjects points to a novel role for PCSK9 in PBMC-adipose cell interactions.

Human Serum PCSK9 Is Elevated at Parturition in Comparison to Nonpregnant Subjects While Serum PCSK9 from Umbilical Cord Blood is Lower Compared to Maternal Blood.

Background. Serum lipids including total cholesterol (TC), triglycerides (TG), and low density lipoprotein cholesterol (LDL-C) are increased in pregnancy. Serum proprotein convertase subtilisin kexin 9 (PCSK9) is a significant player in lipoprotein metabolism. Circulating PCSK9 downregulates the LDL receptor on the surface of the liver, inhibiting clearance of LDL-C. Therefore, our study assessed serum PCSK9 concentrations at parturition (Maternal) compared to a nonpregnant (Control) cohort, as well as between mother and newborn (Maternal and Newborn). Methods. Blood was collected from women at parturition and from umbilical cords. Serum lipids and PCSK9 were measured and data were analysed for significance by Mann-Whitney U test at P < 0.05 and presented as median levels. Spearman's correlations were made at a 95% confidence interval. Results. Serum PCSK9 was significantly higher in Maternal versus Control cohorts (493.1 versus 289.7 ng/mL; P < 0.001, resp.), while the Newborn cohort was significantly lower than Maternal (278.2 versus 493.1 ng/mL; P < 0.0001, resp.). PCSK9 was significantly correlated with TC and HDL-C in Maternal and with TC, LDL-C, and HDL-C in Newborn cohorts. Conclusions. Our study provides the first quantitative report on PCSK9 in pregnancy (at parturition) and in umbilical cord blood. Further research will determine how these changes may affect lipoprotein levels during this physiological state.

Resistance exercise but not aerobic exercise lowers remnant-like lipoprotein particle cholesterol in type 2 diabetes: a randomized controlled trial.

The comparative effects of aerobic and resistance exercise on triglyceride-rich lipoproteins including remnant lipoproteins are controversial. This study examined exercise effect on remnant-like lipoprotein particle cholesterol (RLP-C) in type 2 diabetes. Participants were randomized to control (Control), aerobic (Aerobic), resistance (Resistance), or both (Combined) exercise groups. Baseline and 6-month fasting RLP-C and apolipoprotein B48 concentrations were measured. Data analysis was on an intention-to-treat basis. At 6 months, RLP-C was lower in all groups; ΔRLP-C mg/dl, (95% confidence interval), Control -3.91, (-6.21 to -1.6), p=0.001; Aerobic -3.89, (-6.41 to -1.36), p=0.003, Resistance -7.52, (-9.89 to -5.15), p=0.0001, Combined -7.50, (-9.87 to -5.13), p=0.0001. Total triglycerides were significantly lower in Resistance and Combined groups only; -17.7mg/dl (-32.8 to -2.7), p=0.02 and -27.5 (-42.5 to -11.5), p=0.001, respectively. Inter-group comparisons showed no difference in RLP-C change between Aerobic and Control and a significant difference in RLP-C change only where groups incorporating resistance exercise were compared with those without. There was no significant difference in RLP-C change between Resistance and Combined. Inter-group comparisons of total triglycerides change were significant only between Combined and Control. Changes in apolipoprotein B48 were not significant in inter-group comparisons. In conclusion, our data indicate that resistance exercise training, not aerobic, lowers RLP-C in type 2 diabetes. This effect was not revealed by changes in total triglycerides and apolipoprotein B48. The discordance between changes in RLP-C and apolipoprotein B48 in response to resistance exercise may indicate (a) a decrease in VLDL remnant and not chylomicron remnant particle number and/or (b) a depletion of cholesterol in chylomicron and/or VLDL remnants.

Plasma PCSK9 levels correlate with cholesterol in men but not in women.

Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a secreted glycoprotein that negatively regulates low density lipoprotein receptor (LDLR) levels. Several single nucleotide polymorphisms (SNPs) in PCSK9 have been linked to autosomal dominant hypercholesterolemia (ADH). Conversely, hypocholesterolemia associates with both 'loss of function' nonsense and missense SNPs in PCSK9. We examined the association of plasma PCSK9 with lipoprotein parameters in 182 normolipidemics. For men (n=98) plasma PCSK9 averaged 6.08+/-1.96 microg/ml and Spearman analysis revealed significant correlation between it and total cholesterol (TC), LDLC, and TC/high density lipoprotein (HDLC) (r=0.276, 0.282, and 0.228, respectively). For women (n=84) plasma PCSK9 averaged 6.46+/-1.99 microg/ml having no correlation with TC, LDLC or TC/HDLC. The ratio of plasma PCSK9/LDLC increased in men carrying 'loss of function' PCSK9 variations. Our results suggest a gender difference in PCSK9 regulation and function with PCSK9 correlated to TC and LDLC in men but not women.