- Email: [email protected]
Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults
+Model
ARTICLE IN PRESS
DIABET-749; No. of Pages 4
Available online at
ScienceDirect www.sciencedirect.com Diabetes & Metabolism xxx (2016) xxx–xxx
Short Report
Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults M. Monseu a , S. Dubois b , J. Boursier c , C. Aubé d , F. Gagnadoux e , G. Lefthériotis f , P.-H. Ducluzeau g,∗ a
Department of Internal Medicine, CHU Tours, 2, boulevard Tonnellé, Tours, France b Department of Diabetology, CHU, Angers, France c Department of Hepatology, CHU, Angers, France d Department of Radiology, CHU, Angers, France e Department of Pneumology, CHU, Angers, France f Department of Vascular Explorations, CHU, Angers, France g Inserm UMR1069, “Nutrition, Growth and Cancer”, CHRU Bretonneau, University of Tours, 37044 Tours, France Received 21 December 2015; received in revised form 18 February 2016; accepted 24 February 2016
Abstract Aim. – This study aimed to determine the association between visceral adipose tissue (VAT), liver fat (LF) content, and other markers of the metabolic syndrome (MetS) and osteoprotegerin (OPG) in dysmetabolic adults. Methods. – Subjects from the NUMEVOX cohort were included if they fulfilled at least one MetS criterion. They then underwent a thorough metabolic and cardiovascular evaluation, including arterial stiffness, atherosclerotic plaques, homoeostasis model assessment for insulin resistance (HOMA-IR) indices and OPG. VAT and LF content were measured by magnetic resonance imaging (MRI). Ultrasound examination of arteries and arterial stiffness were recorded, and age- and gender-adjusted paired correlations calculated. Results. – Body mass index, waist circumference and MRI-derived VAT correlated with OPG, whereas abdominal subcutaneous fat did not. OPG levels were strongly correlated with LF content (r = 0.25, P = 0.003), liver markers such as alanine aminotransferase (r = 0.39, P < 0.001) and HOMA-IR index (r = 0.39, P < 0.0001). Plasma OPG also correlated with arterial stiffness and the number of atherosclerotic sites. Conclusion. – Plasma OPG levels are positively associated with both liver markers and increased LF content, but not with subcutaneous fat in dysmetabolic men. These findings suggest that elevated OPG levels may play a role in the link between fatty liver disease and enhanced cardiovascular risk. © 2016 Elsevier Masson SAS. All rights reserved. Keywords: Insulin resistance; Liver steatosis; Metabolic syndrome; Osteoprotegerin; Vascular complications
1. Introduction
Abbreviations: OPG, osteoprotegerin; VAT, visceral adipose tissue; SAT, subcutaneous adipose tissue; LF, liver fat content; IDF, International Diabetes Federation; HOMA-IR, HOMA insulin resistance index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. ∗ Corresponding author. Service de Médecine Interne, CHU Tours, 2, boulevard Tonnellé, Tours, France. E-mail addresses: [email protected] (M. Monseu), [email protected] (S. Dubois), [email protected] (J. Boursier), [email protected] (C. Aubé), [email protected] (F. Gagnadoux), [email protected] (G. Lefthériotis), [email protected] (P.-H. Ducluzeau).
Osteoprotegerin (OPG) is an inflammatory cytokine receptor implicated in bone remodelling. It is a soluble glycoprotein, of the tumour necrosis factor receptor superfamily, responsible for osteoclastogenesis inhibition. OPG is expressed in vivo by vascular smooth muscle cells, hepatic cells and osteoblasts, and its evaluation has been a key factor in understanding the close relationship between bone mineralization and vascular pathology [1]. Clinical studies suggest that OPG might also be a risk factor for progressive atherosclerotic cardiovascular disease [2]. Indeed, OPG has been associated with both
http://dx.doi.org/10.1016/j.diabet.2016.02.004 1262-3636/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model DIABET-749; No. of Pages 4 2
ARTICLE IN PRESS M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
the increased risk and severity of atherosclerotic diseases, and correlates positively with intima–media thickness and coronary artery calcification [3]. Moreover, circulating OPG is an independent predictor of long-term mortality and heart-failure development in patients with acute coronary syndromes [4]. Patients with higher OPG levels have higher major adverse cardiac event rates at midterm follow-up [5]. A prospective study showed that, for each doubling of plasma OPG concentration, the risk of subclinical peripheral atherosclerosis increased by 50% [6]. Altogether, these results suggest that OPG plays a key clinical role in atherosclerosis progression and plaque destabilization. The metabolic syndrome (MetS) comprises a constellation of vascular risk factors, including obesity, dyslipidaemia, hyperglycaemia, hypertension and insulin resistance, leading to an increased cardiovascular risk. Liver steatosis is a key component of MetS progression [7]. Furthermore, little is known of the associations between visceral adipose tissue (VAT), liver fat (LF) content and plasma OPG in dysmetabolic subjects. With OPG being expressed in vascular and hepatic cells, the crosstalk of these cells and their role in MetS and vascular complications need to be further explored. The objective of our present work was to determine the association between plasma OPG levels and intra-abdominal fat depots, including LF content, metabolic and vascular variables, in a French adult cohort displaying at least one of the five MetS criteria. 2. Research design and methods
2.2. Biological measurements Samples were taken during fasting before any medications had been taken. For each patient, fasting glucose, insulin, HbA1c , liver enzymes, creatinine, microalbuminuria, C-reactive protein (CRP), ferritin, leptin, adiponectin, cholesterol and triglyceride (TG) levels were measured. The homoeostasis model assessment for insulin resistance (HOMA-IR) index was calculated as described previously [9]. Serum adiponectin was measured by sandwich enzyme-linked immunosorbent assay (ELISA; DRG Diagnostics, Marburg, Germany); leptin was also measured by sandwich ELISA (EMD Millipore, Billerica, MA, USA), and plasma OPG was measured using highly sensitive quantitative sandwich ELISA (BioVendor R&D, Modˇrice, Czech Republic) [10]. 2.3. Imaging studies Ultrasound examination of arteries and arterial stiffness (pulse wave velocity) were recorded using laser Doppler velocimetry analysis. Plaque score was established by counting the number of arterial sites presenting with atherosclerosis (from 0 to 5). LF content, intra- and retroperitoneal VAT, and subcutaneous adipose tissue (SAT) were assessed from an abdominal MRI scan in a subset of 145 patients. Individuals who did and did not undergo MRI had similar ages and BMI scores. Imaging was carried out with a 1.5-Tesla MRI scanner using a phased-array surface coil. LF proportions (in percentage) as well as VAT and SAT areas (in cm2 at the level of the L3–L4 intervertebral disc) were quantified using previously validated software [7].
2.1. Study protocols and patient selection 2.4. Statistical analyses Subjects from the NUMEVOX cohort were included and transversally analyzed. The objective of this cohort (registered on clinicaltrials.gov: NCT00997165) was to describe the impact of fat distribution on vascular and metabolic progression in patients with at least one MetS criterion. Included patients were referred for metabolic exploration at the Department of Nutrition of the Angers University Hospital, France. Patients excluded from the cohort were those presenting with any of the following criteria: age < 18 or > 80 years; body mass index (BMI) > 40 kg/m2 ; poorly controlled diabetes with HbA1c > 9%; insulin-treated diabetes; uncontrolled severe arterial hypertension (> 180/120 mmHg); severe hypertriglyceridaemia (triglyceride > 10 g/L); severe renal failure (creatinine clearance < 30 mL/min); or any counterindication for magnetic resonance imaging (MRI). No patient presented with obvious acute disease at the time of evaluation. Patients received their usual medications and were treated, if necessary, by antihypertensive medications and statins. The presence of MetS was defined according to International Diabetes Federation (IDF) 2005 criteria [8]. The study was approved by the institutional ethics committee and, for each patient, oral and written consent was obtained in a process validated by the Ethical Research Committee of Angers University Hospital.
All analyses used Open Source R Statistical Programming Language 2.10.0 (downloaded from the Internet), and a twosided P value < 0.05 was considered statistically significant. Spearman correlation coefficients (r) were determined for OPG with markers of MetS, hepatic and vascular variables, or MRIassessed LF, VAT and SAT. All the correlations were adjusted for age and gender. All quantitative variables showed an asymmetrical distribution and were therefore log-transformed before being entered into the models. 3. Results A total of 314 subjects (72% male) were considered in the present analysis. Baseline characteristics of the study population are shown in Table 1. Participants had, on average, 2.86 MetS IDF criteria; 105 patients had no MetS criteria according to the IDF definition, while 70% of patients had at least three criteria and thus presented with MetS. Among these latter patients, 86 also had known type 2 diabetes. Mean plasma OPG was 14.2 ± 5.0 pmol/L (Table 1). After adjusting for age and gender, circulating OPG levels correlated with anthropometric variables such as BMI, and waist and hip circumferences. A positive correlation was also found
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model
ARTICLE IN PRESS
DIABET-749; No. of Pages 4
M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx Table 1 Baseline characteristics of the study population.
3
Table 2 Spearman correlation coefficients (r) between anthropometric, metabolic, hepatic and vascular variables and plasma osteoprotegerin (adjusted for age and gender).
Variables
n
Gender: male/female [n (%)] Age (years) Active smoking [n (%)] Type 2 diabetes [n (%)] IDF criteria (n) Osteoprotegerin (pmol/L)
314 314 314 314 314 314
227 (72%)/87 (28%) 54.3 ± 9.7 56 (18%) 95 (39%) 2.86 ± 1.19 14.2 ± 5.0
Anthropometric variables Body mass index (kg/m2 ) Waist circumference (cm) Hip circumference (cm) VAT (cm2 ) SAT (cm2 )
314 314 314 145 145
31.4 ± 5.3 104.8 ± 12.9 107.7 ± 10.6 186.9 ± 92 242.4 ± 124.3
Metabolic variables Glycaemia (g/L) HbA1c (%) Insulin (mIU/L) HOMA-IR (mU/L) Leptin (g/mL) Adiponectin (g/mL) HDL cholesterol (g/L) LDL cholesterol (g/L) Triglycerides (g/L)
314 314 314 314 297 297 314 314 314
1.1 ± 0.2 6.1 ± 0.7 17.2 ± 10.8 4.8 ± 3.5 20 ± 18.4 7.2 ± 3.4 0.58 ± 0.2 1.12 ± 0.4 1.68 ± 1.0
Hepatic variables Liver fat content (%) ␥-GT (U/L) ALT (U/L) Bilirubin (mol/L) Ferritin (g/mL)
145 314 314 314 314
Vascular variables Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Intima–media thickness Arterial stiffness Plaque score
314 314 296 296 296
Variables
r
P
Anthropometric variables Body mass index Waist circumference Hip circumference VAT SAT
0.195 0.183 0.150 0.241 0.145
0.0006 0.001 0.008 0.004 0.09
Metabolic variables HbA1c Insulin HOMA-IR Leptin Adiponectin HDL cholesterol LDL cholesterol Triglycerides
0.197 0.368 0.394 0.285 −0.114 −0.072 −0.006 0.165
0.0005 < 0.0001 < 0.0001 < 0.0001 0.05 0.21 0.92 0.004
Hepatic variables Liver fat content ␥-GT ALT Bilirubin Ferritin
0.252 0.351 0.392 0.021 0.181
0.003 < 0.0001 < 0.0001 0.72 0.002
13.5 ± 13.4 62.2 ± 70.5 38.5 ± 23.2 4.4 ± 4.0 290.2 ± 251.7
Vascular variables Systolic blood pressure Diastolic blood pressure Intima–media thickness Arterial stiffness Plaque score
0.068 0.051 0.050 0.193 0.125
0.24 0.38 0.40 0.04 0.003
129.5 ± 13.7 77.9 ± 9.6 0.63 ± 0.09 8.46 ± 2.86 2.53 ± 1.94
IDF: International Diabetes Federation; VAT: visceral adipose tissue; SAT: subcutaneous adipose tissue; HOMA-IR: homoeostasis model assessment for insulin resistance; HDL/LDL: high-density/low-density lipoprotein; ␥-GT: gamma-glutamyl transferase; ALT: alanine aminotransferase.
Quantitative variables are expressed as means ± standard deviation unless otherwise specified. IDF: International Diabetes Federation; VAT: visceral adipose tissue; SAT: subcutaneous adipose tissue; HOMA-IR: homoeostasis model assessment for insulin resistance; HDL/LDL: high-density/low-density lipoprotein; ␥-GT: gamma-glutamyl transferase; ALT: alanine aminotransferase.
with MRI-based VAT, but not with SAT (Table 2). HOMAIR was strongly correlated with OPG, as well as leptinaemia, adiponectinaemia and triglyceridaemia. Moreover, circulating OPG levels correlated with vascular variables such as arterial stiffness (pulse wave velocity) and the number of atherosclerotic sites, but not with blood pressure or intima–media thickness. Finally, after adjusting for age and gender, circulating OPG levels correlated strongly with hepatic variables alanine aminotransferase (ALT), gamma-glutamyl transferase (␥-GT) and plasma ferritin levels as well as MRI-assessed LF content (Table 2). 4. Discussion In the present cohort of dysmetabolic subjects, plasma OPG levels were positively associated with VAT, insulin resistance and adipokines. OPG was also associated with arterial stiffness
and the number of atherosclerotic sites. Interestingly, our study has shown, for the first time, that OPG was also associated with liver steatosis markers and LF content. Previous studies that investigated the correlation between OPG and MetS revealed conflicting findings. On the one hand, no relationship was found between OPG and MetS in postmenopausal women [11], and no significant correlations were found between OPG and BMI, waist circumference, systolic and diastolic blood pressure, TG, high-density lipoprotein (HDL) cholesterol, leptin and adiponectin in either obese or non-obese subjects [12]. On the other hand, higher OPG levels were associated with the risk of MetS in type 2 diabetes patients as well as in women with previous gestational diabetes [13]. In such cases, serum OPG levels were associated with BMI, insulin resistance, serum CRP levels and carotid intima–media thickness [13]. In one male cohort, a statistical association between OPG and insulin sensibility, adiponectin and sex steroids was demonstrated for the first time [14]. Our present results show that circulating OPG was associated with three of the five major components of MetS (waist circumference, insulin resistance and hypertriglyceridaemia).
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model DIABET-749; No. of Pages 4 4
ARTICLE IN PRESS M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
OPG is known to regulate bone mineral metabolism, and is associated with cardiovascular disease and mortality. Recent evidence supports a relationship between serum OPG levels and atherosclerosis: Ciccone et al. [5] demonstrated the relationship between intima–media thickness of the common carotid artery and OPG levels in patients with acute coronary syndrome and chronic coronary artery disease. Patients with atheroma plaques had higher OPG levels, as did those with coronary artery calcifications [2]. Major adverse cardiac events were more commonly seen in patients with higher baseline OPG levels [15]. However, our present study found no correlation of OPG with intima–media thickness, although it did highlight a positive correlation with arterial stiffness (as evaluated by Doppler pulse wave velocity) and plaque score in the whole cohort of 314 subjects. Non-alcoholic fatty liver disease (NAFLD) has also been associated with atherosclerotic cardiovascular disease outcomes, although many of those studies were performed predominantly in diabetic populations [16]. However, fewer population-based studies have evaluated the association with both clinical and biological markers of liver steatosis and OPG blood levels. One paper described decreased OPG blood levels in NAFLD patients treated by metformin [17]. To the best of our knowledge, our present study is the first to describe a correlation between OPG and hepatic variables such as ALT and ferritin levels, and suggests that OPG might mediate, at least in part, the previously observed association between elevated liver enzymes and increased risk of cardiovascular disease. In addition, along with endothelial cells and osteoblasts, hepatocytes are able to produce OPG; thus, an increased production of OPG in the presence of fatty liver and its related inflammation may be postulated. However, LF content was measured on MRI scans only for the last 145 subjects entered into our cohort, and showed a positive relationship with OPG levels and percentage of liver TG content after adjusting for age and gender. Also, the size of our sample was too small to adjust for level of VAT, although the strength of the relationship with ALT and ␥-GT argue in favour of a predominant role of liver steatosis in the increased production of OPG observed in MetS. In addition, our study had yet another limitation: hepatic fat content was measured only in a subset of our cohort. Nevertheless, this subgroup with liver MRI scans did not differ from the rest of the cohort. However, the crosssectional nature of our study precludes any demonstration of causality for the relationship observed. 5. Conclusion The significant positive association found between OPG and some of the major components of MetS as well as LF content suggest a possible role of liver steatosis in the overproduction of OGP in metabolic patients. Longitudinal studies now need to be done to determine whether OPG might be a useful biomarker to identify steatosis patients at risk of vascular complications.
Disclosure of interest The authors declare that they have no competing interest. References [1] St˛epie´n E, Fedak D, Klimeczek P, Wilkosz T, Bany´s RP, Starzyk K, et al. Osteoprotegerin, but not osteopontin, as a potential predictor of vascular calcification in normotensive subjects. Hypertens Res 2012;35:531–8. [2] Pérez de Ciriza C, Moreno M, Restituto P, Bastarrika G, Simon I, Colina I, et al. Circulating osteoprotegerin is increased in the metabolic syndrome and associates with subclinical atherosclerosis and coronary arterial calcification. Clin Biochem 2014;47:272–8. [3] Ciccone MM, Scicchitano P, Gesualdo M, Zito A, Carbonara R, Locorotondo M, et al. Serum osteoprotegerin and carotid intima-media thickness in acute/chronic coronary artery diseases. J Cardiovasc Med 2013;14:43–8. [4] Jansson AM, Hartford M, Omland T, Karlsson T, Lindmarker P, Herlitz J, et al. Multimarker risk assessment including osteoprotegerin and CXCL16 in acute coronary syndromes. Arterioscler Thromb Vasc Biol 2012;32:3041–9. [5] Ghaffari S, Yaghoubi A, Baghernejad R, Sepehrvand N, Sokhanvar S, Haghjou AG. The value of serum osteoprotegerin levels in patients with angina like chest pain undergoing diagnostic coronary angiography. Cardiol J 2013;20:261–7. [6] Mogelvang R, Pedersen SH, Flyvbjerg A, Bjerre M, Iversen AZ, Galatius S, et al. Comparison of osteoprotegerin to traditional atherosclerotic risk factors and high-sensitivity C-Reactive Protein for diagnosis of atherosclerosis. Am J Cardiol 2012;109:515–20. [7] Ducluzeau PH, Boursier J, Bertrais S, Dubois S, Gauthier A, Rohmer V, et al. MRI measurement of liver fat content predicts the metabolic syndrome. Diabetes Metab 2013;39:314–21. [8] Alberti KG, Zimmet P, Shaw J. The metabolic syndrome – a new worldwide definition. Lancet 2005;366:1059–62. [9] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [10] Silva TE, Colombo G, Schiavon LL. Adiponectin: a multitasking player in the field of liver diseases. Diabetes Metab 2014;40:95–107. [11] Nabipour I, Kalantarhormozi M, Larijani B, Assadi M, Sanjdideh Z. Osteoprotegerin in relation to type 2 diabetes mellitus and the metabolic syndrome in postmenopausal women. Metabolism 2010;59:742–7. [12] Gannagé-Yared MH, Yaghi C, Habre B, Khalife S, Noun R, GermanosHaddad M, et al. Osteoprotegerin in relation to body weight, lipid parameters insulin sensitivity, adipocytokines, and C-reactive protein in obese and non-obese young individuals: results from both cross-sectional and interventional study. Eur J Endocrinol 2008;158:353–9. [13] Akinci B, Celtik A, Yuksel F, Genc S, Yener S, Secil M, et al. Increased osteoprotegerin levels in women with previous gestational diabetes developing metabolic syndrome. Diabetes Res Clin Pract 2011;91:26–31. [14] Gannagé-Yared MH, Fares F, Semaan M, Khalife S, Jambart S. Circulating osteoprotegerin is correlated with lipid profile, insulin sensitivity, adiponectin and sex steroids in an ageing male population. Clin Endocrinol (Oxf) 2006;64:652–8. [15] Venuraju SM, Yerramasu A, Corder R, Lahiri A. Osteoprotegerin as a predictor of coronary artery disease and cardiovascular mortality and morbidity. J Am Coll Cardiol 2010;55:2049–61. [16] Targher G, Bertolini L, Rodella S, Tessari R, Zenari L, Lippi G, et al. Nonalcoholic fatty liver disease is independently associated with an increased incidence of cardiovascular events in type 2 diabetic patients. Diabetes Care 2007;30:2116–21. [17] Sofer E, Shargorodsky M. Effect of metformin treatment on circulating osteoprotegerin in patients with nonalcoholic fatty liver disease. Hepatol Int 2015;7:121–8.
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
ARTICLE IN PRESS
DIABET-749; No. of Pages 4
Available online at
ScienceDirect www.sciencedirect.com Diabetes & Metabolism xxx (2016) xxx–xxx
Short Report
Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults M. Monseu a , S. Dubois b , J. Boursier c , C. Aubé d , F. Gagnadoux e , G. Lefthériotis f , P.-H. Ducluzeau g,∗ a
Department of Internal Medicine, CHU Tours, 2, boulevard Tonnellé, Tours, France b Department of Diabetology, CHU, Angers, France c Department of Hepatology, CHU, Angers, France d Department of Radiology, CHU, Angers, France e Department of Pneumology, CHU, Angers, France f Department of Vascular Explorations, CHU, Angers, France g Inserm UMR1069, “Nutrition, Growth and Cancer”, CHRU Bretonneau, University of Tours, 37044 Tours, France Received 21 December 2015; received in revised form 18 February 2016; accepted 24 February 2016
Abstract Aim. – This study aimed to determine the association between visceral adipose tissue (VAT), liver fat (LF) content, and other markers of the metabolic syndrome (MetS) and osteoprotegerin (OPG) in dysmetabolic adults. Methods. – Subjects from the NUMEVOX cohort were included if they fulfilled at least one MetS criterion. They then underwent a thorough metabolic and cardiovascular evaluation, including arterial stiffness, atherosclerotic plaques, homoeostasis model assessment for insulin resistance (HOMA-IR) indices and OPG. VAT and LF content were measured by magnetic resonance imaging (MRI). Ultrasound examination of arteries and arterial stiffness were recorded, and age- and gender-adjusted paired correlations calculated. Results. – Body mass index, waist circumference and MRI-derived VAT correlated with OPG, whereas abdominal subcutaneous fat did not. OPG levels were strongly correlated with LF content (r = 0.25, P = 0.003), liver markers such as alanine aminotransferase (r = 0.39, P < 0.001) and HOMA-IR index (r = 0.39, P < 0.0001). Plasma OPG also correlated with arterial stiffness and the number of atherosclerotic sites. Conclusion. – Plasma OPG levels are positively associated with both liver markers and increased LF content, but not with subcutaneous fat in dysmetabolic men. These findings suggest that elevated OPG levels may play a role in the link between fatty liver disease and enhanced cardiovascular risk. © 2016 Elsevier Masson SAS. All rights reserved. Keywords: Insulin resistance; Liver steatosis; Metabolic syndrome; Osteoprotegerin; Vascular complications
1. Introduction
Abbreviations: OPG, osteoprotegerin; VAT, visceral adipose tissue; SAT, subcutaneous adipose tissue; LF, liver fat content; IDF, International Diabetes Federation; HOMA-IR, HOMA insulin resistance index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. ∗ Corresponding author. Service de Médecine Interne, CHU Tours, 2, boulevard Tonnellé, Tours, France. E-mail addresses: [email protected] (M. Monseu), [email protected] (S. Dubois), [email protected] (J. Boursier), [email protected] (C. Aubé), [email protected] (F. Gagnadoux), [email protected] (G. Lefthériotis), [email protected] (P.-H. Ducluzeau).
Osteoprotegerin (OPG) is an inflammatory cytokine receptor implicated in bone remodelling. It is a soluble glycoprotein, of the tumour necrosis factor receptor superfamily, responsible for osteoclastogenesis inhibition. OPG is expressed in vivo by vascular smooth muscle cells, hepatic cells and osteoblasts, and its evaluation has been a key factor in understanding the close relationship between bone mineralization and vascular pathology [1]. Clinical studies suggest that OPG might also be a risk factor for progressive atherosclerotic cardiovascular disease [2]. Indeed, OPG has been associated with both
http://dx.doi.org/10.1016/j.diabet.2016.02.004 1262-3636/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model DIABET-749; No. of Pages 4 2
ARTICLE IN PRESS M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
the increased risk and severity of atherosclerotic diseases, and correlates positively with intima–media thickness and coronary artery calcification [3]. Moreover, circulating OPG is an independent predictor of long-term mortality and heart-failure development in patients with acute coronary syndromes [4]. Patients with higher OPG levels have higher major adverse cardiac event rates at midterm follow-up [5]. A prospective study showed that, for each doubling of plasma OPG concentration, the risk of subclinical peripheral atherosclerosis increased by 50% [6]. Altogether, these results suggest that OPG plays a key clinical role in atherosclerosis progression and plaque destabilization. The metabolic syndrome (MetS) comprises a constellation of vascular risk factors, including obesity, dyslipidaemia, hyperglycaemia, hypertension and insulin resistance, leading to an increased cardiovascular risk. Liver steatosis is a key component of MetS progression [7]. Furthermore, little is known of the associations between visceral adipose tissue (VAT), liver fat (LF) content and plasma OPG in dysmetabolic subjects. With OPG being expressed in vascular and hepatic cells, the crosstalk of these cells and their role in MetS and vascular complications need to be further explored. The objective of our present work was to determine the association between plasma OPG levels and intra-abdominal fat depots, including LF content, metabolic and vascular variables, in a French adult cohort displaying at least one of the five MetS criteria. 2. Research design and methods
2.2. Biological measurements Samples were taken during fasting before any medications had been taken. For each patient, fasting glucose, insulin, HbA1c , liver enzymes, creatinine, microalbuminuria, C-reactive protein (CRP), ferritin, leptin, adiponectin, cholesterol and triglyceride (TG) levels were measured. The homoeostasis model assessment for insulin resistance (HOMA-IR) index was calculated as described previously [9]. Serum adiponectin was measured by sandwich enzyme-linked immunosorbent assay (ELISA; DRG Diagnostics, Marburg, Germany); leptin was also measured by sandwich ELISA (EMD Millipore, Billerica, MA, USA), and plasma OPG was measured using highly sensitive quantitative sandwich ELISA (BioVendor R&D, Modˇrice, Czech Republic) [10]. 2.3. Imaging studies Ultrasound examination of arteries and arterial stiffness (pulse wave velocity) were recorded using laser Doppler velocimetry analysis. Plaque score was established by counting the number of arterial sites presenting with atherosclerosis (from 0 to 5). LF content, intra- and retroperitoneal VAT, and subcutaneous adipose tissue (SAT) were assessed from an abdominal MRI scan in a subset of 145 patients. Individuals who did and did not undergo MRI had similar ages and BMI scores. Imaging was carried out with a 1.5-Tesla MRI scanner using a phased-array surface coil. LF proportions (in percentage) as well as VAT and SAT areas (in cm2 at the level of the L3–L4 intervertebral disc) were quantified using previously validated software [7].
2.1. Study protocols and patient selection 2.4. Statistical analyses Subjects from the NUMEVOX cohort were included and transversally analyzed. The objective of this cohort (registered on clinicaltrials.gov: NCT00997165) was to describe the impact of fat distribution on vascular and metabolic progression in patients with at least one MetS criterion. Included patients were referred for metabolic exploration at the Department of Nutrition of the Angers University Hospital, France. Patients excluded from the cohort were those presenting with any of the following criteria: age < 18 or > 80 years; body mass index (BMI) > 40 kg/m2 ; poorly controlled diabetes with HbA1c > 9%; insulin-treated diabetes; uncontrolled severe arterial hypertension (> 180/120 mmHg); severe hypertriglyceridaemia (triglyceride > 10 g/L); severe renal failure (creatinine clearance < 30 mL/min); or any counterindication for magnetic resonance imaging (MRI). No patient presented with obvious acute disease at the time of evaluation. Patients received their usual medications and were treated, if necessary, by antihypertensive medications and statins. The presence of MetS was defined according to International Diabetes Federation (IDF) 2005 criteria [8]. The study was approved by the institutional ethics committee and, for each patient, oral and written consent was obtained in a process validated by the Ethical Research Committee of Angers University Hospital.
All analyses used Open Source R Statistical Programming Language 2.10.0 (downloaded from the Internet), and a twosided P value < 0.05 was considered statistically significant. Spearman correlation coefficients (r) were determined for OPG with markers of MetS, hepatic and vascular variables, or MRIassessed LF, VAT and SAT. All the correlations were adjusted for age and gender. All quantitative variables showed an asymmetrical distribution and were therefore log-transformed before being entered into the models. 3. Results A total of 314 subjects (72% male) were considered in the present analysis. Baseline characteristics of the study population are shown in Table 1. Participants had, on average, 2.86 MetS IDF criteria; 105 patients had no MetS criteria according to the IDF definition, while 70% of patients had at least three criteria and thus presented with MetS. Among these latter patients, 86 also had known type 2 diabetes. Mean plasma OPG was 14.2 ± 5.0 pmol/L (Table 1). After adjusting for age and gender, circulating OPG levels correlated with anthropometric variables such as BMI, and waist and hip circumferences. A positive correlation was also found
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model
ARTICLE IN PRESS
DIABET-749; No. of Pages 4
M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx Table 1 Baseline characteristics of the study population.
3
Table 2 Spearman correlation coefficients (r) between anthropometric, metabolic, hepatic and vascular variables and plasma osteoprotegerin (adjusted for age and gender).
Variables
n
Gender: male/female [n (%)] Age (years) Active smoking [n (%)] Type 2 diabetes [n (%)] IDF criteria (n) Osteoprotegerin (pmol/L)
314 314 314 314 314 314
227 (72%)/87 (28%) 54.3 ± 9.7 56 (18%) 95 (39%) 2.86 ± 1.19 14.2 ± 5.0
Anthropometric variables Body mass index (kg/m2 ) Waist circumference (cm) Hip circumference (cm) VAT (cm2 ) SAT (cm2 )
314 314 314 145 145
31.4 ± 5.3 104.8 ± 12.9 107.7 ± 10.6 186.9 ± 92 242.4 ± 124.3
Metabolic variables Glycaemia (g/L) HbA1c (%) Insulin (mIU/L) HOMA-IR (mU/L) Leptin (g/mL) Adiponectin (g/mL) HDL cholesterol (g/L) LDL cholesterol (g/L) Triglycerides (g/L)
314 314 314 314 297 297 314 314 314
1.1 ± 0.2 6.1 ± 0.7 17.2 ± 10.8 4.8 ± 3.5 20 ± 18.4 7.2 ± 3.4 0.58 ± 0.2 1.12 ± 0.4 1.68 ± 1.0
Hepatic variables Liver fat content (%) ␥-GT (U/L) ALT (U/L) Bilirubin (mol/L) Ferritin (g/mL)
145 314 314 314 314
Vascular variables Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Intima–media thickness Arterial stiffness Plaque score
314 314 296 296 296
Variables
r
P
Anthropometric variables Body mass index Waist circumference Hip circumference VAT SAT
0.195 0.183 0.150 0.241 0.145
0.0006 0.001 0.008 0.004 0.09
Metabolic variables HbA1c Insulin HOMA-IR Leptin Adiponectin HDL cholesterol LDL cholesterol Triglycerides
0.197 0.368 0.394 0.285 −0.114 −0.072 −0.006 0.165
0.0005 < 0.0001 < 0.0001 < 0.0001 0.05 0.21 0.92 0.004
Hepatic variables Liver fat content ␥-GT ALT Bilirubin Ferritin
0.252 0.351 0.392 0.021 0.181
0.003 < 0.0001 < 0.0001 0.72 0.002
13.5 ± 13.4 62.2 ± 70.5 38.5 ± 23.2 4.4 ± 4.0 290.2 ± 251.7
Vascular variables Systolic blood pressure Diastolic blood pressure Intima–media thickness Arterial stiffness Plaque score
0.068 0.051 0.050 0.193 0.125
0.24 0.38 0.40 0.04 0.003
129.5 ± 13.7 77.9 ± 9.6 0.63 ± 0.09 8.46 ± 2.86 2.53 ± 1.94
IDF: International Diabetes Federation; VAT: visceral adipose tissue; SAT: subcutaneous adipose tissue; HOMA-IR: homoeostasis model assessment for insulin resistance; HDL/LDL: high-density/low-density lipoprotein; ␥-GT: gamma-glutamyl transferase; ALT: alanine aminotransferase.
Quantitative variables are expressed as means ± standard deviation unless otherwise specified. IDF: International Diabetes Federation; VAT: visceral adipose tissue; SAT: subcutaneous adipose tissue; HOMA-IR: homoeostasis model assessment for insulin resistance; HDL/LDL: high-density/low-density lipoprotein; ␥-GT: gamma-glutamyl transferase; ALT: alanine aminotransferase.
with MRI-based VAT, but not with SAT (Table 2). HOMAIR was strongly correlated with OPG, as well as leptinaemia, adiponectinaemia and triglyceridaemia. Moreover, circulating OPG levels correlated with vascular variables such as arterial stiffness (pulse wave velocity) and the number of atherosclerotic sites, but not with blood pressure or intima–media thickness. Finally, after adjusting for age and gender, circulating OPG levels correlated strongly with hepatic variables alanine aminotransferase (ALT), gamma-glutamyl transferase (␥-GT) and plasma ferritin levels as well as MRI-assessed LF content (Table 2). 4. Discussion In the present cohort of dysmetabolic subjects, plasma OPG levels were positively associated with VAT, insulin resistance and adipokines. OPG was also associated with arterial stiffness
and the number of atherosclerotic sites. Interestingly, our study has shown, for the first time, that OPG was also associated with liver steatosis markers and LF content. Previous studies that investigated the correlation between OPG and MetS revealed conflicting findings. On the one hand, no relationship was found between OPG and MetS in postmenopausal women [11], and no significant correlations were found between OPG and BMI, waist circumference, systolic and diastolic blood pressure, TG, high-density lipoprotein (HDL) cholesterol, leptin and adiponectin in either obese or non-obese subjects [12]. On the other hand, higher OPG levels were associated with the risk of MetS in type 2 diabetes patients as well as in women with previous gestational diabetes [13]. In such cases, serum OPG levels were associated with BMI, insulin resistance, serum CRP levels and carotid intima–media thickness [13]. In one male cohort, a statistical association between OPG and insulin sensibility, adiponectin and sex steroids was demonstrated for the first time [14]. Our present results show that circulating OPG was associated with three of the five major components of MetS (waist circumference, insulin resistance and hypertriglyceridaemia).
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004
+Model DIABET-749; No. of Pages 4 4
ARTICLE IN PRESS M. Monseu et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
OPG is known to regulate bone mineral metabolism, and is associated with cardiovascular disease and mortality. Recent evidence supports a relationship between serum OPG levels and atherosclerosis: Ciccone et al. [5] demonstrated the relationship between intima–media thickness of the common carotid artery and OPG levels in patients with acute coronary syndrome and chronic coronary artery disease. Patients with atheroma plaques had higher OPG levels, as did those with coronary artery calcifications [2]. Major adverse cardiac events were more commonly seen in patients with higher baseline OPG levels [15]. However, our present study found no correlation of OPG with intima–media thickness, although it did highlight a positive correlation with arterial stiffness (as evaluated by Doppler pulse wave velocity) and plaque score in the whole cohort of 314 subjects. Non-alcoholic fatty liver disease (NAFLD) has also been associated with atherosclerotic cardiovascular disease outcomes, although many of those studies were performed predominantly in diabetic populations [16]. However, fewer population-based studies have evaluated the association with both clinical and biological markers of liver steatosis and OPG blood levels. One paper described decreased OPG blood levels in NAFLD patients treated by metformin [17]. To the best of our knowledge, our present study is the first to describe a correlation between OPG and hepatic variables such as ALT and ferritin levels, and suggests that OPG might mediate, at least in part, the previously observed association between elevated liver enzymes and increased risk of cardiovascular disease. In addition, along with endothelial cells and osteoblasts, hepatocytes are able to produce OPG; thus, an increased production of OPG in the presence of fatty liver and its related inflammation may be postulated. However, LF content was measured on MRI scans only for the last 145 subjects entered into our cohort, and showed a positive relationship with OPG levels and percentage of liver TG content after adjusting for age and gender. Also, the size of our sample was too small to adjust for level of VAT, although the strength of the relationship with ALT and ␥-GT argue in favour of a predominant role of liver steatosis in the increased production of OPG observed in MetS. In addition, our study had yet another limitation: hepatic fat content was measured only in a subset of our cohort. Nevertheless, this subgroup with liver MRI scans did not differ from the rest of the cohort. However, the crosssectional nature of our study precludes any demonstration of causality for the relationship observed. 5. Conclusion The significant positive association found between OPG and some of the major components of MetS as well as LF content suggest a possible role of liver steatosis in the overproduction of OGP in metabolic patients. Longitudinal studies now need to be done to determine whether OPG might be a useful biomarker to identify steatosis patients at risk of vascular complications.
Disclosure of interest The authors declare that they have no competing interest. References [1] St˛epie´n E, Fedak D, Klimeczek P, Wilkosz T, Bany´s RP, Starzyk K, et al. Osteoprotegerin, but not osteopontin, as a potential predictor of vascular calcification in normotensive subjects. Hypertens Res 2012;35:531–8. [2] Pérez de Ciriza C, Moreno M, Restituto P, Bastarrika G, Simon I, Colina I, et al. Circulating osteoprotegerin is increased in the metabolic syndrome and associates with subclinical atherosclerosis and coronary arterial calcification. Clin Biochem 2014;47:272–8. [3] Ciccone MM, Scicchitano P, Gesualdo M, Zito A, Carbonara R, Locorotondo M, et al. Serum osteoprotegerin and carotid intima-media thickness in acute/chronic coronary artery diseases. J Cardiovasc Med 2013;14:43–8. [4] Jansson AM, Hartford M, Omland T, Karlsson T, Lindmarker P, Herlitz J, et al. Multimarker risk assessment including osteoprotegerin and CXCL16 in acute coronary syndromes. Arterioscler Thromb Vasc Biol 2012;32:3041–9. [5] Ghaffari S, Yaghoubi A, Baghernejad R, Sepehrvand N, Sokhanvar S, Haghjou AG. The value of serum osteoprotegerin levels in patients with angina like chest pain undergoing diagnostic coronary angiography. Cardiol J 2013;20:261–7. [6] Mogelvang R, Pedersen SH, Flyvbjerg A, Bjerre M, Iversen AZ, Galatius S, et al. Comparison of osteoprotegerin to traditional atherosclerotic risk factors and high-sensitivity C-Reactive Protein for diagnosis of atherosclerosis. Am J Cardiol 2012;109:515–20. [7] Ducluzeau PH, Boursier J, Bertrais S, Dubois S, Gauthier A, Rohmer V, et al. MRI measurement of liver fat content predicts the metabolic syndrome. Diabetes Metab 2013;39:314–21. [8] Alberti KG, Zimmet P, Shaw J. The metabolic syndrome – a new worldwide definition. Lancet 2005;366:1059–62. [9] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9. [10] Silva TE, Colombo G, Schiavon LL. Adiponectin: a multitasking player in the field of liver diseases. Diabetes Metab 2014;40:95–107. [11] Nabipour I, Kalantarhormozi M, Larijani B, Assadi M, Sanjdideh Z. Osteoprotegerin in relation to type 2 diabetes mellitus and the metabolic syndrome in postmenopausal women. Metabolism 2010;59:742–7. [12] Gannagé-Yared MH, Yaghi C, Habre B, Khalife S, Noun R, GermanosHaddad M, et al. Osteoprotegerin in relation to body weight, lipid parameters insulin sensitivity, adipocytokines, and C-reactive protein in obese and non-obese young individuals: results from both cross-sectional and interventional study. Eur J Endocrinol 2008;158:353–9. [13] Akinci B, Celtik A, Yuksel F, Genc S, Yener S, Secil M, et al. Increased osteoprotegerin levels in women with previous gestational diabetes developing metabolic syndrome. Diabetes Res Clin Pract 2011;91:26–31. [14] Gannagé-Yared MH, Fares F, Semaan M, Khalife S, Jambart S. Circulating osteoprotegerin is correlated with lipid profile, insulin sensitivity, adiponectin and sex steroids in an ageing male population. Clin Endocrinol (Oxf) 2006;64:652–8. [15] Venuraju SM, Yerramasu A, Corder R, Lahiri A. Osteoprotegerin as a predictor of coronary artery disease and cardiovascular mortality and morbidity. J Am Coll Cardiol 2010;55:2049–61. [16] Targher G, Bertolini L, Rodella S, Tessari R, Zenari L, Lippi G, et al. Nonalcoholic fatty liver disease is independently associated with an increased incidence of cardiovascular events in type 2 diabetic patients. Diabetes Care 2007;30:2116–21. [17] Sofer E, Shargorodsky M. Effect of metformin treatment on circulating osteoprotegerin in patients with nonalcoholic fatty liver disease. Hepatol Int 2015;7:121–8.
Please cite this article in press as: Monseu M, et al. Osteoprotegerin levels are associated with liver fat and liver markers in dysmetabolic adults. Diabetes Metab (2016), http://dx.doi.org/10.1016/j.diabet.2016.02.004