Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein (LDL) cholesterol (LDL-C) metabolism by targeting LDL receptors for degradation. Statins increase serum PCSK9 concentration limiting the potential of statins to reduce LDL-C, whereas ezetimibe, inhibitor of cholesterol absorption, has ambiguous effects on circulating PCSK9 levels. Plant stanols also reduce cholesterol absorption, but their effect on serum PCSK9 concentration is not known. Therefore, we performed a controlled, randomized, double-blind study, in which 92 normo- to moderately hypercholesterolaemic subjects (35 males and 57 females) consumed vegetable-oil spread 20 g/day enriched (plant stanol group, n=46) or not (control group, n=46) with plant stanols 3 g/day as ester for 6 months. Fasting blood samples were drawn at baseline and at the end of the study. Serum PCSK9 concentration was analysed with Quantikine Elisa Immunoassay, serum and lipoprotein lipids enzymatically and serum non-cholesterol sterols with GLC. At baseline, PCSK9 concentration varied from 91 to 716 ng/ml with a mean value of 278±11 (S.E.M.) ng/ml with no gender difference. It correlated with serum and LDL-C, serum triglycerides, age, body mass index (BMI) and plasma glucose concentration, but not with variables of cholesterol metabolism when adjusted to serum cholesterol. Plant stanols reduced LDL-C by 10% from controls (P<0.05), but PCSK9 levels were unchanged and did not differ between the groups. In conclusion, the present study demonstrated for the first time that inhibition of cholesterol absorption with plant stanol esters did not affect serum PCSK9 concentration. Thus, plant stanol esters provide an efficient dietary means to lower LDL-C without interfering with the PCSK9 metabolism and in this regard the LDL receptor-mediated cellular cholesterol uptake and removal.

INTRODUCTION

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism mainly because it binds to low-density lipoprotein (LDL) receptors and promotes their degradation [1]. The important observations related to gain-of-function and loss-of-function mutations in PCSK9 revealed the essential role of PCSK9 as a regulator of circulating LDL cholesterol (LDL-C) levels. The gain-of-function mutations cause severe hypercholesterolaemia and premature coronary artery disease (CAD) [2]. On the other hand, subjects with the loss-of-function mutations have reduced LDL-C concentrations and are protected from CAD, attesting to the importance of the LDL-C, atherosclerosis interaction [3,4]. This mechanism of action of circulating PCSK9 has initiated the development of anti-PCSK9 monoclonal antibodies, which effectively block the PCSK9–LDL receptor interaction at the cell surface and lower LDL-C levels up to 70% [5]. Plasma PCSK9 concentration, having a wide range of ~100-fold in a population sample of over 3000 subjects [6], correlates in several studies with serum and LDL-C and serum triglyceride concentrations, with fasting glucose levels, age, body mass index (BMI) and even with blood pressure in some studies [69]. Statins activate the cholesterol-responsive transcription factor sterol response element-binding protein 2 (SREBP-2) in the liver, which results in up-regulation of PCSK9, but also up-regulation of the LDL receptor and de novo cholesterol synthesis [1,10,11]. Consequently, higher plasma PCSK9 levels have been observed during statin treatment [12]. This elevation attenuates the statin-induced increase in the number of LDL receptors, which may explain why the most effective LDL-C lowering effect occurs with the first dose of statin. Thus, further increases in the dose reduce the LDL-C levels by only ~6% [13].

Regarding other hypocholesterolaemic drugs, reduction in cholesterol absorption with ezetimibe has revealed inconsistent results on the circulating PCSK9 concentrations; ezetimibe either increases [9] or has no effect on PCSK9 concentration [14,15]. Consuming ~2 g/day phytosterols also disrupts cholesterol absorption and reduces LDL-C concentration by ~10% [16]. There is no information however on whether phytosterols have any effect on the circulating PCSK9 levels. To this end, the aim of the present study was to evaluate the effect of 6-month plant stanol ester consumption on serum PCSK9 concentration in a randomized, controlled, double-blind clinical intervention.

MATERIALS AND METHODS

Study population

Ninety-four volunteers were screened and recruited into this intervention called BLOOD FLOW by advertisements on the walls or in the intranet of four large companies employing mainly office workers and in two research institutes in Helsinki, Finland. The detailed design, methods, baseline data [17] and the results of the intervention on serum lipids, arterial stiffness and endothelial function [18] have recently been published. In short, no inclusion or exclusion criteria for serum and lipoprotein lipids were set, but lipid-lowering medication or the consumption of nutrient supplements interfering with serum cholesterol level (red rice or berberine) were included as exclusion criteria. If the subjects had used plant sterol/stanol products, they could be included in the study after 3 weeks’ wash out period. Other exclusion criteria were gravidity or breast feeding, unstable CAD or coronary bypass or angioplasty <6 months, inflammatory bowel disease, alcohol consumption >45 g absolute alcohol/day or abnormal liver, kidney or thyroid function. Possible medication should have remained unchanged for 1 month before the study and, if possible, during the study.

All subjects gave their written informed consent. The study was performed according to the principles of the Declaration of Helsinki. The Ethics Committee of the Department of Medicine, Hospital District of Helsinki and Uusimaa approved the study protocol (ClinicalTrials.govRegister # NCT00738933).

Study design

The study was a randomized, placebo-controlled, double-blind and parallel clinical intervention lasting for 6 months [17,18]. The subjects were randomly divided into plant stanol and control groups. They were advised to continue their habitual diet otherwise unchanged but to replace 20 g/day of their regular spread intake with vegetable oil-based test spread during regular meals. In the plant stanol group, the test spread contained 3 g of plant stanols/day provided as their fatty acid esters, whereas in the control group the same test spread was consumed without added plant stanols. The test products were provided by Raisio Nutrition Ltd. The diet was monitored by 3-day food record kept at baseline and at the end of the study [17,18].

The subjects visited the research centre four times: at baseline (visit 1, randomization) and after 2 (visit 2), 4 (visit 3) and 6 months (visit 4, end of the study). At visits 1 and 4, fasting blood samples were drawn after 12-h-fast at 7:00–9:00 hours. In the present study, we report the results of serum PCSK9, lipids and serum non-cholesterol sterols at baseline and at the end of the intervention.

Methods and measurements

Routine laboratory measurements were analysed with standardized methods at the Central Laboratory of Helsinki University Hospital (HUSLAB). Serum total, LDL-C and HDL (high-density lipoprotein) cholesterol and serum triglycerides were analysed enzymatically using automated analysers (HUSLAB).

Serum PCSK9 concentration was analysed with Quantikine® Elisa Immunoassay using a monoclonal antibody specific for human PCSK9 (www.rndsystems.com/pdf/DPC900.pdf). According to the manufacturer, the intra- and inter-assay CV%s vary from 4.1% to 6.5% and a range for serum values in a random population sample was 177–460 ng/ml, with a mean ± S.D. value of 313±71 ng/ml.

Serum cholesterol, cholesterol precursors (squalene, cholestenol, desmosterol and lathosterol), plant sterols (sitosterol and campesterol), plant stanols (campestanol and sitostanol) and cholestanol were quantified by capillary GLC with a 50-m Ultra 2 capillary column as described previously [19]. Serum concentrations of non-cholesterol sterols were expressed in μmol/l and also as the ratio of 100× μmol/mmol of cholesterol (called ratio in the text) by adjusting the concentrations with the cholesterol value in the same GLC run. Ratios of serum cholesterol precursors to cholesterol reflect whole-body cholesterol synthesis, whereas the ratios of plant sterols and cholestanol reflect cholesterol absorption efficiency [2022]. We also calculated the lathosterol/campesterol ratio, which reflects whole-body cholesterol metabolism.

Statistical analyses

Statistical analyses were performed with SPSS for Windows 19.0 statistics program (SPSS). Sample size calculation was based on the only clinical trial in which serum PCSK9 concentration changed during absorption inhibition (ezetimibe) with significance level α=0.05 and β=0.20. Using these estimates, the required minimal sample size is 70. Normality and homogeneity of variance assumptions were checked before further analyses. Univariate ANOVA was used to compare the baseline values and the changes between groups. ANOVA for repeated measurements (GLM) was used to describe the interaction of time and group, effects of gender and changes over time in between-group comparisons followed by post hoc comparisons with Bonferroni corrections. Spearman correlation coefficients were calculated. Variables not normally distributed even after logarithmic transformation, non-homogenous in variance or non-continuous were tested with Mann–Whitney U-test and Fisher exact test. A P-value of <0.05 was considered statistically significant. The results are given as mean ± S.E.M.

RESULTS

Baseline

Shortly after randomization, two subjects dropped out of the study, one because of personal reasons not related to the study (plant stanol group) and the other because of gastric distress (control group). The baseline characteristics of the 92 subjects completing the study are shown in Table 1 and the baseline lipid characteristics in Table 2. In the total study population, LDL-C varied from 1.5 to 4.5 mmol/l. Twenty-six subjects (28%) had normal LDL-C (≤3.0 mmol/l) values and three had elevated serum triglycerides (≥2.0 mmol/l). There were no significant differences in any of the clinical variables, prevalence of diseases, medications or nutrient intakes regarding cholesterol, fats, proteins, carbohydrates and total fibre [17,18] between the groups. All subjects with a history of hypothyreosis were on thyroxin and were euthyreoid.

Table 1
Baseline characteristics of the study population
VariablesControl groupPlant stanol groupP
n (M/F) 46(14/32) 46(21/25) 0.197 
Age (years) 50.6±1.4 50.9±1.4 0.676 
Weight (kg) 72.4±2.0 76.6±2.2 0.515 
BMI (kg/m225.0±0.5 25.4±0.6 0.915 
SBP (mmHg) 120±2 125±2 0.150 
DBP (mmHg) 75±1 76±1 0.542 
Plasma glucose (mmol/l) 4.87±0.08 4.97±0.08 0.552 
Serum creatinine (mmol/l) 74.1±1.7 75.5±1.4 0.765 
ALT (units) 23.7±1.6 25.9±2.0 0.673 
TSH (units) 1.74±0.13 1.75±0.10 0.618 
hs-CRP (mg/l) 1.08±0.14 1.01±0.12 0.803 
Diseases (n   
Hypertension 0.574 
Type 2 diabetes 
Hypothyreosis 
Medication (n   
Calcium channel blockers 
β-Blockers 0.495 
Diuretics 
ACE- or ATR blockers 
Thyroxin 
Contraceptives or HRT 11 0.623 
VariablesControl groupPlant stanol groupP
n (M/F) 46(14/32) 46(21/25) 0.197 
Age (years) 50.6±1.4 50.9±1.4 0.676 
Weight (kg) 72.4±2.0 76.6±2.2 0.515 
BMI (kg/m225.0±0.5 25.4±0.6 0.915 
SBP (mmHg) 120±2 125±2 0.150 
DBP (mmHg) 75±1 76±1 0.542 
Plasma glucose (mmol/l) 4.87±0.08 4.97±0.08 0.552 
Serum creatinine (mmol/l) 74.1±1.7 75.5±1.4 0.765 
ALT (units) 23.7±1.6 25.9±2.0 0.673 
TSH (units) 1.74±0.13 1.75±0.10 0.618 
hs-CRP (mg/l) 1.08±0.14 1.01±0.12 0.803 
Diseases (n   
Hypertension 0.574 
Type 2 diabetes 
Hypothyreosis 
Medication (n   
Calcium channel blockers 
β-Blockers 0.495 
Diuretics 
ACE- or ATR blockers 
Thyroxin 
Contraceptives or HRT 11 0.623 

Mean ± S.E.M.

Abbreviations: SBP=systolic blood pressure; DBP=diastolic blood pressure; ALT=alanine aminotransferase; TSH=thyroid-stimulating hormone; ACE=angiotensin converting enzyme; ATR=angiotensin receptor; HRT=hormone replacement therapy

Table 2
Serum PCSK9 and serum and lipoprotein lipid concentrations and serum non-cholesterol sterol to cholesterol ratios at baseline and after 6-month intervention in the control and plant stanol ester groups
Control group (n=46)Plant stanol group (n=46)
VariablesBaselineInterventionP*BaselineInterventionP*PP
PCSK9 (ng/ml) 272.3±15.9 309.0±15.7  283.6±15.6 293.1±14.6   0.129 
Serum cholesterol§ 5.57±0.14 5.73±0.15 0.045 5.48±0.12 5.28±0.11 0.009 0.050 <0.001 
LDL cholesterol§ 3.54±0.14 3.60±0.15 0.413 3.52±0.12 3.23±0.12 0.000 0.045 <0.001 
HDL cholesterol§ 1.79±0.07 1.88±0.07  1.76±0.07 1.85±0.08   0.986 
Serum triglycerides§ 0.96±0.07 1.05±0.07  0.89±0.06 0.98±0.07   0.905 
Squalene 12.4±0.6 14.5±0.9  13.1±0.6 14.9±0.8   0.992 
Cholestenol 16.5±0.7 16.8±0.9 0.592 17.2±1.0 19.8±1.0 <0.001 0.026 0.001 
Desmosterol 79.8±2.8 78.3±2.0 0.556 78.6±2.7 87.0±2.5 <0.001 0.007 <0.001 
Lathosterol 113.1±5.6 114.4±4.9 0.758 119.9±7.4 141.9±6.8 <0.001 0.001 <0.001 
Cholestanol 155.3±4.8 152.2±3.9  151.9±4.0 144.8±3.4   0.066 
Campesterol 267.5±14.7 300.6±15.8 0.005 255.8±15.1 181.7±8.4 <0.001 <0.001 <0.001 
Sitosterol 148.8±8.9 150.5±8.6 0.661 138.7±9.2 94.1±4.8 <0.001 <0.001 <0.001 
Sitostanol 8.5±0.3 8.0±0.2 0.216 7.8±0.2 15.5±0.6 <0.001 <0.001 <0.001 
Control group (n=46)Plant stanol group (n=46)
VariablesBaselineInterventionP*BaselineInterventionP*PP
PCSK9 (ng/ml) 272.3±15.9 309.0±15.7  283.6±15.6 293.1±14.6   0.129 
Serum cholesterol§ 5.57±0.14 5.73±0.15 0.045 5.48±0.12 5.28±0.11 0.009 0.050 <0.001 
LDL cholesterol§ 3.54±0.14 3.60±0.15 0.413 3.52±0.12 3.23±0.12 0.000 0.045 <0.001 
HDL cholesterol§ 1.79±0.07 1.88±0.07  1.76±0.07 1.85±0.08   0.986 
Serum triglycerides§ 0.96±0.07 1.05±0.07  0.89±0.06 0.98±0.07   0.905 
Squalene 12.4±0.6 14.5±0.9  13.1±0.6 14.9±0.8   0.992 
Cholestenol 16.5±0.7 16.8±0.9 0.592 17.2±1.0 19.8±1.0 <0.001 0.026 0.001 
Desmosterol 79.8±2.8 78.3±2.0 0.556 78.6±2.7 87.0±2.5 <0.001 0.007 <0.001 
Lathosterol 113.1±5.6 114.4±4.9 0.758 119.9±7.4 141.9±6.8 <0.001 0.001 <0.001 
Cholestanol 155.3±4.8 152.2±3.9  151.9±4.0 144.8±3.4   0.066 
Campesterol 267.5±14.7 300.6±15.8 0.005 255.8±15.1 181.7±8.4 <0.001 <0.001 <0.001 
Sitosterol 148.8±8.9 150.5±8.6 0.661 138.7±9.2 94.1±4.8 <0.001 <0.001 <0.001 
Sitostanol 8.5±0.3 8.0±0.2 0.216 7.8±0.2 15.5±0.6 <0.001 <0.001 <0.001 

Mean ± S.E.M.

P Group by time interaction [repeated measures of variance (general linear model)]

*P from baseline

P from controls

§

mmol/l

100× μmol/mmol of cholesterol

P<0.05 change over time

Serum PCSK9 concentrations were similar between the study groups at baseline (Table 2). In the whole study population, serum PCSK9 concentration varied from 91 to 716 ng/ml with no significant difference between men and women (253.0±15.5 compared with 293.3±14.9 ng/ml, P=0.073). The different disease groups or medications were not related to serum PCSK9 concentration.

Serum lipids and non-cholesterol sterols did not differ between the study groups or between genders, different diseases or medications at baseline. Serum non-cholesterol sterols are shown as ratios to cholesterol in Table 2, but the results were similar when the non-cholesterol sterols were expressed as absolute concentrations (result not shown).

In the whole study population, serum cholesterol precursors correlated with the serum absorption markers (e.g., lathosterol compared with sitosterol, r=−0.606, P=0.008) suggesting that cholesterol homoeostasis was intact. All cholesterol precursors were interrelated [r-values ranged from 0.515 (squalene-cholestenol) to 0.948 (cholestenol-lathosterol); P<0.001 for all]. Similarly, all the absorption markers were interrelated [r-values ranged from 0.769 (cholestanol-campesterol) to 0.941 (campesterol-sitosterol); P<0.001 for all].

Serum PCSK9 concentration correlated with serum and LDL-C concentrations (Table 3). It also correlated with serum triglycerides, age, BMI and plasma glucose concentration, but except for serum and LDL-C and BMI, the other correlation coefficients were modest albeit significant. Serum PCSK9 concentration did not correlate with HDL cholesterol, hs-CRP (high sensitive C-reactive protein) or blood pressure. Regarding cholesterol metabolism, serum PCSK9 level correlated with the concentrations of all cholesterol synthesis markers shown for lathosterol in Table 3. However, all significant correlations were lost when the markers were adjusted to cholesterol. There was no correlation between PCSK9 and cholesterol absorption markers or lathosterol/campesterol ratio.

Table 3
Correlation coefficients at baseline between serum PCSK9 concentration and clinical characteristics, lipids and non-cholesterol sterols in the study population (n=92)
VariablesPCSK9 (ng/ml)
Age (years) 0.230* 
BMI (kg/m20.337* 
Systolic blood pressure (mmHg) 0.039 
Diastolic blood pressure (mmHg) 0.048 
Plasma glucose (mmol/l) 0.230* 
hs-CRP (mmol/l) 0.071 
Serum cholesterol (mmol/l) 0.338 
LDL cholesterol (mmol/l) 0.319 
HDL cholesterol (mmol/l) −0.016 
Serum triglycerides (mmol/l) 0.293* 
Serum marker of cholesterol synthesis  
Lathosterol (μmol/l) 0.269 
Lathosterol (100× μmol/mmol of cholesterol) 0.128 
Serum markers of cholesterol absorption  
Sitosterol (μmol/l) 0.144 
Sitosterol (100× μmol/mmol of cholesterol) −0.009 
Campesterol (μmol/l) 0.076 
Campesterol (100× μmol/mmol of cholesterol) −0.097 
Serum marker of cholesterol metabolism  
Lathosterol/campesterol 0.128 
VariablesPCSK9 (ng/ml)
Age (years) 0.230* 
BMI (kg/m20.337* 
Systolic blood pressure (mmHg) 0.039 
Diastolic blood pressure (mmHg) 0.048 
Plasma glucose (mmol/l) 0.230* 
hs-CRP (mmol/l) 0.071 
Serum cholesterol (mmol/l) 0.338 
LDL cholesterol (mmol/l) 0.319 
HDL cholesterol (mmol/l) −0.016 
Serum triglycerides (mmol/l) 0.293* 
Serum marker of cholesterol synthesis  
Lathosterol (μmol/l) 0.269 
Lathosterol (100× μmol/mmol of cholesterol) 0.128 
Serum markers of cholesterol absorption  
Sitosterol (μmol/l) 0.144 
Sitosterol (100× μmol/mmol of cholesterol) −0.009 
Campesterol (μmol/l) 0.076 
Campesterol (100× μmol/mmol of cholesterol) −0.097 
Serum marker of cholesterol metabolism  
Lathosterol/campesterol 0.128 

*P<0.05; P<0.01

Intervention

During the intervention, no side effects were reported. The nutrient intakes did not differ between the groups [18]. The intake of mono-unsaturated fatty acids increased and the intake of protein decreased similarly in both groups. The absolute PCSK9 and lipid intervention values are shown in Table 2 and percentage changes for selected variables are shown in Figure 1. In the plant stanol group, serum and LDL-C levels were reduced by 7% and 10% compared with the control group (P<0.05 for both, Figure 1 shown for LDL-C). HDL cholesterol and serum triglycerides were unchanged in both groups.

Changes after the intervention

Figure 1
Changes after the intervention

Percent changes in LDL-C and serum PCSK9 (PCSK9) concentrations, serum lathosterol to cholesterol ratio (Lathosterol/C) and serum sitosterol to cholesterol ratio (Sitosterol/C) in subjects consuming control (n=46) and plant stanol ester (n=46) spread for 6 months. *P<0.05 from baseline and from controls.

Figure 1
Changes after the intervention

Percent changes in LDL-C and serum PCSK9 (PCSK9) concentrations, serum lathosterol to cholesterol ratio (Lathosterol/C) and serum sitosterol to cholesterol ratio (Sitosterol/C) in subjects consuming control (n=46) and plant stanol ester (n=46) spread for 6 months. *P<0.05 from baseline and from controls.

Serum PCSK9 concentration was not significantly changed in either of the groups and did not differ between the groups at the end of the intervention (Table 2, Figure 1). Individual changes of PCSK9 from baseline in both groups are illustrated in Figure 2.

Changes in serum PCSK9 concentrations

Figure 2
Changes in serum PCSK9 concentrations

Changes in serum PCSK9 concentrations in the control (n=46) and plant stanol ester (n=46) groups after the 6-month intervention. Individual values.

Figure 2
Changes in serum PCSK9 concentrations

Changes in serum PCSK9 concentrations in the control (n=46) and plant stanol ester (n=46) groups after the 6-month intervention. Individual values.

In the plant stanol group, cholestenol, desmosterol and lathosterol ratios to cholesterol were increased by 12%–29% compared with the control group or from the baseline values (P<0.01 for all, Table 2, Figure 1 shown for lathosterol). Plant sterol ratios to cholesterol decreased by 20%–40% compared with the control group or by 15%–28% from the baseline values (P<0.001 for all). Serum sitostanol increased by 96.0±8.7% from baseline (P<0.001). The increased serum sitostanol and the decreased plant sterol levels indicated good compliance. Serum campestanol was undetectable.

In the control group, the only change in the non-cholesterol sterols was the significant increase in serum campesterol to cholesterol ratio from baseline probably resulting from the rapeseed oil-containing test spread (Table 2). In addition, there was a significant change over time in serum squalene and cholestanol ratios to cholesterol in the whole study population.

At the end of the intervention, in the plant stanol group, serum PCSK9 concentration correlated with serum and LDL cholesterol concentrations (r=0.342 and r=340, P<0.05 for both). In the control group, the respective associations did not reach significance (e.g., PCSK9-serum cholesterol r=0.233, P=0.170). Serum PCSK9 did not correlate with the non-cholesterol sterols in either of the groups.

The change in serum PCSK9 concentration associated inversely with its baseline value in both groups (plant stanol group: r=−0.473, P=0.002; control group: r=−0.360, P=0.028). The change in serum PCSK9 concentration associated also with the changes in serum squalene and desmosterol ratios to cholesterol in both groups (e.g., squalene to cholesterol ratio, plant stanol group, r=0.417, P<0.001 and control group r=0.379, P<0.01).

DISCUSSION

The main novel finding in this controlled 6-month intervention was that the reduction in cholesterol absorption with plant stanol esters did not affect the circulating PCSK9 concentration in adult normo-to moderately hypercholesterolaemic subjects. This result was observed in a setting in which the reduction in LDL-C concentration was as expected according to earlier studies [16]. Accordingly, LDL-C concentration was reduced by 10% compared with the control group and serum plant sterols were reduced up to 40% from the control values indicating reduced cholesterol absorption [22]. Cholesterol synthesis was up-regulated as demonstrated by the increase in all cholesterol precursor sterols. In addition, at baseline there was nothing unexpected related to the serum PCSK9 concentration and its correlation with the demographic and metabolic variables; the serum levels were within the limits reported earlier [7] and the correlations between serum PCSK9 concentration and variables including age, BMI, serum and LDL-C and serum triglyceride concentrations and plasma glucose levels were similar to those described in earlier studies [69]. The changes in circulating PCSK9 level were related with the baseline value as described before [15]. The lack of gender difference in serum PCSK9 concentration in the present study has also been observed before [9]. The study population (n=92) was large enough to address the objectives of the study.

To the best of our knowledge, previous interventions evaluating the behaviour of circulating PCSK9 concentration during absorption inhibition of cholesterol with plant stanols or sterols have not been published. Thus, the finding that plant stanol esters did not change serum PCSK9 concentration has to be compared with earlier results obtained with ezetimibe. Like in our study, the circulating PCSK9 levels were unchanged in two out of three ezetimibe studies [14,15], whereas the circulating PCSK9 level was increased in the third study [9]. In the study by Lakoski et al. [14], ezetimibe 10 mg/day, simvastatin 10 mg/day or their combination did not change the median PCSK9 levels in 215 men with mild to moderate hypercholesterolaemia in spite of significant LDL-C lowering by 19% (ezetimibe), 25% (simvastatin) and 41% (their combination). In the study by Berthold et al. [15], 24 healthy male subjects consumed ezetimibe 10 mg/day, simvastatin 40 mg/day or their combination. Ezetimibe decreased LDL-C by 22%, simvastatin by 41% and the combination by 60% and all reductions were significant. During ezetimibe, the circulating PSCK9 concentration was unchanged, significantly increased by 68% during simvastatin, but was unchanged when ezetimibe was added with simvastatin. On the other hand, in the study by Dubuc et al. [9], the plasma PCSK9 concentration increased significantly by ~20% when adding ezetimibe to statin-treated hypercholesterolaemic patients. In an in vitro study by the same group, ezetimibe had no effect on PCSK9 protein secretion by HepG2 or Caco-2 cells [23]. In an animal study, 3-day ezetimibe consumption significantly increased control-related plasma PCSK9 concentration in rats before any lipid changes could be observed [24]. On the whole, the reason for these contradictory results extending from in vitro studies to clinical interventions remains open.

The reduction in cholesterol absorption depletes the hepatic cholesterol stores and up-regulates cholesterol synthesis [22,25], observed also in the present study. Cholesterol synthesis is up-regulated by the activation of SREBP-2, which, when activated, also concomitantly up-regulates the expressions of PCSK9 and LDL receptors [1,10,11]. Accordingly, the increased cholesterol synthesis indicated that plant stanol consumption activated SREPB-2 but did not increase the circulating levels of PCSK9. It was recently demonstrated that high intake of linoleic acid reduced the circulating PCSK9 concentration, which was assumed to reduce LDL-C level [26]. In the present study however, the intake of polyunsaturated fatty acids including linoleic acid were similar between the plant stanol and control groups and did not change during the intervention, so that dietary changes could not explain the unchangeable serum PCSK9 concentration in the present intervention. Regarding the possible LDL receptor activation during plant stanol consumption, in two kinetic studies plant stanols did not affect the fractional catabolic rate of LDL apoprotein B100 in spite of significantly reduced LDL-C and very-low-density lipoprotein (VLDL) cholesterol concentrations [27,28]. Instead, the production rate of LDL apoprotein B100 was reduced. Thus, it is possible that the LDL receptor was activated and removed preferably VLDL instead of LDL particles in these study populations consisting of patients with type 2 diabetes [27,28]. In a plant sterol study, there were no changes in the kinetic variables of apoprotein B100 in lipoproteins VLDL, intermediate-density lipoprotein (IDL) and LDL, but actually there were no changes in the lipoprotein cholesterol levels either so that the kinetic results were expected [29]. Consequently, serum PCSK9 concentration may be the only downstream variable not markedly changed by SREPB-2 activation during plant stanol consumption. One possible reason might arise from the weak correlation between the circulating PCSK9 and LDL-C concentrations; it was suggested that the circulating PCSK9 level reflects only part of the total activity of PCSK9 [6]. Alternatively, in vitro studies in HepG2 cells revealed that when the cells were dose-dependently deprived of cholesterol by increasing doses of statin, the PCSK9 and LDL receptor expressions increased in a dose–response manner according to the severity of cholesterol depletion [30]. On the other hand, mevalonate added to the media reversed the up-regulation of the PCSK9 and LDL receptor genes. PCSK9 responded more sensitively than the LDL receptors to the addition of mevalonate, so that the authors concluded that PCSK9 is more tightly regulated by cholesterol than the LDLR gene [30]. These results might suggest that during plant stanol consumption, the synthesized cholesterol precursors prevented the up-regulation of PCSK9. At the same time, being not so sensitive to the increased concentrations of cholesterol precursors, the LDL receptor was, in fact, activated.

The question whether serum PCSK9 concentration correlates with cholesterol metabolism seems to be complicated. In a large cohort including both patients with familial combined hyperlipidaemia and their normolipidaemic relatives, plasma PCSK9 concentration and lathosterol were interrelated [31]. In the present study, the absolute concentrations of cholesterol precursors correlated with serum PCSK9 levels at baseline, but the significances were lost when the precursors were adjusted to cholesterol. Obviously, these correlations reflected only the interplay between PCSK9 and serum cholesterol and there was no real association between serum PCSK9 concentrations and markers of cholesterol synthesis. However, during intervention the change in serum PCSK9 concentration was related to the changes in squalene and desmosterol ratios to cholesterol in both control and plant stanol groups. These findings suggest that there is a positive functional interplay between serum PCSK9 concentration and markers of cholesterol synthesis; if cholesterol synthesis is changed, serum PCSK9 concentration is also changed in the same direction. In fact, during a 48 h fast, Browning et al. [32] demonstrated a steady decline in plasma PCSK9 levels, which were mirrored by lathosterol to cholesterol ratio. However, the correlation between lathosterol to cholesterol ratio and plasma PCSK9 level was not significant. The authors concluded that a given concentration of circulating PCSK9 does not seem to predict the absolute rate of cholesterol synthesis [32].

Regarding the clinical perspectives, the present study demonstrated for the first time that reduction in cholesterol absorption with plant stanol esters did not affect serum PCSK9 concentration in this 6-month controlled, randomized intervention in normo-to moderately hypercholesterolaemic subjects. Thus, plant stanol esters provide an efficient dietary means to lower LDL-C without interfering with PCSK9 metabolism and in this regard the LDL receptor-mediated cellular cholesterol uptake and removal.

AUTHOR CONTRIBUTION

Piia Simonen was involved in the planning of the study, participated in the acquisition of the clinical data, analysed and interpreted the data and drafted and finalized the manuscript. Ulf-Håkan Stenman was involved in the planning of the study, was responsible for the analyses and interpretation of serum PCSK9 data and critically reviewed and revised the manuscript. Helena Gylling was involved in the planning of the study, participated in the acquisition of the clinical data, was responsible for the analyses of serum sterols and critically reviewed and revised the manuscript. All authors have approved this final version to be submitted.

Dr Maarit Hallikainen is acknowledged for statistical consultations. Ms Leena Kaipiainen is acknowledged for excellent technical assistance. Part of the present study has been presented as an abstract at the American Heart Association Scientific Sessions 2014, Chicago, Illinois.

DEDICATION

This manuscript is dedicated to the memory of Professor Tatu A. Miettinen.

FUNDING

This study was supported by the Raisio Nutrition Ltd. [grant number CL2010-028].

Abbreviations

     
  • BMI

    body mass index

  •  
  • CAD

    coronary artery disease

  •  
  • HDL

    high-density lipoprotein

  •  
  • hs-CRP

    high sensitive C-reactive protein

  •  
  • LDL

    low-density lipoprotein

  •  
  • LDL-C

    LDL cholesterol

  •  
  • PCSK9

    proprotein convertase subtilisin/kexin type 9

  •  
  • SREBP-2

    sterol response element-binding protein 2

  •  
  • VLDL

    very-low-density lipoprotein

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