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PCSK9 levels in abdominally obese men: Association with cardiometabolic risk profile and effects of a one-year lifestyle modification program
Atherosclerosis 236 (2014) 321e326
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PCSK9 levels in abdominally obese men: Association with cardiometabolic risk profile and effects of a one-year lifestyle modification program ras a, Benoit J. Arsenault a, b, *, Emilie Pelletier-Beaumont a, c, Natalie Alme a, c a, d e s a, c , Jean Bergeron , Jean-Pierre Despre Angelo Tremblay , Paul Poirier Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Qu ebec, Canada Department of Medicine, Faculty of Medicine, Universit e Laval, Qu ebec, Canada Department of Kinesiology, Faculty of Medicine, Universit e Laval, Qu ebec, Canada d Faculty of Pharmacy, Universit e Laval, Qu ebec, Canada e Lipid Research Centre, CHU de Qu ebec Research Centre, Qu ebec, Canada a
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 May 2014 Received in revised form 1 July 2014 Accepted 13 July 2014 Available online 26 July 2014
Objectives: Studies performed in rodents have suggested a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in insulin resistance and impaired body fat distribution. Our objective was to examine the relationships between markers of adiposity and insulin resistance and plasma PCSK9 levels in humans. In addition, we explored the effect of a one-year lifestyle modification program on plasma PCSK9 levels in abdominally obese, dyslipidemic men. Methods: Plasma PCSK9 levels were measured by ELISA in 175 abdominally obese, dyslipidemic sedentary men. Of these abdominally obese men, 117 non-diabetic individuals completed a one-year lifestyle modification program aiming at increasing cardiorespiratory fitness levels and improving nutritional quality. Results: We found no association between plasma PCSK9 levels and body mass index, waist circumference, fat and fat-free mass, or visceral and subcutaneous adipose tissue measured by computed tomography. Compared to men with the lowest PCSK9 levels (bottom tertile), those with the highest PCSK9 levels (top tertile) had the most detrimental lipoprotein-lipid profile including lower LDL particle size (253.6 ± 4.0 vs. 251.6 ± 4.0 Å, p < 0.05) and higher apolipoprotein C-III levels (36.8 ± 10.6 vs. 32.3 ± 32.3, p < 0.05). These men were also characterized by higher HOMA-IR indices (6.78 ± 3.01 vs. 5.54 ± 2.91, p < 0.05). After one year, study participants lost on average 6.7 ± 4.6 kg (p < 0.0001). Plasma PCSK9 decreased by 9.2 ± 53.7 ng/ml (3.8%, p ¼ 0.07). Conclusions: Plasma PCSK9 levels are not associated with body fat distribution indices, modestly associated with markers of insulin resistance and LDL particle size and are slightly affected by a lifestyle modification program in abdominally obese men. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: PCSK9 Physical activity Nutrition Obesity Insulin resistance
1. Introduction In 2003, Abifadel and colleagues performed a genetic linkage association study in French families with abnormally elevated levels of low-density lipoprotein (LDL) cholesterol and identified the gene encoding for proprotein convertase subtilisin/kexin type 9
* Corresponding author. Centre de recherche de l'Institut universitaire de carbec, Y-2110, Pavillon Marguerite D'Youville, 2725 diologie et de pneumologie de Que bec, QC G1V 4G5, Canada. Tel.: þ1 418 656 8711x3498. chemin Ste-Foy, Que E-mail address: [email protected] (B.J. Arsenault). http://dx.doi.org/10.1016/j.atherosclerosis.2014.07.010 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
(PCSK9) as a susceptibility locus for familial hypercholesterolemia (FH) [1]. In the following years, several groups around the world confirmed this finding and it is nowadays well accepted that approximately 2% of FH cases may be attributable to gain-offunction mutations at the PCSK9 locus [2]. The role of PCSK9 on coronary artery disease (CAD) risk has been documented by genome-wide association and Mendelian randomization studies, which have confirmed that individuals carrying a common singlenucleotide polymorphism (SNP) at the PCSK9 locus were simultaneously characterized by low LDL cholesterol levels and a decreased CAD risk [3e5]. Several investigations using the Pcsk9/ mouse model have been performed to better understand the mechanisms
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that linked PCSK9 with LDL cholesterol levels and CAD risk. These studies confirmed that PCSK9 is a secreted protein which binds with high affinity to the epidermal growth factor like domain-A of the LDL receptor (LDLR) at the surface of hepatocytes and other cell types, thereby flagging the LDLR for lysosomal degradation thus halting its recycling at the cell surface [6,7]. Reduced LDLR concentration at the surface of the hepatocyte hampers LDL particle uptake and is thereby associated with high LDL cholesterol levels and increased atherosclerosis burden, as suggested by a recent study using the Pcsk9/ mouse [8]. Recent investigations on Pcsk9/ mice also lead to peculiar findings. For instance, a recent study documented that Pcsk9/ mice had 80% more visceral fat compared to wild-type mice and suggested that by targeting the very low-density lipoprotein receptor (VLDLR) in adipocytes, PCSK9 may be an important regulatory factor of visceral adipocytes maturation [9]. Another study using the same model showed that Pcsk9/ mice were characterized by pancreatic islets that exhibited signs of malformation, apoptosis and inflammation leading to a severely glucose intolerant state [10]. Another study however, did not find any features of insulin resistance in Pcsk9/ mice. [11] Although some studies have shown that plasma PCSK9 may be associated positively with plasma insulin levels in humans, most studies conducted on the topic had cross-sectional designs [12,13]. Additionally, there have been no studies to our knowledge on the impact of PCSK9 levels on body fat distribution (or vice-versa) in humans. Our objective was to determine the association between plasma PCSK9 levels and markers of lipoprotein-lipid metabolism, glucoseinsulin homeostasis, body fat distribution, inflammation and cardiorespiratory fitness in abdominally obese men. We also aimed at documenting the directionality of these associations by studying the impact of a lifestyle modification program that significantly improved insulin sensitivity, fitness levels and adiposity on plasma PCSK9 levels in these men. 2. Materials and methods 2.1. Study participants A sample of 175 men, aged between 30 and 65 years, were bec City metropolitan area. recruited through the media in the Que Participants had to be sedentary, which was defined as less than 30 min of continuous and vigorous physical activity per week performed over the past two months and to have an elevated waist circumference (90 cm) combined with the presence of high triglycerides (1.69 mmol/L) and/or low HDL cholesterol levels (<1.03 mmol/L) [14]. In order to avoid the effect of concomitant medication on the metabolic risk profile, participants on lipidlowering-, blood pressure-lowering- or glucose-lowering therapy were not included in the study. Aspirin use was allowed. Each participant signed a consent form approved by the local medical ethics committees. 2.2. Intervention The intervention consisted of a lifestyle modification program in which study participants were offered a personalized healthy eating and physical activity counseling performed by a nutritionist and a kinesiologist, respectively, as previously described [15]. For the first four months, counseling was scheduled every two weeks followed by monthly visits for the following eight months. The nutritional counseling was designed to induce a 500 kcal daily energy deficit for one year. The physical activity program was individualized for each participant according to his physical activity history and preferences. The objective was to reach 160 min per
week of aerobic activity of moderate intensity, along with an increase in occupational physical activity. 2.3. Anthropometric/body composition measurements Body weight and height were measured according to the procedures recommended at the Airlie Conference [16] whereas waist and hip circumferences were measured using standardized procedures, as previously described [17]. Body composition was assessed by dual energy X-ray absorptiometry (DEXA, Lunar Prodigy, GE, Madison, WI, USA). Total, visceral (VAT) and subcutaneous (SCAT) abdominal adipose tissue at the L2eL3 and L4eL5 cross-sectional areas were assessed by CT, with a Somatom DRH scanner (Siemens, Erlanger, Germany), using previously described procedures [18,19] and partial volumes of VAT and SCAT (between L2eL3 and L4eL5) were calculated. Briefly, participants were examined while being in the supine position with both arms stretched above the head. Using specially designed image-analysis software (Slice-O-Matic, Tomovision, Montreal, Canada) adipose tissue areas were calculated using an attenuation range of 190 to 30 Hounsfield units. 2.4. Cardiorespiratory fitness Cardiorespiratory fitness was assessed using a submaximal standardized exercise test on a TMX 425 treadmill (Trackmaster, Newton, KS) linked to a QuarkB2 monitor (Cosmed, Rome, Italy). In the present study, two variables were retained as fitness endpoints to evaluate cardiorespiratory fitness: (i) the subject's heart rate (mean of the last 3 min) at a standardized treadmill stage (3.5 mph, 2% slope) and (ii) the estimated metabolic equivalent of task (MET) reached by the subject at a heart rate of 150 beats/min. 2.5. Plasma lipid/lipoprotein profile and apolipoproteins Participants were asked to fast for 12 h before blood sampling. A catheter was inserted into a forearm vein and a fasting blood sample was taken into vacutainer tubes containing EDTA (Miles Pharmaceuticals, Rexdale, Ontario, Canada) to determine plasma levels of lipids and lipoproteins. Triglycerides and cholesterol levels were determined in plasma and lipoprotein fractions using automated techniques [20,21]. The high-density lipoprotein fraction (HDL) was obtained after precipitation of low-density lipoprotein (LDL) in the infranatant (density 1.006 g/mL) with heparin and manganese chloride. Triglycerides and cholesterol concentrations were measured before and after the precipitation step. Apolipoprotein B and AI levels were obtained in plasma by nephelometry using polyclonal antibodies on the Behring BN ProSpec (Dade Behring, Marburg, Germany). LDL peak particle size and the proportion of small, medium and large LDL particles as well as HDL mean particle size were measured, respectively, by 2e16% and 4e30% polyacrylamide gradient-gel electrophoresis (GGE), as previously described [22,23]. Plasma apoC-III levels were measured using a commercially available electroimmunoassay (Hydragel LP C-III; Sebia, Issey-les-Moulineaux, France) and PCSK9 levels were measured by ELISA (R&D systems, Mineapolis, MN). 2.6. Oral glucose tolerance test After a 12-h overnight fast, participants were subjected to a 3 h, 75 g oral glucose tolerance test (OGTT). Plasma glucose was measured enzymatically, whereas plasma insulin and C-peptide were determined by radioimmunoassay. The HOMA-IR, ISI-Matsuda indices, as well as the area under the curve of glucose and
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insulin during the OGTT were computed as previously described [24]. 2.7. Adipokines and inflammatory markers Plasma leptin and adiponectin concentrations (B-Bridge, CA) as well as plasma interleukin-6 and tumor-necrosis factor-a levels (R&D Systems, Minneapolis, MN) were determined by ELISA on frozen plasma samples (80 C). Highly sensitive C-reactive protein (hsCRP) levels were measured by immunoassay (Dade Behring, Munich, Germany). 2.8. Statistical analyses The normality of variables was assessed by the ShapiroeWilk procedure and skewed variables were log-transformed before each statistical test. Differences between baseline characteristics of study participants across PCSK9 tertiles were tested by ANOVA with the general linear model procedure. As PCSK9 were among the variables with a skewed distribution, Spearman rank correlations were performed. Paired T-tests were used to assess the differences in each variable before and after the intervention program. All statistical analyses were performed with SAS (v9.3, Cary, NC, USA). 3. Results The baseline characteristics of the 175 study participants separated on the basis of plasma PCSK9 levels tertiles are shown in Table 1. Overall, patients with the highest PCSK9 levels were characterized by higher triglyceride, apoC-III and insulin levels (at baseline and at 2 h during the OGTT). Men with the highest PCSK9 levels were characterized by lower LDL particle size (Fig. 1A). Additionally, after further characterization of the migration profile of LDL particles, we found that men with higher PCSK9 levels did not have higher cholesterol in large or medium size LDL particles, but had higher cholesterol levels in small LDL particles (Fig. 1BeD). Men in the highest PCSK9 levels were also characterized by higher insulin levels during the oral glucose tolerance test (Fig. 2). Body fat distribution and cardiorespiratory fitness did not vary across PCSK9 tertiles. From the 175 men initially included in the study, 26 of them were newly diagnosed, untreated type 2 diabetics and were not included in the lifestyle modification program. The differences in plasma PCSK9 levels in these 26 men were comparable to the PCSK9 levels of the participants included in the lifestyle modification program. Of the 149 men enrolled in the program, 117 were assigned to the lifestyle modification program and 32 were assigned to a control group. Post-1 year PCSK9 values were available for 23 men of the control group. Mean level of the participants of the lifestyle modification program and the control group before and after the one-year intervention are shown in Fig. 3, which shows that the lifestyle modification program induced a borderline-significant reduction of 3.8% of PCSK9 levels. The effects of the lifestyle modification program on other parameters listed in Table 1 have been previously published [25]. In order to determine whether changes in PCSK9 levels were associated with changes in other cardiometabolic risk markers, we have divided participants included in the lifestyle modification program in two groups, based on mean changes in plasma PCSK9 levels (below or equal or above median). Table 2 shows mean changes in cardiometabolic risk markers in patients with PCSK9 changes below (lower responders) or equal or above (higher responders) the median change in PCSK9 level (9.23 ng/ml). Changes in plasma PCSK9 levels were not correlated with changes in indices of adiposity but were significantly associated with changes in fasting insulin levels (r ¼ 0.20, p ¼ 0.04) and HOMA-IR
323
Table 1 Baseline characteristics of the study participants classified on the basis of plasma PCSK9 levels. PCSK9 range, ng/ml
Number of participants
Tertile 1
Tertile 2
Tertile 3
<212.5
212.5e<256.0
256.0
58
58
59
PCSK9, ng/ml 182.2 Age, years 47.5 Body mass index, kg/m2 31.4 Waist circumference, cm 108.8 Systolic blood 117 pressure, mm Hg Diastolic blood 78 pressure, mm Hg 3 Adipose tissue, cm Total 3682 Subcutaneous 1821 Visceral 1870 Fat-free mass 66.3 Fat mass 29.8 Exercise output 7.64 at 150 BPM, METs Heart rate, 116 3.5mph/2%, BPM Plasma lipoproteins Total cholesterol, mmol/L 4.86 VLDL cholesterol, mmol/L 0.90 LDL cholesterol, mmol/L 3.04 HDL cholesterol, mmol/L 0.96 Triglycerides, mmol/L 2.05 Apolipoprotein B, g/L 1.04 Apolipoprotein A-I, g/L 1.11 Apolipoprotein C-III, mg/L 32.3 HDL size, Å 82.1 Plasma glucose-insulin homeostasis Fasting glucose, mmol/L 6.02 120-min OGTT-glucose, 7.83 mmol/L Fasting insulin, pmol/L 143.8 120-min OGTT-insulin, 930.4 pmol/L HOMA-IR 5.54 ISI-Matsuda 2.45 Adipokines/Inflammatory markers C-reactive protein, mg/L 3.58 Adiponectin, mg/ml 3.77 Leptin, ng/ml 10.86 Interleukin-6, pg/ml 1.42 Tumor-necrosis 1.17 factor-a, pg/ml
(26.2) (9.1) (3.1) (9.2) (9) (7)
(846) (559) (523) (6.5) (6.3) (1.55)
233.8 48.8 31.0 107.8 118
(12.8)a (7.7) (3.6) (9.6) (12)
80 (8)
3733 1759 1993 65.1 29.4 7.49
(970) (633) (488) (7.4) (8.0) (1.59)
303.2 46.3 30.8 107.6 117
(43.9)a,b (9.3) (2.6) (7.2) (12)
78 (8)
3627 1771 1856 64.6 28.7 7.45
(782) (650) (378) (7.5) (6.3) (1.21)
(13)
117 (15)
118 (12)
(0.83) (0.47) (0.74) (0.20) (0.84) (0.19) (0.15) (13.4) (2.5)
5.12 0.99 3.18 0.95 2.38 1.09 1.14 32.4 81.6
5.29 1.14 3.16 0.98 2.73 1.13 1.18 36.8 81.7
(0.44) (1.69)
5.95 (0.44) 7.59 (1.75)
6.02 (0.52) 7.79 (1.58)
(77.1) (576.9)
154.1 (70.0) 1080.6 (673.0)
174.3 (72.7)a 1181.7 (670.5)a
(2.91) (4.93)
5.91 (2.88) 1.62 (0.92)
(8.79) (1.38) (7.36) (1.20) (0.52)
2.35 3.68 11.53 1.31 1.07
(0.75) (0.35) (0.67) (0.18) (0.69)a (0.16) (0.16) (8.5) (2.0)
(2.04) (1.48) (8.24) (0.79) (0.69)
(0.77) (0.48) (0.67) (0.17) (0.97)a (0.17)a (0.15)a (10.6)a,b (2.2)
6.78 (3.01)b 1.45 (1.03)a 2.67 3.68 13.00 1.23 1.42
(4.38) (1.47) (8.36) (0.64) (2.38)
PCSK9 indicates proprotein convertase subtilisin/kexin type 9, BPM indicates beats per minute, MET indicates metabolic equivalent, VLDL indicates very low-density lipoprotein, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein, OGTT indicates oral glucose tolerance test, HOMA-IR indicates homeostatic model of insulin resistance, ISI indicates insulin sensitivity index and DI indicates disposition index. Data are presented as mean (±SD). a Significantly different than tertile 1. b Significantly different than fertile 2.
index (r ¼ 0.19, p ¼ 0.04). We also found that changes in PCSK9 levels were not associated with cholesterol levels in large or medium-sized particles but were modestly correlated with changes in cholesterol levels in small LDL particles (r ¼ 0.16, p ¼ 0.08). 4. Discussion Results of the present study show that in initially sedentary and abdominally obese men, plasma PCSK9 levels are associated with several cardiometabolic risk markers such as glucose-insulin homeostasis parameters and markers of the lipoprotein profile including plasma triglyceride, apolipoprotein B, apolipoprotein A-I and apolipoprotein C-III levels. Plasma PCSK9 levels were not associated with circulating LDL cholesterol levels but were
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Fig. 1. Association between plasma levels of PCSK9 and LDL particle size (A), cholesterol levels in large (B), medium (C) and small (D) particles.
positively associated with cholesterol levels in small LDL particles. We found no association between plasma PCSK9 levels and cardiorespiratory fitness or body fat distribution indices. We also found that our lifestyle modification program had a trivial clinical impact on PCSK9 levels. Previous studies that have examined the impact of plasma PCSK9 levels on insulin levels reported modest associations with PCSK9 levels and insulin levels or the HOMA-IR index. Our study confirms this finding and also suggests that PCSK9 levels are also associated with the insulin sensitivity index. Indeed, participants in the top PCSK9 levels tertile have approximately 40% lower ISIs compared to participants in the bottom tertile. There is however little evidence in the literature on the directionality of this association. Kappelle and colleagues [26] investigated the impact of 24-h insulin infusions on PCSK9 levels in healthy participants and patients with type 2 diabetes and found no impact on PCSK9 levels. This is in accordance with the results of our study, which suggested that although our intervention had a strong impact on insulin sensitivity, the impact of the intervention on PCSK9 levels was
modest. However, patients with the highest PCSK9 decreases during the intervention had more important increases in insulin sensitivity. Interestingly, a study of French-Canadian patients showed that carriers a common polymorphism in the PCSK9 gene conferring low LDL cholesterol levels had higher HOMA-IR indices compared to non-carriers [27]. The results of an earlier report by Costet et al. [28] suggested that hepatic PCSK9 expression could be regulated by insulin via the sterol regulatory element-binding protein 1c, thereby providing a molecular connection between PCSK9 and insulin metabolism. Abdominally obese patients are commonly characterized by an insulin-resistance state. Under these circumstances, visceral adipocytes are plunged in a pro-inflammatory milieu, which enables them to secrete several adipokines that are detrimental for the glucose-insulin homeostasis such as interleukin-6, tumor-necrosis factor-a, interleukin-1b, resistin, leptin and many more [29]. Visceral adipocytes secrete less of the insulin-sensitizing hormone adiponectin as visceral adipose tissue expands [30]. In our study, we found no association whatsoever between plasma PCSK9 levels
Fig. 2. Plasma levels of PCSK9 insulin levels during an oral glucose tolerance test (A) and insulin area under the curve during the oral glucose tolerance test (B) in men according to PCSK9 tertiles.
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325
Table 2 Changes in cardiometabolic risk markers in men with higher vs. lower responses in PCSK9 during the one-year lifestyle modification program. Number of participants
Fig. 3. Impact of the lifestyle modification program on plasma PCSK9 levels in men of the intervention and the control groups.
and these adipokines. We also found no association between visceral adipose tissue accumulation and PCSK9 levels, which suggest that the relationship between PCSK9 and markers of insulin resistance may occur via mechanisms that are independent of body fat distribution. Our results also suggest that unlike the recently reported association between loss of PCSK9 and increased visceral fat in mice, [9] PCSK9 does not appear to be influenced by body fat distribution in humans. However, we have not studied the impact of loss- or gain-of-function genetic mutations at the PCSK9 locus, on body fat distribution and believe that our results should be replicated in the context of more severe PCSK9 variations. The impact of PCSK9 inhibition on glucose-insulin homeostasis and body fat distribution should also be investigated. Several studies have shown that PCSK9 contributes to a certain extent to the variation in plasma lipoprotein-lipid levels. In our study, we found no associations between PCSK9 levels and LDL cholesterol levels. In other populations, circulating PCSK9 levels has been shown to be modestly associated with LDL cholesterol levels. However, the strength of the association between PCSK9 and LDL cholesterol levels was at best modest. To our knowledge, our study is the first to suggest that PCSK9 levels may not be associated with cholesterol levels in large LDL particles but may be more closely associated with cholesterol levels in small LDL particles. The mechanism underlying this observation is not completely understood. However, the association between PCSK9 levels with both triglyceride and apoC-III levels that we have observed may suggest that PCSK9 levels influences the catabolism of triglyceride-rich lipoproteins. Previous results from our group has shown that cholesterol levels in small LDL particles were closely association with cardiovascular risk while cholesterol levels in large LDL particles were not associated with cardiovascular risk in a large European prospective study [22]. Statin therapy is the first-line therapy for the prevention and treatment of CAD. By lowering intracellular cholesterol concentrations, statins also upregulate the LDLR, which further contributes to the uptake of LDL particles at the cell surface. However, the statin-dependent increase in LDL particle clearance mostly favor the uptake of large LDL particles and statins are not as efficient as clearing smaller LDL particles. On the other hand, statins upregulate PCSK9 expression and secretion, which may limit their efficiency in terms of LDL-lowering [31]. This observation, combined with our study results, could be another explanation on why statin therapy has a null impact on the small LDL phenotype. Given the fact that PCSK9 inhibitors are currently being tested for their potential to reduce cardiovascular outcomes in high-risk study population, it
Lower responders
Higher responders
59
58
PCSK9, ng/ml 31.5 Body mass index, kg/m2 2.3 Waist circumference, cm 8.9 Systolic blood 1.1 pressure, mm Hg Diastolic blood 4.9 pressure, mm Hg 3 Adipose tissue, cm Total 890 Subcutaneous 355 Visceral 523 Fat-free mass 1.05 Fat mass 6.26 Exercise output 1.36 at 150 BPM, METs 14.0 Heart rate, 3.5mph/2%, BPM Plasma lipoproteins Total cholesterol, mmol/L 0.01 VLDL cholesterol, mmol/L 0.22 LDL cholesterol, mmol/L 0.11 HDL cholesterol, mmol/L 0.10 Triglycerides, mmol/L 0.38 Apolipoprotein B, g/L 0.03 Apolipoprotein A-I, g/L 0.16 Apolipoprotein C-III, mg/L 1.15 LDL size, Å 1.08 LDL-C<255 Å, mmol/L 0.01 LDL-C255-260 Å, mmol/L 0.07 LDL-C>260 Å, mmol/L 0.02 HDL size, Å 2.38 Plasma glucose-insulin homeostasis Fasting glucose, mmol/L 0.10 120-min OGTT-glucose, 1.03 mmol/L Fasting insulin, pmol/L 39.8 120-min OGTT-insulin, 445.7 pmol/L HOMA-IR 1.62 ISI-Matsuda 0.47 Adipokines/Inflammatory markers C-reactive protein, mg/L 0.77 Adiponectin, mg/ml 0.58 Leptin, ng/ml 3.02 Interleukin-6, pg/ml 0.11 Tumor-necrosis 0.06 factor-a, pg/ml
(28.6)a (1.7)a (5.7)a (9.8) (7.5)a
50.7 2.0 8.4 3.2
(39.6)a (1.3)a (5.1)a (9.9)a
4.5 (6.8)a
P-value
<0.0001 0.44 0.64 0.26 0.77
(598)a (257)a (394)a (1.89)a (3.98)a (1.43)a
762 460 302 0.46 5.70 1.34
(512)a (292)a (266)a (1.66)a (3.60)a (1.23)a
0.24 0.30 0.34 0.08 0.89 0.56
(13.4)a
12.3 (8.59)a
0.45
(0.70) (0.34)a (0.60) (0.12)a (0.69)a (0.16) (0.10)a (11.20) (3.43)a (0.30) (0.16)a (0.30) (3.60)a
0.05 0.34 0.13 0.16 0.67 0.06 0.19 1.78 1.76 0.11 0.10 0.04 2.17
(0.14) (0.42)a (0.54) (0.14)a (0.97)a (0.14)a (0.14)a (8.85) (3.06)a (0.40)a (0.19)a (0.19) (3.17)a
0.76 0.10 0.84 0.03 0.07 0.28 0.18 0.72 0.27 0.08 0.28 0.59 0.77
(0.37)a (2.11)a
0.11 (0.39)a 1.07 (1.54)a
0.88 0.94
(64.2)a (465.1)a
72.0 (70.0)a 558.5 (527.3)a
0.02 0.22
(2.70)a (2.29)a
2.89 (2.87)a 1.73 (5.76)a
0.07 0.07
(2.08)a (0.99)a (4.03)a (0.82) (0.46)
0.63 1.00 3.91 0.18 0.24
(1.84)a (1.16)a (4.13)a (0.72)a (1.62)
0.78 0.18 0.36 0.47 0.80
PCSK9 indicates proprotein convertase subtilisin/kexin type 9, BPM indicates beats per minute, MET indicates metabolic equivalent, VLDL indicates very low-density lipoprotein, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein, OGTT indicates oral glucose tolerance test, HOMA-IR indicates homeostatic model of insulin resistance, ISI indicates insulin sensitivity index and DI indicates disposition index. Data are presented as mean (±SD). a Indicates a significant changes from baseline values.
would be important to document the impact of PCSK9 inhibitor on LDL size and on the concentration of cholesterol in small LDL particles to determine whether it would be a valuable add-on therapy to statins. Our study has limitations. First, we do not know if any of the study participants had mutations at the PCSK9 locus and whether some of the examples of phenotypic variation reported here could be secondary to such mutations. Additionally, we have investigated the impact of PCSK9 variations in a relatively homogenous population of initially sedentary and abdominally obese men, which might explain why our reported associated were relatively modest. We believe that further studies in women, other ethnic groups and in samples with a wider BMI range could be useful to confirm and
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extend our findings. Finally, although our study is the first to our knowledge to document the impact of weight loss on PCSK9 levels, the weight loss that was achieved was relatively modest. On the other hand, our intervention has been proven sufficient to induce a significant amount of total and visceral fat and to improve insulin sensitivity [32]. In conclusion, our study provides evidence that plasma PCSK9 levels are associated with several parameters of the glucose-insulin homeostasis and parameters of the lipoprotein-lipid profile, including small, dense LDL particles. We documented that lifestyle modification therapy had a modest impact on PCSK9 levels. Whether pharmacological intervention leading to a more robust inhibition of PCSK9 influences body fat distribution, insulin resistance and small LDL particles should be further explored in ongoing PCSK9 inhibition trials. Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This study was supported by the Canadian Institutes of Health Research (MOP-64439). We would like to thank Sylvain Pouliot for his technical help. B.J.A. holds a junior scholar award from the bec: Sante (FRQS). P.P. holds a senior Fonds de recherche du Que scholar award from the FRQS. References [1] Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003;34:154e6. [2] Nordestgaard BG, Chapman MJ, Humphries SE, Ginsberg HN, Masana L, Descamps OS, et al., European atherosclerosis society consensus P. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European atherosclerosis society. Eur Heart J 2013;34:3478e3490a. [3] Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet 2013;45:25e33. [4] Cohen JC, Boerwinkle E, Mosley Jr TH, Hobbs HH. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264e72. [5] Benn M, Nordestgaard BG, Grande P, Schnohr P, Tybjaerg-Hansen A. PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses. J Am Coll Cardiol 2010;55: 2833e42. [6] Lagace TA, Curtis DE, Garuti R, McNutt MC, Park SW, Prather HB, et al. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest 2006;116:2995e3005. [7] Zhang DW, Lagace TA, Garuti R, Zhao Z, McDonald M, Horton JD, et al. Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factorlike repeat a of low density lipoprotein receptor decreases receptor recycling and increases degradation. J Biol Chem 2007;282:18602e12. [8] Denis M, Marcinkiewicz J, Zaid A, Gauthier D, Poirier S, Lazure C, et al. Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice. Circulation 2012;125:894e901. [9] Roubtsova A, Munkonda MN, Awan Z, Marcinkiewicz J, Chamberland A, Lazure C, et al. Circulating proprotein convertase subtilisin/kexin 9 (pcsk9) regulates VLDLR protein and triglyceride accumulation in visceral adipose tissue. Arterioscler Thromb Vasc Biol 2011;31:785e91.
[10] Mbikay M, Sirois F, Mayne J, Wang GS, Chen A, Dewpura T, et al. PCSK9deficient mice exhibit impaired glucose tolerance and pancreatic islet abnormalities. FEBS Lett 2010;584:701e6. [11] Langhi C, Le May C, Gmyr V, Vandewalle B, Kerr-Conte J, Krempf M, et al. PCSK9 is expressed in pancreatic delta-cells and does not alter insulin secretion. Biochem Biophys Res Commun 2009;390:1288e93. [12] Chernogubova E, Strawbridge R, Mahdessian H, Malarstig A, Krapivner S, Gigante B, et al. Common and low-frequency genetic variants in the PCSK9 locus influence circulating PCSK9 levels. Arterioscler Thromb Vasc Biol 2012;32:1526e34. [13] Baass A, Dubuc G, Tremblay M, Delvin EE, O'Loughlin J, Levy E, et al. Plasma PCSK9 is associated with age, sex, and multiple metabolic markers in a population-based sample of children and adolescents. Clin Chem 2009;55: 1637e45. ras N, et al. [14] Lemieux I, Pascot A, Couillard C, Lamarche B, Tchernof A, Alme Hypertriglyceridemic waist: a marker of the atherogenic metabolic triad (hyperinsulinemia; hyperapolipoprotein B; small, dense LDL) in men? Circulation 2000;102:179e84. ras N, Tremblay A, Bergeron J, et al. Changes [15] Nazare JA, Smith J, Borel AL, Alme in both global diet quality and physical activity level synergistically reduce visceral adiposity in men with features of metabolic syndrome. J Nutr 2013;143:1074e83. [16] Lohman TRA, Martorel R, editors. The airlie (va) consensus conference: standardization of anthropometric measurements. Champaign, IL: Human Kinetics Publishers; 1988. p. 39e80. [17] van der Kooy K, Leenen R, Seidell JC, Deurenberg P, Visser M. Abdominal diameters as indicators of visceral fat: comparison between magnetic resonance imaging and anthropometry. Br J Nutr 1993;70:47e58. s JP, Tremblay A, Pinault S, Nadeau A, Moorjani S, et al. [18] Ferland M, Despre Assessment of adipose tissue distribution by computed axial tomography in obese women: association with body density and anthropometric measurements. Br J Nutr 1989;61:139e48. s JP, Ross R, Boka G, Alme ras N, Lemieux I. Effect of rimonabant on the [19] Despre high-triglyceride/ low-HDL-cholesterol dyslipidemia, intraabdominal adiposity, and liver fat: the adagio-lipids trial. Arterioscler Thromb Vasc Biol 2009;29:416e23. [20] Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470e5. [21] Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982;28:2077e80. s JP, Wareham NJ, Luben R, Kastelein JJ, et al. [22] Arsenault BJ, Lemieux I, Despre Cholesterol levels in small LDL particles predict the risk of coronary heart disease in the epic-norfolk prospective population study. Eur Heart J 2007;28: 2770e7. russe M, Pascot A, Despre s JP, Couillard C, Lamarche B. A new method for [23] Pe HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma. J Lipid Res 2001;42:1331e4. ras N, Tremblay A, et al. Improved [24] Borel AL, Boulet G, Nazare JA, Smith J, Alme plasma FFA/insulin homeostasis is independently associated with improved glucose tolerance after a 1-year lifestyle intervention in viscerally obese men. Diabetes Care 2013;36:3254e61. ras N, Bergeron J, Tremblay A, [25] Pelletier-Beaumont E, Arsenault BJ, Alme Poirier P, et al. Normalization of visceral adiposity is required to normalize plasma apolipoprotein B levels in response to a healthy eating/physical activity lifestyle modification program in viscerally obese men. Atherosclerosis 2012;221:577e82. [26] Kappelle PJ, Lambert G, Dullaart RP. Plasma proprotein convertase subtilisinkexin type 9 does not change during 24h insulin infusion in healthy subjects and type 2 diabetic patients. Atherosclerosis 2011;214:432e5. [27] Awan Z, Delvin EE, Levy E, Genest J, Davignon J, Seidah NG, et al. Regional distribution and metabolic effect of PCSK9 insleu and R46L gene mutations and APOE genotype. Can J Cardiol 2013;29:927e33. [28] Costet P, Cariou B, Lambert G, Lalanne F, Lardeux B, Jarnoux AL, et al. Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c. J Biol Chem 2006;281:6211e8. s JP, Larose E. Mapping body fat distribu[29] Arsenault BJ, Beaumont EP, Despre tion: a key step towards the identification of the vulnerable patient? Ann Med 2012;44:758e72. ^ te M, Maurie ge P, Bergeron J, Alme ras N, Tremblay A, Lemieux I, et al. [30] Co Adiponectinemia in visceral obesity: impact on glucose tolerance and plasma lipoprotein and lipid levels in men. J Clin Endocrinol Metab 2005;90:1434e9. [31] Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L, et al. Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 2004;24:1454e9. [32] Borel AL, Nazare JA, Smith J, Almeras N, Tremblay A, Bergeron J, et al. Visceral and not subcutaneous abdominal adiposity reduction drives the benefits of a 1-year lifestyle modification program. Obes (Silver Spring) 2012;20:1223e33.
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PCSK9 levels in abdominally obese men: Association with cardiometabolic risk profile and effects of a one-year lifestyle modification program ras a, Benoit J. Arsenault a, b, *, Emilie Pelletier-Beaumont a, c, Natalie Alme a, c a, d e s a, c , Jean Bergeron , Jean-Pierre Despre Angelo Tremblay , Paul Poirier Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Qu ebec, Canada Department of Medicine, Faculty of Medicine, Universit e Laval, Qu ebec, Canada Department of Kinesiology, Faculty of Medicine, Universit e Laval, Qu ebec, Canada d Faculty of Pharmacy, Universit e Laval, Qu ebec, Canada e Lipid Research Centre, CHU de Qu ebec Research Centre, Qu ebec, Canada a
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 May 2014 Received in revised form 1 July 2014 Accepted 13 July 2014 Available online 26 July 2014
Objectives: Studies performed in rodents have suggested a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in insulin resistance and impaired body fat distribution. Our objective was to examine the relationships between markers of adiposity and insulin resistance and plasma PCSK9 levels in humans. In addition, we explored the effect of a one-year lifestyle modification program on plasma PCSK9 levels in abdominally obese, dyslipidemic men. Methods: Plasma PCSK9 levels were measured by ELISA in 175 abdominally obese, dyslipidemic sedentary men. Of these abdominally obese men, 117 non-diabetic individuals completed a one-year lifestyle modification program aiming at increasing cardiorespiratory fitness levels and improving nutritional quality. Results: We found no association between plasma PCSK9 levels and body mass index, waist circumference, fat and fat-free mass, or visceral and subcutaneous adipose tissue measured by computed tomography. Compared to men with the lowest PCSK9 levels (bottom tertile), those with the highest PCSK9 levels (top tertile) had the most detrimental lipoprotein-lipid profile including lower LDL particle size (253.6 ± 4.0 vs. 251.6 ± 4.0 Å, p < 0.05) and higher apolipoprotein C-III levels (36.8 ± 10.6 vs. 32.3 ± 32.3, p < 0.05). These men were also characterized by higher HOMA-IR indices (6.78 ± 3.01 vs. 5.54 ± 2.91, p < 0.05). After one year, study participants lost on average 6.7 ± 4.6 kg (p < 0.0001). Plasma PCSK9 decreased by 9.2 ± 53.7 ng/ml (3.8%, p ¼ 0.07). Conclusions: Plasma PCSK9 levels are not associated with body fat distribution indices, modestly associated with markers of insulin resistance and LDL particle size and are slightly affected by a lifestyle modification program in abdominally obese men. © 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: PCSK9 Physical activity Nutrition Obesity Insulin resistance
1. Introduction In 2003, Abifadel and colleagues performed a genetic linkage association study in French families with abnormally elevated levels of low-density lipoprotein (LDL) cholesterol and identified the gene encoding for proprotein convertase subtilisin/kexin type 9
* Corresponding author. Centre de recherche de l'Institut universitaire de carbec, Y-2110, Pavillon Marguerite D'Youville, 2725 diologie et de pneumologie de Que bec, QC G1V 4G5, Canada. Tel.: þ1 418 656 8711x3498. chemin Ste-Foy, Que E-mail address: [email protected] (B.J. Arsenault). http://dx.doi.org/10.1016/j.atherosclerosis.2014.07.010 0021-9150/© 2014 Elsevier Ireland Ltd. All rights reserved.
(PCSK9) as a susceptibility locus for familial hypercholesterolemia (FH) [1]. In the following years, several groups around the world confirmed this finding and it is nowadays well accepted that approximately 2% of FH cases may be attributable to gain-offunction mutations at the PCSK9 locus [2]. The role of PCSK9 on coronary artery disease (CAD) risk has been documented by genome-wide association and Mendelian randomization studies, which have confirmed that individuals carrying a common singlenucleotide polymorphism (SNP) at the PCSK9 locus were simultaneously characterized by low LDL cholesterol levels and a decreased CAD risk [3e5]. Several investigations using the Pcsk9/ mouse model have been performed to better understand the mechanisms
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that linked PCSK9 with LDL cholesterol levels and CAD risk. These studies confirmed that PCSK9 is a secreted protein which binds with high affinity to the epidermal growth factor like domain-A of the LDL receptor (LDLR) at the surface of hepatocytes and other cell types, thereby flagging the LDLR for lysosomal degradation thus halting its recycling at the cell surface [6,7]. Reduced LDLR concentration at the surface of the hepatocyte hampers LDL particle uptake and is thereby associated with high LDL cholesterol levels and increased atherosclerosis burden, as suggested by a recent study using the Pcsk9/ mouse [8]. Recent investigations on Pcsk9/ mice also lead to peculiar findings. For instance, a recent study documented that Pcsk9/ mice had 80% more visceral fat compared to wild-type mice and suggested that by targeting the very low-density lipoprotein receptor (VLDLR) in adipocytes, PCSK9 may be an important regulatory factor of visceral adipocytes maturation [9]. Another study using the same model showed that Pcsk9/ mice were characterized by pancreatic islets that exhibited signs of malformation, apoptosis and inflammation leading to a severely glucose intolerant state [10]. Another study however, did not find any features of insulin resistance in Pcsk9/ mice. [11] Although some studies have shown that plasma PCSK9 may be associated positively with plasma insulin levels in humans, most studies conducted on the topic had cross-sectional designs [12,13]. Additionally, there have been no studies to our knowledge on the impact of PCSK9 levels on body fat distribution (or vice-versa) in humans. Our objective was to determine the association between plasma PCSK9 levels and markers of lipoprotein-lipid metabolism, glucoseinsulin homeostasis, body fat distribution, inflammation and cardiorespiratory fitness in abdominally obese men. We also aimed at documenting the directionality of these associations by studying the impact of a lifestyle modification program that significantly improved insulin sensitivity, fitness levels and adiposity on plasma PCSK9 levels in these men. 2. Materials and methods 2.1. Study participants A sample of 175 men, aged between 30 and 65 years, were bec City metropolitan area. recruited through the media in the Que Participants had to be sedentary, which was defined as less than 30 min of continuous and vigorous physical activity per week performed over the past two months and to have an elevated waist circumference (90 cm) combined with the presence of high triglycerides (1.69 mmol/L) and/or low HDL cholesterol levels (<1.03 mmol/L) [14]. In order to avoid the effect of concomitant medication on the metabolic risk profile, participants on lipidlowering-, blood pressure-lowering- or glucose-lowering therapy were not included in the study. Aspirin use was allowed. Each participant signed a consent form approved by the local medical ethics committees. 2.2. Intervention The intervention consisted of a lifestyle modification program in which study participants were offered a personalized healthy eating and physical activity counseling performed by a nutritionist and a kinesiologist, respectively, as previously described [15]. For the first four months, counseling was scheduled every two weeks followed by monthly visits for the following eight months. The nutritional counseling was designed to induce a 500 kcal daily energy deficit for one year. The physical activity program was individualized for each participant according to his physical activity history and preferences. The objective was to reach 160 min per
week of aerobic activity of moderate intensity, along with an increase in occupational physical activity. 2.3. Anthropometric/body composition measurements Body weight and height were measured according to the procedures recommended at the Airlie Conference [16] whereas waist and hip circumferences were measured using standardized procedures, as previously described [17]. Body composition was assessed by dual energy X-ray absorptiometry (DEXA, Lunar Prodigy, GE, Madison, WI, USA). Total, visceral (VAT) and subcutaneous (SCAT) abdominal adipose tissue at the L2eL3 and L4eL5 cross-sectional areas were assessed by CT, with a Somatom DRH scanner (Siemens, Erlanger, Germany), using previously described procedures [18,19] and partial volumes of VAT and SCAT (between L2eL3 and L4eL5) were calculated. Briefly, participants were examined while being in the supine position with both arms stretched above the head. Using specially designed image-analysis software (Slice-O-Matic, Tomovision, Montreal, Canada) adipose tissue areas were calculated using an attenuation range of 190 to 30 Hounsfield units. 2.4. Cardiorespiratory fitness Cardiorespiratory fitness was assessed using a submaximal standardized exercise test on a TMX 425 treadmill (Trackmaster, Newton, KS) linked to a QuarkB2 monitor (Cosmed, Rome, Italy). In the present study, two variables were retained as fitness endpoints to evaluate cardiorespiratory fitness: (i) the subject's heart rate (mean of the last 3 min) at a standardized treadmill stage (3.5 mph, 2% slope) and (ii) the estimated metabolic equivalent of task (MET) reached by the subject at a heart rate of 150 beats/min. 2.5. Plasma lipid/lipoprotein profile and apolipoproteins Participants were asked to fast for 12 h before blood sampling. A catheter was inserted into a forearm vein and a fasting blood sample was taken into vacutainer tubes containing EDTA (Miles Pharmaceuticals, Rexdale, Ontario, Canada) to determine plasma levels of lipids and lipoproteins. Triglycerides and cholesterol levels were determined in plasma and lipoprotein fractions using automated techniques [20,21]. The high-density lipoprotein fraction (HDL) was obtained after precipitation of low-density lipoprotein (LDL) in the infranatant (density 1.006 g/mL) with heparin and manganese chloride. Triglycerides and cholesterol concentrations were measured before and after the precipitation step. Apolipoprotein B and AI levels were obtained in plasma by nephelometry using polyclonal antibodies on the Behring BN ProSpec (Dade Behring, Marburg, Germany). LDL peak particle size and the proportion of small, medium and large LDL particles as well as HDL mean particle size were measured, respectively, by 2e16% and 4e30% polyacrylamide gradient-gel electrophoresis (GGE), as previously described [22,23]. Plasma apoC-III levels were measured using a commercially available electroimmunoassay (Hydragel LP C-III; Sebia, Issey-les-Moulineaux, France) and PCSK9 levels were measured by ELISA (R&D systems, Mineapolis, MN). 2.6. Oral glucose tolerance test After a 12-h overnight fast, participants were subjected to a 3 h, 75 g oral glucose tolerance test (OGTT). Plasma glucose was measured enzymatically, whereas plasma insulin and C-peptide were determined by radioimmunoassay. The HOMA-IR, ISI-Matsuda indices, as well as the area under the curve of glucose and
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insulin during the OGTT were computed as previously described [24]. 2.7. Adipokines and inflammatory markers Plasma leptin and adiponectin concentrations (B-Bridge, CA) as well as plasma interleukin-6 and tumor-necrosis factor-a levels (R&D Systems, Minneapolis, MN) were determined by ELISA on frozen plasma samples (80 C). Highly sensitive C-reactive protein (hsCRP) levels were measured by immunoassay (Dade Behring, Munich, Germany). 2.8. Statistical analyses The normality of variables was assessed by the ShapiroeWilk procedure and skewed variables were log-transformed before each statistical test. Differences between baseline characteristics of study participants across PCSK9 tertiles were tested by ANOVA with the general linear model procedure. As PCSK9 were among the variables with a skewed distribution, Spearman rank correlations were performed. Paired T-tests were used to assess the differences in each variable before and after the intervention program. All statistical analyses were performed with SAS (v9.3, Cary, NC, USA). 3. Results The baseline characteristics of the 175 study participants separated on the basis of plasma PCSK9 levels tertiles are shown in Table 1. Overall, patients with the highest PCSK9 levels were characterized by higher triglyceride, apoC-III and insulin levels (at baseline and at 2 h during the OGTT). Men with the highest PCSK9 levels were characterized by lower LDL particle size (Fig. 1A). Additionally, after further characterization of the migration profile of LDL particles, we found that men with higher PCSK9 levels did not have higher cholesterol in large or medium size LDL particles, but had higher cholesterol levels in small LDL particles (Fig. 1BeD). Men in the highest PCSK9 levels were also characterized by higher insulin levels during the oral glucose tolerance test (Fig. 2). Body fat distribution and cardiorespiratory fitness did not vary across PCSK9 tertiles. From the 175 men initially included in the study, 26 of them were newly diagnosed, untreated type 2 diabetics and were not included in the lifestyle modification program. The differences in plasma PCSK9 levels in these 26 men were comparable to the PCSK9 levels of the participants included in the lifestyle modification program. Of the 149 men enrolled in the program, 117 were assigned to the lifestyle modification program and 32 were assigned to a control group. Post-1 year PCSK9 values were available for 23 men of the control group. Mean level of the participants of the lifestyle modification program and the control group before and after the one-year intervention are shown in Fig. 3, which shows that the lifestyle modification program induced a borderline-significant reduction of 3.8% of PCSK9 levels. The effects of the lifestyle modification program on other parameters listed in Table 1 have been previously published [25]. In order to determine whether changes in PCSK9 levels were associated with changes in other cardiometabolic risk markers, we have divided participants included in the lifestyle modification program in two groups, based on mean changes in plasma PCSK9 levels (below or equal or above median). Table 2 shows mean changes in cardiometabolic risk markers in patients with PCSK9 changes below (lower responders) or equal or above (higher responders) the median change in PCSK9 level (9.23 ng/ml). Changes in plasma PCSK9 levels were not correlated with changes in indices of adiposity but were significantly associated with changes in fasting insulin levels (r ¼ 0.20, p ¼ 0.04) and HOMA-IR
323
Table 1 Baseline characteristics of the study participants classified on the basis of plasma PCSK9 levels. PCSK9 range, ng/ml
Number of participants
Tertile 1
Tertile 2
Tertile 3
<212.5
212.5e<256.0
256.0
58
58
59
PCSK9, ng/ml 182.2 Age, years 47.5 Body mass index, kg/m2 31.4 Waist circumference, cm 108.8 Systolic blood 117 pressure, mm Hg Diastolic blood 78 pressure, mm Hg 3 Adipose tissue, cm Total 3682 Subcutaneous 1821 Visceral 1870 Fat-free mass 66.3 Fat mass 29.8 Exercise output 7.64 at 150 BPM, METs Heart rate, 116 3.5mph/2%, BPM Plasma lipoproteins Total cholesterol, mmol/L 4.86 VLDL cholesterol, mmol/L 0.90 LDL cholesterol, mmol/L 3.04 HDL cholesterol, mmol/L 0.96 Triglycerides, mmol/L 2.05 Apolipoprotein B, g/L 1.04 Apolipoprotein A-I, g/L 1.11 Apolipoprotein C-III, mg/L 32.3 HDL size, Å 82.1 Plasma glucose-insulin homeostasis Fasting glucose, mmol/L 6.02 120-min OGTT-glucose, 7.83 mmol/L Fasting insulin, pmol/L 143.8 120-min OGTT-insulin, 930.4 pmol/L HOMA-IR 5.54 ISI-Matsuda 2.45 Adipokines/Inflammatory markers C-reactive protein, mg/L 3.58 Adiponectin, mg/ml 3.77 Leptin, ng/ml 10.86 Interleukin-6, pg/ml 1.42 Tumor-necrosis 1.17 factor-a, pg/ml
(26.2) (9.1) (3.1) (9.2) (9) (7)
(846) (559) (523) (6.5) (6.3) (1.55)
233.8 48.8 31.0 107.8 118
(12.8)a (7.7) (3.6) (9.6) (12)
80 (8)
3733 1759 1993 65.1 29.4 7.49
(970) (633) (488) (7.4) (8.0) (1.59)
303.2 46.3 30.8 107.6 117
(43.9)a,b (9.3) (2.6) (7.2) (12)
78 (8)
3627 1771 1856 64.6 28.7 7.45
(782) (650) (378) (7.5) (6.3) (1.21)
(13)
117 (15)
118 (12)
(0.83) (0.47) (0.74) (0.20) (0.84) (0.19) (0.15) (13.4) (2.5)
5.12 0.99 3.18 0.95 2.38 1.09 1.14 32.4 81.6
5.29 1.14 3.16 0.98 2.73 1.13 1.18 36.8 81.7
(0.44) (1.69)
5.95 (0.44) 7.59 (1.75)
6.02 (0.52) 7.79 (1.58)
(77.1) (576.9)
154.1 (70.0) 1080.6 (673.0)
174.3 (72.7)a 1181.7 (670.5)a
(2.91) (4.93)
5.91 (2.88) 1.62 (0.92)
(8.79) (1.38) (7.36) (1.20) (0.52)
2.35 3.68 11.53 1.31 1.07
(0.75) (0.35) (0.67) (0.18) (0.69)a (0.16) (0.16) (8.5) (2.0)
(2.04) (1.48) (8.24) (0.79) (0.69)
(0.77) (0.48) (0.67) (0.17) (0.97)a (0.17)a (0.15)a (10.6)a,b (2.2)
6.78 (3.01)b 1.45 (1.03)a 2.67 3.68 13.00 1.23 1.42
(4.38) (1.47) (8.36) (0.64) (2.38)
PCSK9 indicates proprotein convertase subtilisin/kexin type 9, BPM indicates beats per minute, MET indicates metabolic equivalent, VLDL indicates very low-density lipoprotein, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein, OGTT indicates oral glucose tolerance test, HOMA-IR indicates homeostatic model of insulin resistance, ISI indicates insulin sensitivity index and DI indicates disposition index. Data are presented as mean (±SD). a Significantly different than tertile 1. b Significantly different than fertile 2.
index (r ¼ 0.19, p ¼ 0.04). We also found that changes in PCSK9 levels were not associated with cholesterol levels in large or medium-sized particles but were modestly correlated with changes in cholesterol levels in small LDL particles (r ¼ 0.16, p ¼ 0.08). 4. Discussion Results of the present study show that in initially sedentary and abdominally obese men, plasma PCSK9 levels are associated with several cardiometabolic risk markers such as glucose-insulin homeostasis parameters and markers of the lipoprotein profile including plasma triglyceride, apolipoprotein B, apolipoprotein A-I and apolipoprotein C-III levels. Plasma PCSK9 levels were not associated with circulating LDL cholesterol levels but were
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Fig. 1. Association between plasma levels of PCSK9 and LDL particle size (A), cholesterol levels in large (B), medium (C) and small (D) particles.
positively associated with cholesterol levels in small LDL particles. We found no association between plasma PCSK9 levels and cardiorespiratory fitness or body fat distribution indices. We also found that our lifestyle modification program had a trivial clinical impact on PCSK9 levels. Previous studies that have examined the impact of plasma PCSK9 levels on insulin levels reported modest associations with PCSK9 levels and insulin levels or the HOMA-IR index. Our study confirms this finding and also suggests that PCSK9 levels are also associated with the insulin sensitivity index. Indeed, participants in the top PCSK9 levels tertile have approximately 40% lower ISIs compared to participants in the bottom tertile. There is however little evidence in the literature on the directionality of this association. Kappelle and colleagues [26] investigated the impact of 24-h insulin infusions on PCSK9 levels in healthy participants and patients with type 2 diabetes and found no impact on PCSK9 levels. This is in accordance with the results of our study, which suggested that although our intervention had a strong impact on insulin sensitivity, the impact of the intervention on PCSK9 levels was
modest. However, patients with the highest PCSK9 decreases during the intervention had more important increases in insulin sensitivity. Interestingly, a study of French-Canadian patients showed that carriers a common polymorphism in the PCSK9 gene conferring low LDL cholesterol levels had higher HOMA-IR indices compared to non-carriers [27]. The results of an earlier report by Costet et al. [28] suggested that hepatic PCSK9 expression could be regulated by insulin via the sterol regulatory element-binding protein 1c, thereby providing a molecular connection between PCSK9 and insulin metabolism. Abdominally obese patients are commonly characterized by an insulin-resistance state. Under these circumstances, visceral adipocytes are plunged in a pro-inflammatory milieu, which enables them to secrete several adipokines that are detrimental for the glucose-insulin homeostasis such as interleukin-6, tumor-necrosis factor-a, interleukin-1b, resistin, leptin and many more [29]. Visceral adipocytes secrete less of the insulin-sensitizing hormone adiponectin as visceral adipose tissue expands [30]. In our study, we found no association whatsoever between plasma PCSK9 levels
Fig. 2. Plasma levels of PCSK9 insulin levels during an oral glucose tolerance test (A) and insulin area under the curve during the oral glucose tolerance test (B) in men according to PCSK9 tertiles.
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325
Table 2 Changes in cardiometabolic risk markers in men with higher vs. lower responses in PCSK9 during the one-year lifestyle modification program. Number of participants
Fig. 3. Impact of the lifestyle modification program on plasma PCSK9 levels in men of the intervention and the control groups.
and these adipokines. We also found no association between visceral adipose tissue accumulation and PCSK9 levels, which suggest that the relationship between PCSK9 and markers of insulin resistance may occur via mechanisms that are independent of body fat distribution. Our results also suggest that unlike the recently reported association between loss of PCSK9 and increased visceral fat in mice, [9] PCSK9 does not appear to be influenced by body fat distribution in humans. However, we have not studied the impact of loss- or gain-of-function genetic mutations at the PCSK9 locus, on body fat distribution and believe that our results should be replicated in the context of more severe PCSK9 variations. The impact of PCSK9 inhibition on glucose-insulin homeostasis and body fat distribution should also be investigated. Several studies have shown that PCSK9 contributes to a certain extent to the variation in plasma lipoprotein-lipid levels. In our study, we found no associations between PCSK9 levels and LDL cholesterol levels. In other populations, circulating PCSK9 levels has been shown to be modestly associated with LDL cholesterol levels. However, the strength of the association between PCSK9 and LDL cholesterol levels was at best modest. To our knowledge, our study is the first to suggest that PCSK9 levels may not be associated with cholesterol levels in large LDL particles but may be more closely associated with cholesterol levels in small LDL particles. The mechanism underlying this observation is not completely understood. However, the association between PCSK9 levels with both triglyceride and apoC-III levels that we have observed may suggest that PCSK9 levels influences the catabolism of triglyceride-rich lipoproteins. Previous results from our group has shown that cholesterol levels in small LDL particles were closely association with cardiovascular risk while cholesterol levels in large LDL particles were not associated with cardiovascular risk in a large European prospective study [22]. Statin therapy is the first-line therapy for the prevention and treatment of CAD. By lowering intracellular cholesterol concentrations, statins also upregulate the LDLR, which further contributes to the uptake of LDL particles at the cell surface. However, the statin-dependent increase in LDL particle clearance mostly favor the uptake of large LDL particles and statins are not as efficient as clearing smaller LDL particles. On the other hand, statins upregulate PCSK9 expression and secretion, which may limit their efficiency in terms of LDL-lowering [31]. This observation, combined with our study results, could be another explanation on why statin therapy has a null impact on the small LDL phenotype. Given the fact that PCSK9 inhibitors are currently being tested for their potential to reduce cardiovascular outcomes in high-risk study population, it
Lower responders
Higher responders
59
58
PCSK9, ng/ml 31.5 Body mass index, kg/m2 2.3 Waist circumference, cm 8.9 Systolic blood 1.1 pressure, mm Hg Diastolic blood 4.9 pressure, mm Hg 3 Adipose tissue, cm Total 890 Subcutaneous 355 Visceral 523 Fat-free mass 1.05 Fat mass 6.26 Exercise output 1.36 at 150 BPM, METs 14.0 Heart rate, 3.5mph/2%, BPM Plasma lipoproteins Total cholesterol, mmol/L 0.01 VLDL cholesterol, mmol/L 0.22 LDL cholesterol, mmol/L 0.11 HDL cholesterol, mmol/L 0.10 Triglycerides, mmol/L 0.38 Apolipoprotein B, g/L 0.03 Apolipoprotein A-I, g/L 0.16 Apolipoprotein C-III, mg/L 1.15 LDL size, Å 1.08 LDL-C<255 Å, mmol/L 0.01 LDL-C255-260 Å, mmol/L 0.07 LDL-C>260 Å, mmol/L 0.02 HDL size, Å 2.38 Plasma glucose-insulin homeostasis Fasting glucose, mmol/L 0.10 120-min OGTT-glucose, 1.03 mmol/L Fasting insulin, pmol/L 39.8 120-min OGTT-insulin, 445.7 pmol/L HOMA-IR 1.62 ISI-Matsuda 0.47 Adipokines/Inflammatory markers C-reactive protein, mg/L 0.77 Adiponectin, mg/ml 0.58 Leptin, ng/ml 3.02 Interleukin-6, pg/ml 0.11 Tumor-necrosis 0.06 factor-a, pg/ml
(28.6)a (1.7)a (5.7)a (9.8) (7.5)a
50.7 2.0 8.4 3.2
(39.6)a (1.3)a (5.1)a (9.9)a
4.5 (6.8)a
P-value
<0.0001 0.44 0.64 0.26 0.77
(598)a (257)a (394)a (1.89)a (3.98)a (1.43)a
762 460 302 0.46 5.70 1.34
(512)a (292)a (266)a (1.66)a (3.60)a (1.23)a
0.24 0.30 0.34 0.08 0.89 0.56
(13.4)a
12.3 (8.59)a
0.45
(0.70) (0.34)a (0.60) (0.12)a (0.69)a (0.16) (0.10)a (11.20) (3.43)a (0.30) (0.16)a (0.30) (3.60)a
0.05 0.34 0.13 0.16 0.67 0.06 0.19 1.78 1.76 0.11 0.10 0.04 2.17
(0.14) (0.42)a (0.54) (0.14)a (0.97)a (0.14)a (0.14)a (8.85) (3.06)a (0.40)a (0.19)a (0.19) (3.17)a
0.76 0.10 0.84 0.03 0.07 0.28 0.18 0.72 0.27 0.08 0.28 0.59 0.77
(0.37)a (2.11)a
0.11 (0.39)a 1.07 (1.54)a
0.88 0.94
(64.2)a (465.1)a
72.0 (70.0)a 558.5 (527.3)a
0.02 0.22
(2.70)a (2.29)a
2.89 (2.87)a 1.73 (5.76)a
0.07 0.07
(2.08)a (0.99)a (4.03)a (0.82) (0.46)
0.63 1.00 3.91 0.18 0.24
(1.84)a (1.16)a (4.13)a (0.72)a (1.62)
0.78 0.18 0.36 0.47 0.80
PCSK9 indicates proprotein convertase subtilisin/kexin type 9, BPM indicates beats per minute, MET indicates metabolic equivalent, VLDL indicates very low-density lipoprotein, LDL indicates low-density lipoprotein, HDL indicates high-density lipoprotein, OGTT indicates oral glucose tolerance test, HOMA-IR indicates homeostatic model of insulin resistance, ISI indicates insulin sensitivity index and DI indicates disposition index. Data are presented as mean (±SD). a Indicates a significant changes from baseline values.
would be important to document the impact of PCSK9 inhibitor on LDL size and on the concentration of cholesterol in small LDL particles to determine whether it would be a valuable add-on therapy to statins. Our study has limitations. First, we do not know if any of the study participants had mutations at the PCSK9 locus and whether some of the examples of phenotypic variation reported here could be secondary to such mutations. Additionally, we have investigated the impact of PCSK9 variations in a relatively homogenous population of initially sedentary and abdominally obese men, which might explain why our reported associated were relatively modest. We believe that further studies in women, other ethnic groups and in samples with a wider BMI range could be useful to confirm and
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extend our findings. Finally, although our study is the first to our knowledge to document the impact of weight loss on PCSK9 levels, the weight loss that was achieved was relatively modest. On the other hand, our intervention has been proven sufficient to induce a significant amount of total and visceral fat and to improve insulin sensitivity [32]. In conclusion, our study provides evidence that plasma PCSK9 levels are associated with several parameters of the glucose-insulin homeostasis and parameters of the lipoprotein-lipid profile, including small, dense LDL particles. We documented that lifestyle modification therapy had a modest impact on PCSK9 levels. Whether pharmacological intervention leading to a more robust inhibition of PCSK9 influences body fat distribution, insulin resistance and small LDL particles should be further explored in ongoing PCSK9 inhibition trials. Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This study was supported by the Canadian Institutes of Health Research (MOP-64439). We would like to thank Sylvain Pouliot for his technical help. B.J.A. holds a junior scholar award from the bec: Sante (FRQS). P.P. holds a senior Fonds de recherche du Que scholar award from the FRQS. References [1] Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003;34:154e6. [2] Nordestgaard BG, Chapman MJ, Humphries SE, Ginsberg HN, Masana L, Descamps OS, et al., European atherosclerosis society consensus P. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European atherosclerosis society. Eur Heart J 2013;34:3478e3490a. [3] Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, et al. Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet 2013;45:25e33. [4] Cohen JC, Boerwinkle E, Mosley Jr TH, Hobbs HH. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264e72. [5] Benn M, Nordestgaard BG, Grande P, Schnohr P, Tybjaerg-Hansen A. PCSK9 R46L, low-density lipoprotein cholesterol levels, and risk of ischemic heart disease: 3 independent studies and meta-analyses. J Am Coll Cardiol 2010;55: 2833e42. [6] Lagace TA, Curtis DE, Garuti R, McNutt MC, Park SW, Prather HB, et al. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest 2006;116:2995e3005. [7] Zhang DW, Lagace TA, Garuti R, Zhao Z, McDonald M, Horton JD, et al. Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factorlike repeat a of low density lipoprotein receptor decreases receptor recycling and increases degradation. J Biol Chem 2007;282:18602e12. [8] Denis M, Marcinkiewicz J, Zaid A, Gauthier D, Poirier S, Lazure C, et al. Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice. Circulation 2012;125:894e901. [9] Roubtsova A, Munkonda MN, Awan Z, Marcinkiewicz J, Chamberland A, Lazure C, et al. Circulating proprotein convertase subtilisin/kexin 9 (pcsk9) regulates VLDLR protein and triglyceride accumulation in visceral adipose tissue. Arterioscler Thromb Vasc Biol 2011;31:785e91.
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