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Plasma proprotein convertase subtilisin–kexin type 9 does not change during 24 h insulin infusion in healthy subjects and type 2 diabetic patients
Atherosclerosis 214 (2011) 432–435
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Plasma proprotein convertase subtilisin–kexin type 9 does not change during 24 h insulin infusion in healthy subjects and type 2 diabetic patients Paul J.W.H. Kappelle a,1 , Gilles Lambert b,c,1 , Robin P.F. Dullaart a,∗ a
Department of Endocrinology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands The Heart Research Institute, Sydney, Australia c Université de Nantes, Faculté de Médecine, Nantes, France b
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 18 October 2010 Accepted 22 October 2010 Available online 2 November 2010 Key words: Apolipoprotein B Insulin Hyperinsulinemia Proprotein convertase subtilisin–kexin type 9 Type 2 diabetes mellitus
a b s t r a c t Purpose: Proprotein convertase subtilisin–kexin type 9 (PCSK9) promotes low density lipoprotein (LDL) receptor degradation, thereby providing a key pathway for LDL metabolism. PCSK9 mRNA expression may be upregulated by insulin in murine models. Here we examined effects of exogenous hyperinsulinemia on plasma PCSK9 levels in humans without and with type 2 diabetes mellitus. Methods: A 24 h moderately hyperinsulinemic glucose clamp (30 mU/kg/h) was performed in 8 healthy men and 8 male type 2 diabetic patients. Plasma PCSK9 was measured using a sandwich enzyme-linked immunosorbent assay. Results: Plasma LDL cholesterol and apolipoprotein B were lowered by insulin in healthy subjects and diabetic patients (P < 0.01 for all), whereas triglycerides were also decreased in healthy subjects (P < 0.01). Plasma PCSK9 levels remained unchanged in healthy subjects (median (interquartile range) change, −23 (−63 to 25) %, P = 0.50) and in diabetic patients (change, 4 (−17 to 44) %, P = 0.20). Individual absolute and relative changes in LDL cholesterol, apolipoprotein B and triglycerides after 24 h of insulin were unrelated to changes in PCSK9 (P > 0.15 for all). Conclusion: Plasma PCSK9 levels are not increased by exposure to moderate 24 h hyperinsulinemia in healthy and type 2 diabetic individuals. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Proprotein convertase subtilisin–kexin type 9 (PCSK9) plays a key role in low density lipoprotein (LDL) receptor processing [1–4]. PCSK9 promotes LDL receptor degradation by complex mechanisms which involve intracellular targeting of internalized LDL receptors towards the lysosomal compartment [1,2]. PCSK9 is produced mainly by the liver, targets the LDL receptor towards intracellular degradation and prevents LDL receptor recycling to the cell surface [1,2]. Plasma apolipoprotein B (apoB)-containing lipoprotein levels are correlated positively with PCSK9 levels in humans [5–7]. Moreover, the LDL apoB fractional catabolic rate is correlated inversely with plasma PCSK9 levels [8]. Variations in plasma PCSK9 concentrations are, therefore, likely to have physiological relevance [1,2]. PCSK9 metabolism is regulated by nutritional status in murine models. Prolonged fasting reduces, whereas insulin stim-
∗ Corresponding author at: Department of Endocrinology, University Medical Center Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands. Tel.: +31 50 361 3731; fax: +31 50 361 9392. E-mail address: [email protected] (R.P.F. Dullaart). 1 Contributed equally to the manuscript. 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.10.028
ulates PCSK9 transcription, although posttranscriptional effects are unclear at present [9,10]. Hepatic PCSK9 protein levels are decreased in severely hyperglycemic, insulinopenic rats after streptozotocin-induced diabetes [11]. Interestingly, plasma PCSK9 levels are correlated positively with insulin and insulin resistance in adult and adolescent subjects [6,7], with higher levels being observed in diabetic patients as well [6]. In the absence of human data concerning effects of exogenous hyperinsulinemia on PCSK9 regulation, we determined whether plasma PCSK9 is affected by insulin infusion in healthy subjects and in type 2 diabetic patients.
2. Materials and methods 2.1. Subjects The protocol was approved by the medical ethics committee of the University Medical Centre Groningen. Non-smoking men were recruited by advertisement and provided written informed consent. Eight healthy control subjects and 8 subjects with previously diagnosed type 2 diabetes mellitus, who did not use lipid lowering drug treatment, were selected for the study. Their medical history did not reveal prior cardiovascular, thyroid or renal
P.J.W.H. Kappelle et al. / Atherosclerosis 214 (2011) 432–435
diseases or familial hyperlipidemia. At the screening visit, physical examination did not show abnormalities. Blood pressure was <160 mm Hg systolic and <95 mm Hg diastolic in all participants. Serum thyreotropin, transaminase levels and creatinine concentrations were within their respective reference ranges. Body mass index (BMI, in kg/m2 ) was calculated as weight divided by height squared. 2.2. Study protocol The participants did not use alcohol and fasted from 8 p.m. onwards on the day before the study. They were admitted at the metabolic ward at 7 a.m., and remained in the supine position during the infusion study. The diabetic patients did not use their glucose lowering drugs during the study day. In healthy subjects, a 24 h euglycemic–hyperinsulinemic clamp was performed. Target blood glucose was approximately 0.4 mmol/l below the fasting level to inhibit endogenous insulin secretion. In diabetic subjects, a 24 h isoglycemic–hyperinsulinemic clamp was performed, i.e. glucose was maintained at the individual fasting baseline level. This was done in the expectation that achievement of normoglycemia would require much higher insulin levels in diabetic compared to control subjects. The cannula of one hand vein was kept open with a saline infusion (154 mmol/l NaCl; 30 ml/h). This hand was placed in a thermoregulated box with an ambient temperature of 55 ◦ C to obtain arterialized venous blood [12]. An antecubital vein of the contralateral arm was used for administration of insulin and glucose. Insulin was given at a rate of 30 mU/kg/h, preceded by an insulin bolus of 5 mU/kg body weight. Blood glucose was measured at 10 min intervals. A variable glucose infusion (20% wt/wt), with potassium chloride (20 mmol/l of glucose) being added to prevent hypokalemia, was given to maintain the blood glucose level. After 1 h rest, i.e. just prior to the start of the glucose clamp, baseline blood samples for measurement of plasma insulin, lipids, apoB and PCSK9 levels were obtained. Subsequent blood samples were taken after 8 and 24 h of insulin infusion. The participants consumed a 1000 kcal diet during the study day. 2.3. Laboratory analyses EDTA-containing plasma samples (1.5 mg/ml) were stored at −80 ◦ C until analysis. Blood glucose was measured with a glu-
433
cose analyzer (APEC Inc., Danvers, MA, USA). Cholesterol and triglycerides were assayed by routine enzymatic methods. HDL cholesterol (HDL-C) was measured after removal of apolipoprotein B (apoB)-containing lipoproteins with polyethylene glycol-6000 precipitation [13]. LDL-C was calculated with the Friedewald formula. ApoB was measured by immunoturbidimetry (Boehringer Mannheim, Almere, The Netherlands, cat. no. 726494). PCSK9 was measured using a sandwich ELISA exactly as described [8]. The inter-assay CV was 5.6%. HbA1c was measured by high performance liquid chromatography (Biorad, Veenendaal, The Netherlands, upper normal value 6.1%). 2.4. Statistical analysis Data are given as median (interquartile range). Between group differences in variables were analyzed with Mann–Whitney Utests. Changes in variables in response to insulin were analyzed by Friedman two-way analysis of variance; Duncan’s method was applied to correct for multiple comparisons. Correlation coefficients were calculated using Spearman’s rank analysis. Two-sided P-values <0.05 were considered to be significant. 3. Results Age was not different between control and diabetic subjects (Table 1). Three diabetic patients used sulfonylurea alone and 5 patients also used metformin. Other medications were not used. BMI, systolic and diastolic blood pressure and baseline plasma total cholesterol levels were higher in diabetic patients (Table 1). HDL-C was not different between diabetic and control subjects. Baseline glucose was higher in diabetic patients (Table 1). Glucose decreased slightly in control subjects and remained unchanged in diabetic patients. Plasma insulin levels rose similarly in both groups during insulin infusion (Table 1). LDL-C, plasma triglycerides and apoB levels were higher in diabetic patients compared to control subjects at baseline and during insulin infusion (Table 2). LDL-C and apoB levels were decreased in both groups after insulin (Table 2). Triglycerides were decreased in control subjects insulin, but did not change significantly in diabetic patients (P = 0.20). HDL-C dropped in both groups after insulin (P < 0.01 for both, not shown). PCSK9 levels were not significantly
Table 1 Clinical characteristics, plasma total cholesterol, high density lipoprotein cholesterol (HDL-C), as well as glucose and insulin during 24 h of insulin infusion in 8 control subjects and 8 type 2 diabetic patients.
Age (years) Diabetes duration (years) BMI (kg/m2 ) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) HbA1c (%) Total cholesterol (mmol/l) HDL-C (mmol/l) 24 h insulin infusion study Glucose (mmol/l) Baseline 8h 24 h Insulin (mU/l) Baseline 8h 24 h
Control subjects (n = 8)
Diabetic patients (n = 8)
P-value for difference between diabetic and control subjects
50 (43–59)
0.10
24.7 (21.6–25.9) 122 (118–137) 81 (78–87) 5.2 (4.9–5.3) 4.56 (4.10–4.70) 1.00 (0.91–1.34)
59 (55–62) 4 (3–6) 27.7 (26.7–28.8) 139 (134–145) 89 (88–91) 7.1 (6.6–7.8) 5.80 (5.29–6.02) 0.99 (0.86–1.09)
0.01 0.05 0.06 0.001 0.01 0.71
4.6 (4.3–4.9) 4.3 (4.1–4.7)* 4.3 (4.0–4.7)*
8.2 (6.3–9.1) 8.2 (6.1–9.2) 8.0 (6.0–9.4)
0.01 0.01 0.01
13 (9–16) 38 (27–47)** 32 (28–39)**
18 (12–37) 48 (35–65)† 40 (33–50)†
0.13 0.25 0.12
Data in median (interquartile range). BMI, body mass index. * P < 0.05 from baseline in control subjects. ** P < 0.01 from baseline in control subjects. † P < 0.01 from baseline in diabetic patients.
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Table 2 Changes in plasma low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein B (apoB) and proprotein convertase subtilisin–kexin type 9 (PCSK9) in response to 24 h insulin infusion in 8 control subjects and 8 type 2 diabetic patients. LDL-C (mmol/l) Control subjects Baseline 2.89 (2.61–3.18) 8h 2.93 (2.48–3.17) 24 h 2.74 (2.28–3.20)*
Triglycerides (mmol/l)
ApoB (g/l)
Diabetic patients
Control subjects
Diabetic patients
Control subjects
Diabetic patients
PCSK9 (g/l)
4.00 (3.57–4.29) 3.64 (3.32–4.14) 3.86 (3.36–4.06)†
0.83 (0.66–1.19) 1.03 (0.63–1.46) 0.70 (0.47–1.05)**
1.81 (1.08–2.13) 1.64 (1.32–2.79) 1.40 (1.02–1.93)
0.55 (0.52–0.61) 0.55 (0.52–0.61) 0.53 (0.45–0.60)**
0.74 (0.68–0.85) 114 (87–147) 0.72 (0.65–0.78) 91 (82–150) 0.68 (0.63–0.83)†† 95 (79–126)
Control subjects Diabetic patients 146 (110–152) 112 (91–143) 115 (103–199)
Data in median (interquartile range). LDL-C, triglycerides and apoB levels were higher in diabetic patients vs. control subjects at baseline, as well as after 8 h and 24 h insulin infusion (P < 0.05 for all comparisons). * P < 0.05 from baseline in control subjects. ** P < 0.01 from baseline in control subjects. † P < 0.05 from baseline in diabetic patients. †† P < 0.01 from baseline in diabetic patients.
different between diabetic patients and control subjects at baseline (P = 0.35), and did not significantly change in control subjects (P = 0.50) and diabetic patients during insulin (P = 0.20; Table 2). The absolute and relative changes in plasma PCSK9 in response to insulin were not different between control subjects (−20 (−35 to 28) g/l and −23 (−63 to 25) %, respectively) and diabetic patients (−5 (−27 to 49) g/l, P = 0.54 and −4 (−17 to 44) %, P = 0.34, respectively). In both groups combined, there was also no significant change in plasma PCSK9 after insulin (P = 0.60). In the combined subjects, individual absolute changes in PCSK9 after 24 h insulin were inversely related to the baseline level (n = 16, r = −0.506, P = 0.05). No consistent trend for relationships between either absolute or relative changes in plasma PCSK9 and changes in LDL-C, triglycerides and apoB after 24 h were observed (P > 0.15 for all comparisons, not shown).
unrelated to changes in PCSK9. Obviously, we cannot exclude effects of chronic supra-physiological insulin concentrations on circulating PCSK9. Furthermore, diabetes-associated elevated PCSK9 levels, as demonstrated in the Dallas Heart Study [6], could contribute to impaired LDL receptor availability in the diabetic state [20]. In conclusion, plasma PCSK9 does not increase after exposure to moderate 24 h hyperinsulinemia in healthy and type 2 diabetic individuals. Acknowledgments This study is in part supported by a grant from the Dutch Diabetes Research Foundation (to RPFD), and a grant in aid G-08S-3700 from the Heart Research Foundation of Australia (to GL). We thank Ms. Francine Petrides for excellent technical assistance.
4. Discussion References This study demonstrates that prolonged insulin infusion, aimed at reaching high physiological insulin concentrations, decreases plasma LDL cholesterol and apoB levels, but does not affect plasma PCSK9 in small groups of healthy subjects and type 2 diabetic patients. We suggest that moderate hyperinsulinemia does not have a major impact on circulating PCSK9 in humans. Our findings make it also less plausible that a potential contribution of insulin on LDL receptor expression in vivo is to a considerable extent exerted via the PCSK9 pathway, as far as extracellular levels are involved. Insulin has multiple effects on hepatic lipoprotein metabolism [14,15]. Insulin acutely decreases apoB and triglyceride production. This effect is in part attributable to diminished availability of free fatty acids (FFA) that are required for hepatic triglyceride synthesis [15]. The extent to which hepatic secretion of large very low density lipoprotein particles is affected by hyperinsulinemia closely relates to the degree of FFA suppression by insulin [16]. Thus, impaired FFA suppression may explain the lack of significant decrease in plasma triglycerides in response to insulin in (obese) diabetic subjects [17; present report]. Additionally, insulin may stimulate LDL receptor activity in vitro [18,19]. Chronic insulin treatment normalizes the reduced cell surface LDL receptor expression observed in type 2 diabetes [20], which probably contributes to an increase in LDL catabolism after insulin [21]. An inverse relation of extracellular PCSK9 with LDL receptor abundancy and LDL catabolism has been proposed [1,2,8]. Consequently, an increase in plasma PCSK9 levels during insulin infusion would not directly concur with the hypothesis that the PCSK9 pathway, as judged from its plasma concentration, provides a predominant regulatory mechanism to explain stimulatory effects of insulin on LDL receptor regulation [18,19]. Albeit in a limited number of subjects, the higher the PCSK9 level at baseline, the more pronounced was the drop after insulin, although individual changes in LDL-C, apoB and triglycerides were
[1] Horton JD, Cohen JC, Hobbs HH. PCSK9: a convertase that coordinates LDL catabolism. J Lipid Res 2009;50(Suppl.):S172–7. [2] Lambert G, Charlton F, Rye KA, Piper DE. Molecular basis of PCSK9 function. Atherosclerosis 2009;203:1–7. [3] Abifadel M, Varret M, Rabès JP, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003;34:154–6. [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;354:1264–72. [5] Lambert G, Ancellin N, Charlton F, et al. Plasma PCSK9 concentrations correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment. Clin Chem 2008;54:1034–45. [6] Lakoski SG, Lagace TA, Cohen JC, Horton JD, Hobbs HH. Genetic and metabolic determinants of plasma PCSK9 levels. J Clin Endocrinol Metab 2009;94:2537–43. [7] Baass A, Dubuc G, Tremblay M, 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;55:1637–45. [8] Chan DC, Lambert G, Barrett PH, et al. Plasma proprotein convertase subtilisin/kexin type 9: a marker of LDL apolipoprotein B-100 catabolism? Clin Chem 2009;55:2049–52. [9] Costet P, Cariou B, Lambert G, et al. Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c. J Biol Chem 2006;281:6211–8. [10] Persson L, Gälman C, Angelin B, Rudling M. Importance of proprotein convertase subtilisin/kexin type 9 in the hormonal and dietary regulation of rat liver lowdensity lipoprotein receptors. Endocrinology 2009;150:1140–6. [11] Niesen M, Bedi M, Lopez D. Diabetes alters LDL receptor and PCSK9 expression in rat liver. Arch Biochem Biophys 2008;470:111–5. [12] Jensen MD, Heiling VJ. Heated hand vein blood is satisfactory for measurements during free fatty acid kinetic studies. Metabolism 1991;40:406–9. ´ Laar A, Jansen AP. A study [13] Demacker PN, Hijmans AG, Vos-Janssen HE, Vant of the use of polyethylene glycol in estimating cholesterol in high density lipoprotein. Clin Chem 1980;26:1775–9. [14] Marsh JB, Welty FK, Lichtenstein AH, Lamon-Fava S, Schaefer EJ. Apolipoprotein B metabolism in humans: studies with stable isotope-labeled amino acid precursors. Atherosclerosis 2002;162:227–44. [15] Taskinen MR. Diabetic dyslipidaemia: from basic research to clinical practice. Diabetologia 2003;46:733–49. [16] Adiels M, Westerbacka J, Soro-Paavonen A, et al. Acute suppression of VLDL1 secretion rate by insulin is associated with hepatic fat content and insulin resistance. Diabetologia 2007;50:2356–65.
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LDL receptor activity and diminishes its suppression by exogenous LDL. Eur J Biochem 1988;174:213–8. [20] Duvillard L, Florentin E, Lizard G, et al. Cell surface expression of LDL receptor is decreased in type 2 diabetic patients and is normalized by insulin therapy. Diabetes Care 2003;26:1540–4. [21] Duvillard L, Pont F, Florentin E, Gambert P, Vergès B. Significant improvement of apolipoprotein B-containing lipoprotein metabolism by insulin treatment in patients with non-insulin-dependent diabetes mellitus. Diabetologia 2000;43:27–35.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Plasma proprotein convertase subtilisin–kexin type 9 does not change during 24 h insulin infusion in healthy subjects and type 2 diabetic patients Paul J.W.H. Kappelle a,1 , Gilles Lambert b,c,1 , Robin P.F. Dullaart a,∗ a
Department of Endocrinology, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands The Heart Research Institute, Sydney, Australia c Université de Nantes, Faculté de Médecine, Nantes, France b
a r t i c l e
i n f o
Article history: Received 16 September 2010 Received in revised form 18 October 2010 Accepted 22 October 2010 Available online 2 November 2010 Key words: Apolipoprotein B Insulin Hyperinsulinemia Proprotein convertase subtilisin–kexin type 9 Type 2 diabetes mellitus
a b s t r a c t Purpose: Proprotein convertase subtilisin–kexin type 9 (PCSK9) promotes low density lipoprotein (LDL) receptor degradation, thereby providing a key pathway for LDL metabolism. PCSK9 mRNA expression may be upregulated by insulin in murine models. Here we examined effects of exogenous hyperinsulinemia on plasma PCSK9 levels in humans without and with type 2 diabetes mellitus. Methods: A 24 h moderately hyperinsulinemic glucose clamp (30 mU/kg/h) was performed in 8 healthy men and 8 male type 2 diabetic patients. Plasma PCSK9 was measured using a sandwich enzyme-linked immunosorbent assay. Results: Plasma LDL cholesterol and apolipoprotein B were lowered by insulin in healthy subjects and diabetic patients (P < 0.01 for all), whereas triglycerides were also decreased in healthy subjects (P < 0.01). Plasma PCSK9 levels remained unchanged in healthy subjects (median (interquartile range) change, −23 (−63 to 25) %, P = 0.50) and in diabetic patients (change, 4 (−17 to 44) %, P = 0.20). Individual absolute and relative changes in LDL cholesterol, apolipoprotein B and triglycerides after 24 h of insulin were unrelated to changes in PCSK9 (P > 0.15 for all). Conclusion: Plasma PCSK9 levels are not increased by exposure to moderate 24 h hyperinsulinemia in healthy and type 2 diabetic individuals. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Proprotein convertase subtilisin–kexin type 9 (PCSK9) plays a key role in low density lipoprotein (LDL) receptor processing [1–4]. PCSK9 promotes LDL receptor degradation by complex mechanisms which involve intracellular targeting of internalized LDL receptors towards the lysosomal compartment [1,2]. PCSK9 is produced mainly by the liver, targets the LDL receptor towards intracellular degradation and prevents LDL receptor recycling to the cell surface [1,2]. Plasma apolipoprotein B (apoB)-containing lipoprotein levels are correlated positively with PCSK9 levels in humans [5–7]. Moreover, the LDL apoB fractional catabolic rate is correlated inversely with plasma PCSK9 levels [8]. Variations in plasma PCSK9 concentrations are, therefore, likely to have physiological relevance [1,2]. PCSK9 metabolism is regulated by nutritional status in murine models. Prolonged fasting reduces, whereas insulin stim-
∗ Corresponding author at: Department of Endocrinology, University Medical Center Groningen, PO Box 30001, 9700 RB Groningen, The Netherlands. Tel.: +31 50 361 3731; fax: +31 50 361 9392. E-mail address: [email protected] (R.P.F. Dullaart). 1 Contributed equally to the manuscript. 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.10.028
ulates PCSK9 transcription, although posttranscriptional effects are unclear at present [9,10]. Hepatic PCSK9 protein levels are decreased in severely hyperglycemic, insulinopenic rats after streptozotocin-induced diabetes [11]. Interestingly, plasma PCSK9 levels are correlated positively with insulin and insulin resistance in adult and adolescent subjects [6,7], with higher levels being observed in diabetic patients as well [6]. In the absence of human data concerning effects of exogenous hyperinsulinemia on PCSK9 regulation, we determined whether plasma PCSK9 is affected by insulin infusion in healthy subjects and in type 2 diabetic patients.
2. Materials and methods 2.1. Subjects The protocol was approved by the medical ethics committee of the University Medical Centre Groningen. Non-smoking men were recruited by advertisement and provided written informed consent. Eight healthy control subjects and 8 subjects with previously diagnosed type 2 diabetes mellitus, who did not use lipid lowering drug treatment, were selected for the study. Their medical history did not reveal prior cardiovascular, thyroid or renal
P.J.W.H. Kappelle et al. / Atherosclerosis 214 (2011) 432–435
diseases or familial hyperlipidemia. At the screening visit, physical examination did not show abnormalities. Blood pressure was <160 mm Hg systolic and <95 mm Hg diastolic in all participants. Serum thyreotropin, transaminase levels and creatinine concentrations were within their respective reference ranges. Body mass index (BMI, in kg/m2 ) was calculated as weight divided by height squared. 2.2. Study protocol The participants did not use alcohol and fasted from 8 p.m. onwards on the day before the study. They were admitted at the metabolic ward at 7 a.m., and remained in the supine position during the infusion study. The diabetic patients did not use their glucose lowering drugs during the study day. In healthy subjects, a 24 h euglycemic–hyperinsulinemic clamp was performed. Target blood glucose was approximately 0.4 mmol/l below the fasting level to inhibit endogenous insulin secretion. In diabetic subjects, a 24 h isoglycemic–hyperinsulinemic clamp was performed, i.e. glucose was maintained at the individual fasting baseline level. This was done in the expectation that achievement of normoglycemia would require much higher insulin levels in diabetic compared to control subjects. The cannula of one hand vein was kept open with a saline infusion (154 mmol/l NaCl; 30 ml/h). This hand was placed in a thermoregulated box with an ambient temperature of 55 ◦ C to obtain arterialized venous blood [12]. An antecubital vein of the contralateral arm was used for administration of insulin and glucose. Insulin was given at a rate of 30 mU/kg/h, preceded by an insulin bolus of 5 mU/kg body weight. Blood glucose was measured at 10 min intervals. A variable glucose infusion (20% wt/wt), with potassium chloride (20 mmol/l of glucose) being added to prevent hypokalemia, was given to maintain the blood glucose level. After 1 h rest, i.e. just prior to the start of the glucose clamp, baseline blood samples for measurement of plasma insulin, lipids, apoB and PCSK9 levels were obtained. Subsequent blood samples were taken after 8 and 24 h of insulin infusion. The participants consumed a 1000 kcal diet during the study day. 2.3. Laboratory analyses EDTA-containing plasma samples (1.5 mg/ml) were stored at −80 ◦ C until analysis. Blood glucose was measured with a glu-
433
cose analyzer (APEC Inc., Danvers, MA, USA). Cholesterol and triglycerides were assayed by routine enzymatic methods. HDL cholesterol (HDL-C) was measured after removal of apolipoprotein B (apoB)-containing lipoproteins with polyethylene glycol-6000 precipitation [13]. LDL-C was calculated with the Friedewald formula. ApoB was measured by immunoturbidimetry (Boehringer Mannheim, Almere, The Netherlands, cat. no. 726494). PCSK9 was measured using a sandwich ELISA exactly as described [8]. The inter-assay CV was 5.6%. HbA1c was measured by high performance liquid chromatography (Biorad, Veenendaal, The Netherlands, upper normal value 6.1%). 2.4. Statistical analysis Data are given as median (interquartile range). Between group differences in variables were analyzed with Mann–Whitney Utests. Changes in variables in response to insulin were analyzed by Friedman two-way analysis of variance; Duncan’s method was applied to correct for multiple comparisons. Correlation coefficients were calculated using Spearman’s rank analysis. Two-sided P-values <0.05 were considered to be significant. 3. Results Age was not different between control and diabetic subjects (Table 1). Three diabetic patients used sulfonylurea alone and 5 patients also used metformin. Other medications were not used. BMI, systolic and diastolic blood pressure and baseline plasma total cholesterol levels were higher in diabetic patients (Table 1). HDL-C was not different between diabetic and control subjects. Baseline glucose was higher in diabetic patients (Table 1). Glucose decreased slightly in control subjects and remained unchanged in diabetic patients. Plasma insulin levels rose similarly in both groups during insulin infusion (Table 1). LDL-C, plasma triglycerides and apoB levels were higher in diabetic patients compared to control subjects at baseline and during insulin infusion (Table 2). LDL-C and apoB levels were decreased in both groups after insulin (Table 2). Triglycerides were decreased in control subjects insulin, but did not change significantly in diabetic patients (P = 0.20). HDL-C dropped in both groups after insulin (P < 0.01 for both, not shown). PCSK9 levels were not significantly
Table 1 Clinical characteristics, plasma total cholesterol, high density lipoprotein cholesterol (HDL-C), as well as glucose and insulin during 24 h of insulin infusion in 8 control subjects and 8 type 2 diabetic patients.
Age (years) Diabetes duration (years) BMI (kg/m2 ) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) HbA1c (%) Total cholesterol (mmol/l) HDL-C (mmol/l) 24 h insulin infusion study Glucose (mmol/l) Baseline 8h 24 h Insulin (mU/l) Baseline 8h 24 h
Control subjects (n = 8)
Diabetic patients (n = 8)
P-value for difference between diabetic and control subjects
50 (43–59)
0.10
24.7 (21.6–25.9) 122 (118–137) 81 (78–87) 5.2 (4.9–5.3) 4.56 (4.10–4.70) 1.00 (0.91–1.34)
59 (55–62) 4 (3–6) 27.7 (26.7–28.8) 139 (134–145) 89 (88–91) 7.1 (6.6–7.8) 5.80 (5.29–6.02) 0.99 (0.86–1.09)
0.01 0.05 0.06 0.001 0.01 0.71
4.6 (4.3–4.9) 4.3 (4.1–4.7)* 4.3 (4.0–4.7)*
8.2 (6.3–9.1) 8.2 (6.1–9.2) 8.0 (6.0–9.4)
0.01 0.01 0.01
13 (9–16) 38 (27–47)** 32 (28–39)**
18 (12–37) 48 (35–65)† 40 (33–50)†
0.13 0.25 0.12
Data in median (interquartile range). BMI, body mass index. * P < 0.05 from baseline in control subjects. ** P < 0.01 from baseline in control subjects. † P < 0.01 from baseline in diabetic patients.
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P.J.W.H. Kappelle et al. / Atherosclerosis 214 (2011) 432–435
Table 2 Changes in plasma low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein B (apoB) and proprotein convertase subtilisin–kexin type 9 (PCSK9) in response to 24 h insulin infusion in 8 control subjects and 8 type 2 diabetic patients. LDL-C (mmol/l) Control subjects Baseline 2.89 (2.61–3.18) 8h 2.93 (2.48–3.17) 24 h 2.74 (2.28–3.20)*
Triglycerides (mmol/l)
ApoB (g/l)
Diabetic patients
Control subjects
Diabetic patients
Control subjects
Diabetic patients
PCSK9 (g/l)
4.00 (3.57–4.29) 3.64 (3.32–4.14) 3.86 (3.36–4.06)†
0.83 (0.66–1.19) 1.03 (0.63–1.46) 0.70 (0.47–1.05)**
1.81 (1.08–2.13) 1.64 (1.32–2.79) 1.40 (1.02–1.93)
0.55 (0.52–0.61) 0.55 (0.52–0.61) 0.53 (0.45–0.60)**
0.74 (0.68–0.85) 114 (87–147) 0.72 (0.65–0.78) 91 (82–150) 0.68 (0.63–0.83)†† 95 (79–126)
Control subjects Diabetic patients 146 (110–152) 112 (91–143) 115 (103–199)
Data in median (interquartile range). LDL-C, triglycerides and apoB levels were higher in diabetic patients vs. control subjects at baseline, as well as after 8 h and 24 h insulin infusion (P < 0.05 for all comparisons). * P < 0.05 from baseline in control subjects. ** P < 0.01 from baseline in control subjects. † P < 0.05 from baseline in diabetic patients. †† P < 0.01 from baseline in diabetic patients.
different between diabetic patients and control subjects at baseline (P = 0.35), and did not significantly change in control subjects (P = 0.50) and diabetic patients during insulin (P = 0.20; Table 2). The absolute and relative changes in plasma PCSK9 in response to insulin were not different between control subjects (−20 (−35 to 28) g/l and −23 (−63 to 25) %, respectively) and diabetic patients (−5 (−27 to 49) g/l, P = 0.54 and −4 (−17 to 44) %, P = 0.34, respectively). In both groups combined, there was also no significant change in plasma PCSK9 after insulin (P = 0.60). In the combined subjects, individual absolute changes in PCSK9 after 24 h insulin were inversely related to the baseline level (n = 16, r = −0.506, P = 0.05). No consistent trend for relationships between either absolute or relative changes in plasma PCSK9 and changes in LDL-C, triglycerides and apoB after 24 h were observed (P > 0.15 for all comparisons, not shown).
unrelated to changes in PCSK9. Obviously, we cannot exclude effects of chronic supra-physiological insulin concentrations on circulating PCSK9. Furthermore, diabetes-associated elevated PCSK9 levels, as demonstrated in the Dallas Heart Study [6], could contribute to impaired LDL receptor availability in the diabetic state [20]. In conclusion, plasma PCSK9 does not increase after exposure to moderate 24 h hyperinsulinemia in healthy and type 2 diabetic individuals. Acknowledgments This study is in part supported by a grant from the Dutch Diabetes Research Foundation (to RPFD), and a grant in aid G-08S-3700 from the Heart Research Foundation of Australia (to GL). We thank Ms. Francine Petrides for excellent technical assistance.
4. Discussion References This study demonstrates that prolonged insulin infusion, aimed at reaching high physiological insulin concentrations, decreases plasma LDL cholesterol and apoB levels, but does not affect plasma PCSK9 in small groups of healthy subjects and type 2 diabetic patients. We suggest that moderate hyperinsulinemia does not have a major impact on circulating PCSK9 in humans. Our findings make it also less plausible that a potential contribution of insulin on LDL receptor expression in vivo is to a considerable extent exerted via the PCSK9 pathway, as far as extracellular levels are involved. Insulin has multiple effects on hepatic lipoprotein metabolism [14,15]. Insulin acutely decreases apoB and triglyceride production. This effect is in part attributable to diminished availability of free fatty acids (FFA) that are required for hepatic triglyceride synthesis [15]. The extent to which hepatic secretion of large very low density lipoprotein particles is affected by hyperinsulinemia closely relates to the degree of FFA suppression by insulin [16]. Thus, impaired FFA suppression may explain the lack of significant decrease in plasma triglycerides in response to insulin in (obese) diabetic subjects [17; present report]. Additionally, insulin may stimulate LDL receptor activity in vitro [18,19]. Chronic insulin treatment normalizes the reduced cell surface LDL receptor expression observed in type 2 diabetes [20], which probably contributes to an increase in LDL catabolism after insulin [21]. An inverse relation of extracellular PCSK9 with LDL receptor abundancy and LDL catabolism has been proposed [1,2,8]. Consequently, an increase in plasma PCSK9 levels during insulin infusion would not directly concur with the hypothesis that the PCSK9 pathway, as judged from its plasma concentration, provides a predominant regulatory mechanism to explain stimulatory effects of insulin on LDL receptor regulation [18,19]. Albeit in a limited number of subjects, the higher the PCSK9 level at baseline, the more pronounced was the drop after insulin, although individual changes in LDL-C, apoB and triglycerides were
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