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
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
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
(PCSK9) as a susceptibility locus for familial hypercholesterolemia (FH) . 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 . 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 . 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 . 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 . Another study however, did not ﬁnd any features of insulin resistance in Pcsk9/ mice.  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) . 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 . 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  whereas waist and hip circumferences were measured using standardized procedures, as previously described . 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 . 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 . 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
Table 1 Baseline characteristics of the study participants classiﬁed on the basis of plasma PCSK9 levels. PCSK9 range, ng/ml
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  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 . The results of an earlier report by Costet et al.  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 . Visceral adipocytes secrete less of the insulin-sensitizing hormone adiponectin as visceral adipose tissue expands . 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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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,  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 . 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 . 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
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 . 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  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.  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.  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.  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.  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.  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.  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.  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.  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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