Acta Diabetol DOI 10.1007/s00592-014-0576-0
ORIGINAL ARTICLE
High circulating irisin levels are associated with insulin resistance and vascular atherosclerosis in a cohort of nondiabetic adult subjects G. Sesti • F. Andreozzi • T. V. Fiorentino • G. C. Mannino • A. Sciacqua • M. A. Marini F. Perticone
•
Received: 6 February 2014 / Accepted: 20 February 2014 Ó Springer-Verlag Italia 2014
Abstract Irisin, a novel myokine, was proposed to be able to regulate glucose homeostasis and obesity in mice. Whether irisin levels are associated with cardio-metabolic variables, insulin sensitivity, and vascular atherosclerosis in humans remain unsettled. To determine the associations between circulating irisin levels, cardio-metabolic variables, insulin sensitivity, and common carotid intimamedia thickness (IMT), an indicator of vascular atherosclerosis, a cross-sectional evaluation of circulating irisin levels and cardio-metabolic variables in 192 White adults was conducted. Insulin sensitivity and insulin clearance were assessed by euglycemic–hyperinsulinemic clamp. Common carotid IMT was measured by ultrasound. After adjusting for age and gender, irisin levels were positively correlated with body fat mass (r = 0.12, P \ 0.05), fasting (r = 0.17, P \ 0.01), 2 h post-load insulin (r = 0.15, P \ 0.02) levels, and IMT (r = 0.29, P \ 0.0001) and were negatively correlated with insulin-stimulated glucose disposal (r = -0.18, P = 0.007), Matsuda index (r = -0.13, P \ 0.04), disposition index (r = -0.278, P \ 0.0001), and insulin clearance (r = -0.26, P \ 0.0001). After adjusting for age, gender, and BMI, individuals in the highest tertile of irisin levels exhibited
Managed by Antonio Secchi. G. Sesti (&) F. Andreozzi T. V. Fiorentino G. C. Mannino A. Sciacqua F. Perticone Dipartimento Scienze Mediche e Chirurgiche, Universita` ‘‘Magna-Græcia’’ di Catanzaro, Viale Europa, 88100 Catanzaro, Italy e-mail: [email protected] M. A. Marini Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
higher body fat mass (P \ 0.01), fasting (P \ 0.05), 2 h post-load (P \ 0.01) insulin levels, carotid IMT (P \ 0.001), lower insulin-stimulated glucose disposal (P \ 0.001), Matsuda index (P \ 0.01), disposition index (P \ 0.01), and insulin clearance (P \ 0.001) as compared with subjects in the lowest tertile of circulating irisin levels. Irisin is inversely associated with insulin sensitivity and positively associated with carotid IMT in humans, suggesting either increased release by adipose/ muscle tissue in response to deterioration of insulin sensitivity or a compensatory increase in irisin to overcome an underlying irisin resistance. Keywords Irisin Insulin sensitivity Intima-media thickness Vascular atherosclerosis
Introduction Obesity is a serious threat to public health and is reaching epidemic proportions worldwide [1]. It is a component of the metabolic syndrome, a condition characterized by a clustering of metabolic and atherosclerotic risk factors including visceral adiposity, dyslipidemia, hypertension, and hyperglycemia [2] and is associated with the development of type 2 diabetes mellitus and cardiovascular disease [3–6]. Insulin resistance in skeletal muscle, the major site of insulin-stimulated glucose disposal, is a key feature of obesity, metabolic syndrome, and type 2 diabetes [7]. Sedentary lifestyle is considered a central contributory factor for obesity and obesity-induced metabolic disorders including insulin resistance, metabolic syndrome, type 2 diabetes, and lifestyle interventions that include increased physical activity lead to improvement in insulin sensitivity [8]. Increasing evidence suggests that skeletal muscle acts
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as a secretory organ, releasing a variety of cytokines, termed myokines, especially during or immediately after the physical activity, which mediate some of the beneficial effects of exercise on metabolism [9]. Irisin is a novel myokine released upon cleavage of the plasma membrane protein fibronectin type III domain-containing protein 5 (FNDC5) [10]. In mice, muscle FNDC5 gene is induced by transgenic expression of peroxisome proliferator-activated receptor-a coactivator-1 (PGC1)-a and, more physiologically, by 3 weeks of free wheel running [10]. Irisin induces the browning of subcutaneous white adipocytes and stimulates thermogenic genes including uncoupling protein 1 (UCP1) both in stromal vascular fraction (SVF) cells and in mouse models [10]. In addition, mice injected with adenoviral vectors containing full-length FNDC5 exhibited a threefold–fourfold increase in plasma irisin levels, which led to lower weight gain, improvement in glucose tolerance, and insulin resistance induced by high-fat diet [10]. Although these results have provided evidence that irisin may mediate some of the positive effects of training on body weight, insulin sensitivity, and glucose homeostasis, studies in humans have led to mixed results. Thus, some studies have reported a positive correlation between body mass index (BMI) and circulating irisin levels [12–15] or muscle FNDC5 mRNA expression [11, 16], while other studies have reported a negative correlation between BMI and circulating irisin levels [16]. In addition, weight loss induced by bariatric surgery decreased both circulating irisin and muscle FNDC5 expression [12]. Likewise, conflicting results have been reported in studies investigating the correlation between glucose levels and FNDC5 mRNA expression in skeletal muscle or circulating irisin levels. Although three studies have reported that individuals with type 2 diabetes have lower levels of irisin as compared with nondiabetic controls [15–17], others have reported a positive association between circulating irisin and fasting plasma glucose [12, 13] or muscle FNDC5 mRNA expression and 2 h glucose during an oral glucose tolerance test (OGTT) [18]. In addition, whether circulating irisin levels are associated with insulin sensitivity in humans is an object of debate with divergent results reported by previous studies [11, 13, 16–18]. So far, no prior investigation has assessed the relationship between irisin and insulin sensitivity, cardio-metabolic risk factors, and subclinical atherosclerosis after adjusting for potential confounders. Exploring these relationships is important in order to gain insight into potential pathophysiological roles of irisin in the development of cardio-metabolic disease. To examine this, we have measured, by ultrasonography, intima-media thickness (IMT) of common carotid artery, a well-recognized index of subclinical vascular
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atherosclerosis, in a cohort of 192 nondiabetic individuals in whom insulin sensitivity was assessed by the euglycemic–hyperinsulinemic clamp technique.
Subjects, materials and methods The study group included 192 nondiabetic offspring of European ancestry participating in the EUGENE2 project [19, 20] who had only one of the parents with type 2 diabetes. Participants were consecutively recruited at the Department of Systems Medicine of the University of Rome Tor Vergata and at the Department of Medical and Surgical Sciences of the University ‘‘Magna Graecia’’ of Catanzaro [21]. Subjects were excluded if they had history of cardiovascular disease including myocardial infarction, stoke, peripheral atherosclerosis, chronic gastrointestinal diseases associated with malabsorption, chronic pancreatitis, history of any malignant disease, history of alcohol or drug abuse, positivity for antibodies to hepatitis C virus (HCV) or hepatitis B surface antigen (HBsAg), and liver or kidney failure. All individuals were studied according to a previously described protocol [22]. All anthropometric and biochemical measurements were taken in the morning after a 12 h fast using standardized methods. Weight was measured with subjects in undergarments, height was measured by stadiometer, and BMI was calculated as body weight (kg) divided by the square of height (m). Waist circumference was measured as the narrowest circumference between the lower rib margin and the anterior superior iliac spine, and body composition evaluated by bioelectrical impedance. Brachial blood pressure was measured in the left arm of the supine subjects, after 5 min of quiet rest, with a digital electronic tensiometer (regular or large adult cuffs were used according to arm circumference). Values were calculated as the average of the last two of three consecutive measurements obtained at 3-min intervals. A 75-g OGTT was performed with 0-, 30-, 60-, 90-, and 120-min sampling for plasma glucose and insulin assay. Insulin sensitivity was assessed by euglycemic–hyperinsulinemic clamp study, as previously described [23]. Briefly, a continuous insulin infusion was initiated at the rate of 40 mU/m2 of body surface area per min, after a priming dose, in order to reach and maintain steady-state plasma insulin of 80 ± 5 lU/ml. Plasma glucose was assessed at 5-min intervals during the 2 h clamp study by a glucose analyzer. In the study subjects, mean plasma glucose concentration during the last hour of the clamp was 93 ± 5 mg/dl. Plasma insulin concentrations were measured every 20 min during the insulin infusion. The study was approved by Institutional Ethics Committees of the
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University of Rome Tor Vergata and Catanzaro, and written informed consent was obtained from each participant in accordance with principles of the Declaration of Helsinki.
Insulin secretion was estimated by insulinogenic index I30I0/G30-G0 (DI30/DG30). To evaluate b-cell function, the disposition index was calculated as DI30/DG30 9 MFFM/ SSPI.
Ultrasound measurement of IMT of the common carotid artery
Definitions of metabolic status
High-resolution B-mode ultrasound was used to measure IMT of the common carotid artery using an ATL HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothell, WA) equipped with a 5-MHz linear array transducer. The thickness of the intima-media complex was assessed as previously described [23]. Manual measurements were taken in plaque-free portions of the 10-mm linear segment proximal to the carotid bulb. For each individual, two measurements were taken bilaterally and the values were averaged. Ultrasound measurement of IMT was taken by a trained examiner who was unaware of the subjects’ anthropometric and metabolic findings. Analytical determinations Glucose, triglyceride, and total and HDL-C concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Circulating irisin concentrations were measured using commercial enzyme-linked immunosorbent assays (ELISA, Catalog No. EK-067-52, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). Serum insulin concentration was determined by a chemiluminescencebased assay (ImmuliteÒ, Siemens, Italy). Calculation Glucose disposal (M) was calculated as the mean rate of glucose infusion measured during the last 60 min of the clamp examination (steady-state) and is expressed as milligrams per minute per kilogram fat-free mass (MFFM) measured with the use of electrical bioimpedance. MFFM value was corrected for steady-state plasma insulin concentration during the last hour of the clamp (MFFM/SSPI). Insulin clearance was calculated by dividing the rate of insulin infusion by the mean steady-state plasma insulin concentration during the euglycemic clamp [24]. The Matsuda index was calculated as follows [25]:
10; 000=
Subjects were classified into glucose tolerance status according to the American Diabetes Association (ADA) criteria [26]: NGT with fasting plasma glucose (FPG) \100 mg/dl and 2 h post-load \140 mg/dl, isolated impaired fasting glucose (IFG) with FPG ranging from 100 to 125 mg/dl and 2 h post-load \140 mg/dl, isolated impaired glucose tolerance (IGT) with FPG \ 100 mg/dl and 2 h post-load [140 and \200 mg/dl, and combined IFG/IGT with FPG ranging from 100 to 125 mg/dl and 2 h post-load [140 and \200 mg/dl. Subjects were divided into three groups on the basis of their BMI: normal weight (BMI \ 25.0 kg/m2), overweight (BMI 25.0–29.9 kg/m2), and obese (BMI [ 30 kg/m2). Statistical analyses Variables with skewed distribution including triglyceride, fasting, and 2 h post-load insulin were natural log-transformed for statistical analyses. Continuous variables are expressed as mean ± SD. Categorical variables were compared by v2 test. Unpaired Student’s t test was used to compare differences of continuous variables between two groups. Partial correlation coefficients adjusted for age, gender, and BMI were computed between variables. Subjects were stratified into tertile according to their circulating irisin values. Anthropometric and metabolic differences between groups were tested after adjusting for gender, age, and BMI using a general linear model with post hoc Bonferroni correction for multiple comparisons. For all analyses, a P value B0.05 was considered to be statistically significant. All analyses were performed using SPSS software version 16.0 for Windows. Results Clinical characteristics and laboratory findings of the study group are shown in Table 1. Of the 192 subjects examined, 149 (77.6 %) had NGT, 17 had IFG (8.9 %), and 26
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Acta Diabetol Table 1 Clinical characteristics of the study cohort Overall cohort
Men
Women
P value
Number
192
101
91
–
Age (years)
39 ± 10
40 ± 10
38 ± 9
0.06
Body weight (kg)
83 ± 18
89 ± 16
77 ± 19
\0.0001
2
BMI (kg/m )
29.6 ± 6.1
29.5 ± 4.7
29.7 ± 7.4
0.76
Waist circumference (cm)
97 ± 13
100 ± 11
93 ± 15
\0.0001
Fat body mass (kg)
28 ± 15
25 ± 11
30 ± 16
0.01
Fat-free mass (kg)
55 ± 12
63 ± 10
46 ± 9
\0.0001
SBP (mmHg)
127 ± 15
131 ± 13
122 ± 13
\0.0001
DBP (mmHg)
82 ± 10
85 ± 10
78 ± 10
\0.0001
Total cholesterol (mg/dl)
198 ± 37
199 ± 36
1977 ± 38
0.63
HDL cholesterol (mg/dl) Triglyceride (mg/dl)
50 ± 14 120 ± 71
45 ± 11 137 ± 77
57 ± 14 102 ± 58
\0.0001 \0.0001
Fasting glucose (mg/dl)
89 ± 9
91 ± 9
86 ± 9
\0.0001
2 h Glucose (mg/dl)
112 ± 27
112 ± 24
113 ± 30
0.95 0.42
Fasting insulin (lU/ml)
12 ± 8
12 ± 8
11 ± 8
2 h Insulin (lU/ml)
83 ± 80
82 ± 74
85 ± 84
0.74
Irisin (ng/ml)
156 ± 81
153 ± 72
156 ± 87
0.81
Matsuda index
78 ± 47
74 ± 46
83 ± 47
0.21
DI30/DG30
26 ± 19
27 ± 22
25 ± 15
0.44
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
11.3 ± 7.2
9.0 ± 4.9
13.8 ± 8.3
\0.0001
Disposition index
261 ± 206
222 ± 193
304 ± 234
0.01
Insulin clearance (ml/min 9 m2)
718 ± 476
643 ± 274
764 ± 513
0.04
Intima-media thickness (mm)
0.68 ± 0.16
0.70 ± 0.17
0.66 ± 0.15
0.13
NGT/IFG/IGT (n)
149/17/26
74/4/13
75/13/13
0.11
Normal weight/overweight/obese
47/64/81
19/39/43
28/25/38
0.10
Data are mean ± SD. Triglyceride, fasting, and 2 h insulin levels were log-transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Differences between means were compared using unpaired Student’s t test. Categorical variables were compared by v2 test SBP systolic blood pressure, DBP diastolic blood pressure, NGT normal glucose tolerance, IGT impaired glucose tolerance, and IFG impaired fasting glucose
(13.5 %) had isolated IGT or combined IFG/IGT. In addition, 81 (42.2 %) subjects were obese and 64 (33.3 %) were overweight with no gender-specific differences in the distribution of BMI categories. Men were slightly older and exhibited a worse cardiometabolic risk profile including higher fat body mass, blood pressure, triglyceride, and fasting plasma glucose levels, and lower HDL cholesterol, insulin sensitivity, assessed by euglycemic–hyperinsulinemic clamp, and disposition index. No gender-specific differences were observed in circulating irisin levels between men and women. Univariate correlations between circulating irisin levels and anthropometric and metabolic variables are shown in Table 2. Circulating irisin levels were significantly correlated with age. After adjusting for age and gender, circulating irisin levels were positively correlated with body weight, body fat mass, fasting, 2 h post-load insulin levels,
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and carotid IMT and were negatively correlated with insulin-stimulated glucose disposal, Matsuda index of insulin sensitivity, disposition index, and insulin clearance. These correlations remained significant after further adjusting for BMI in addition to gender and age (Table 2). When univariate correlations between circulating irisin levels and anthropometric measures were analyzed in the obese subgroup (n = 81), irisin was positively correlated with body weight (r = 0.29, P = 0.005), BMI (r = 0.21, P = 0.02), waist circumference (r = 0.24, P = 0.01), and body fat mass (r = 0.28, P = 0.006) after adjusting for age and gender. Additionally, the correlations between circulating irisin levels, insulin sensitivity, disposition index, insulin clearance, and carotid IMT remained statistically significant when analysis was performed separately in men and women (Table 3). Anthropometric and metabolic characteristics of the study group stratified according to tertile of circulating
Acta Diabetol Table 2 Univariate correlations between circulating irisin levels and anthropometric and biochemical variables
Age (years)
Age- and gender-adjusted correlations between circulating irisin levels and metabolic variables
Age-, gender-, and BMI-adjusted correlations between circulating irisin levels and metabolic variables
Pearson’s correlation coefficient (r)
Pearson’s correlation coefficient (r)
-0.19
P 0.003*
-0.19
P 0.003*
Body weight (kg)
0.12
0.05
–
–
BMI (kg/m2)
0.04
0.28
–
–
Waist circumference (cm)
0.05
0.23
–
–
Fat body mass (kg)
0.12
0.05
–
–
Fat-free mass (kg)
0.06
0.47
–
–
SBP (mmHg)
0.05
0.27
0.03
0.31
DBP (mmHg)
0.10
0.08
0.05
0.24
Total cholesterol (mg/dl)
0.06
0.21
0.06
0.22
HDL cholesterol (mg/dl) Triglycerides (mg/dl)
-0.09 0.01
0.09 0.46
-0.11 0.02
0.06 0.38
Fasting glucose (mg/dl)
0.06
0.18
0.07
0.17
2 h Glucose (mg/dl)
0.11
0.07
0.10
0.08
Fasting insulin (lU/ml)
0.17
0.01
0.18
0.007
2 h Insulin (lU/ml)
0.15
0.02
0.14
0.03
Matsuda index
-0.13
0.04
-0.12
0.05
DI30/DG30
-0.11
0.09
-0.12
0.06
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
-0.18
0.007
-0.17
0.007
Disposition index
-0.27
\0.0001
-0.26
\0.0001
-0.26
\0.0001
-0.25
\0.0001
0.29
\0.0001
0.28
\0.0001
2
Insulin clearance (ml/min 9 m ) Intima-media thickness (mm)
BMI body mass index, SBP systolic blood pressure, and DBP diastolic blood pressure * P values refer to results after analyses with adjustment for gender
irisin levels are shown in Table 4. After adjusting for age, gender, and BMI, individuals in the highest tertile of irisin levels exhibited higher body fat mass, fasting, 2 h post-load insulin levels, carotid IMT, lower insulin-stimulated glucose disposal, Matsuda index of insulin sensitivity, disposition index, and insulin clearance as compared with subjects in the lowest tertile of circulating irisin levels.
Discussion Irisin is a novel myokine whose expression in skeletal muscle is stimulated by exercise; it exerts beneficial effects on metabolism by inducing the browning of subcutaneous white adipocytes [10]. Mildly, overexpression of irisin in mice fed a high-fat diet resulted in an improvement of obesity, glucose tolerance, and insulin resistance [10]. Although these initial promising results point to a potential beneficial role of irisin in regulating body weight, insulin sensitivity, and glucose homeostasis, studies in humans have led to divergent results [11–18]. Furthermore, the role
of irisin in cardiovascular disease remains still unsettled. These observations coupled with the accessibility of a carefully characterized cohort of nondiabetic adult individuals have provided the rationale for examining the relationship between circulating irisin levels, glucose homeostasis, insulin sensitivity, cardio-metabolic risk factors, and subclinical atherosclerosis. In the present crosssectional study, we report an inverse relationship between circulating irisin levels and insulin sensitivity, measured either by the euglycemic–hyperinsulinemic clamp or by the Matsuda index, a validated OGTT-derived insulin sensitivity index [25]. These findings obtained using gold standard techniques are consistent with previous studies showing both a positive association between circulating irisin levels and insulin resistance measured by the homeostasis model assessment for insulin resistance (HOMA-IR) index [13] and an inverse relationship between human myotube FNDC5 expression and Matsuda index of insulin sensitivity [18]. Accordingly, an association between insulin sensitivity and a single nucleotide polymorphism (SNP rs726344) in the FNDC5 locus,
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Acta Diabetol Table 3 Univariate correlations between circulating irisin levels and anthropometric and biochemical variables
Age (years)
Age-adjusted correlations between circulating irisin levels and metabolic variables in men
Age-adjusted correlations between circulating irisin levels and metabolic variables in women
Pearson’s correlation coefficient (r)
P
Pearson’s correlation coefficient (r)
-0.10
0.08*
-0.31
P 0.001*
Body weight (kg)
0.12
0.05
0.13
0.10
BMI (kg/m2)
0.04
0.36
0.08
0.23
Waist circumference (cm)
0.08
0.22
0.07
0.26
Fat body mass (kg)
0.12
0.06
0.11
0.07
Fat-free mass (kg)
0.07
0.25
0.04
0.46
SBP (mmHg)
0.10
0.14
0.03
0.49
DBP (mmHg)
0.06
0.28
0.04
0.37
Total cholesterol (mg/dl)
0.01
0.48
0.22
0.02
HDL cholesterol (mg/dl) Triglycerides (mg/dl)
-0.02 0.02
0.43 0.44
0.02 0.03
0.39 0.37
Fasting glucose (mg/dl)
0.09
0.19
0.07
0.24
2 h Glucose (mg/dl)
0.10
0.17
0.14
0.09
Fasting insulin (lU/ml)
0.10
0.10
0.24
0.01
2 h Insulin (lU/ml)
0.24
0.01
0.08
0.25
Matsuda index
-0.27
0.004
-0.11
0.19
DI30/DG30
-0.07
0.52
-0.18
0.06
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
-0.16
0.05
-0.24
0.01
Disposition index
-0.23
0.01
-0.34
0.001
-0.18
0.04
-0.31
0.18
0.04
0.47
2
Insulin clearance (ml/min 9 m ) Intima-media thickness (mm)
0.001 \0.0001
BMI body mass index, SBP systolic blood pressure, and DBP diastolic blood pressure * P values refer to results after analyses without adjustment
encoding the irisin precursor, has been reported. Interestingly, it was observed a trend for an association of the insulin-desensitizing minor A allele of SNP rs726344 with higher FNDC5 mRNA contents (P = 0.19) in human myotubes [18]. We also observed a positive association between circulating irisin levels, fasting, and 2 h post-load insulin levels, in agreement with most [12, 14, 18], but not all prior studies [16]. It has been reported that impaired insulin clearance is a major determinant of hyperinsulinemia after an oral glucose load [27, 28], and therefore, it is possible that the increases in fasting and 2 h post-load insulin levels observed in individuals with higher circulating irisin levels may be due to differences in insulin clearance. Accordingly, we observed an inverse relationship between insulin clearance and circulating irisin levels as well as a lower insulin clearance in the individuals in the highest tertile of irisin levels. A decrease in insulin clearance may represent a compensatory mechanism to reduce the demand on b-cells as insulin resistance worsens [29]. Interestingly, we found that
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subjects in the highest tertile of irisin levels exhibit both a decrease in disposition index, which reflects b-cell adequacy to adjust to prevailing insulin resistance, and diminished insulin clearance. Thus, it is possible to hypothesize that the reduced insulin clearance observed in subjects with elevated levels of irisin represents an adaptive mechanism to preserve b-cells from the overburden to compensate for impaired insulin sensitivity. Importantly, the inverse relationship between circulating irisin levels, fasting, 2 h post-load insulin levels, insulin sensitivity, disposition index, and insulin clearance remains significant after adjustment for confounders potentially affecting circulating irisin including age, gender, and BMI. In fact, there is evidence that circulating irisin levels [12] or muscle FNDC5 gene expression [16] is inversely associated with age, and accordingly, an inverse association has been also observed in the present study (Table 2). In addition, a positive correlation between BMI or body fat mass and circulating irisin levels [12–15] or muscle FNDC5 mRNA expression [11, 16] has been reported in
Acta Diabetol Table 4 Anthropometric and metabolic characteristics of the study subjects stratified according to tertile of circulating irisin levels
Gender (male/female)
Tertile 1 (n = 64)
Tertile 2 (n = 64)
Tertile 3 (n = 64)
34/30
34/30
33/31
P
0.97 b
0.03*
Age (years)
41 ± 9
38 ± 10
37 ± 9
Body weight (kg)
79 ± 15
85 ± 20a
86 ± 19b
0.03§
BMI (kg/m )
28.8 ± 5.1
30.3 ± 6.7
29.8 ± 6.4
0.17§
Waist circumference (cm)
95 ± 11
98 ± 13
98 ± 16
0.20§
2
b
b
0.02§ 0.40§ 0.31
Fat body mass (kg)
24 ± 9
30 ± 17
29 ± 16
Fat-free mass (kg) SBP (mmHg)
55 ± 11 126 ± 13
54 ± 14 125 ± 15
56 ± 13 128 ± 17
DBP (mmHg)
82 ± 11
81 ± 9
82 ± 10
0.30
Total cholesterol (mg/dl)
202 ± 33
191 ± 34
200 ± 39
0.07
HDL cholesterol (mg/dl)
52 ± 14
48 ± 13
49 ± 12
0.40
Triglyceride (mg/dl)
124 ± 67
115 ± 56
125 ± 86
0.63
Fasting glucose (mg/dl)
90 ± 9
90 ± 8
89 ± 11
0.40
2 h Glucose (mg/dl)
112 ± 25
112 ± 26
114 ± 30
0.76
Fasting insulin (lU/ml)
9±6
13 ± 9a
14 ± 8a
0.05
2 h Insulin (lU/ml)
66 ± 65
92 ± 90
90 ± 69b
0.02
Irisin (ng/ml)
78 ± 27
141 ± 15
246 ± 63
\0.0001
Matsuda index
91 ± 54
75 ± 44a
70 ± 40b
0.02
DI30/DG30
26 ± 10
28 ± 16
23 ± 16
0.50
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
13.0 ± 7.5
11.3 ± 8.1
9.5 ± 5.2c
0.01
Disposition index
304 ± 211
288 ± 268
193 ± 130b
0.01
579 ± 223c
0.001
0.74 ± 0.13c
0.005
2
b
Insulin clearance (ml/min 9 m )
906 ± 583
669 ± 281
Intima-media thickness (mm)
0.64 ± 0.16
0.69 ± 0.17
Data are mean ± SD. Triglyceride, fasting, and 2 h insulin levels were log-transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Categorical variables were compared by v2 test. Comparisons between the three groups were made using a general linear model SBP systolic blood pressure, DBP diastolic blood pressure P values refer to results after analyses with adjustment for age, gender, and BMI. * P values refer to results after analyses with adjustment for gender. § P values refer to results after analyses with adjustment for age and gender a
P \ 0.05 versus tertile 1 group
b
P \ 0.01 versus tertile 1 group
c
P \ 0.001 versus tertile 1 group
most, but not all studies [16]. Accordingly, we found that circulating irisin levels were positively associated with body fat mass in the whole cohort and with BMI in the subgroup of obese individuals. These findings may explain some of the divergent results observed in prior studies in relation to insulin sensitivity and related traits and highlight the importance to consider confounders potentially affecting circulating irisin including age, and adiposity when comparing different groups of individuals. Next, we report a positive relationship between circulating irisin levels and carotid IMT, a validated measure of vascular atherosclerosis [30]. To the best of our knowledge, this is the first evidence linking higher circulating irisin levels with signs of vascular atherosclerosis. These data are consistent with the previous observation that circulating
irisin levels were associated with an increased 10-year risk of cardiovascular diseases, assessed by the general Framingham risk profile for cardiovascular diseases risk assessment tool [13]. It is well established that impaired insulin action and sustained hyperinsulinemia due to decreased insulin clearance may have adverse effects on target tissues of insulin action including vasculature [22– 24, 31]. Thus, it has been reported that exposure of vascular cells to high insulin concentrations induces proatherogenic effects. In cultured endothelial cells, elevated insulin concentrations induce the secretion of endothelin-1 (ET-1), a potent vasoconstrictor protein [32], the expression of vascular cell adhesion molecule (VCAM)-1, and monocytes adhesion to cultured endothelial cells [32, 33]. It is conceivable that impaired insulin sensitivity, elevated fasting,
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and postprandial insulinemia due to decreased insulin clearance may have adverse effects on vasculature in individuals with elevated circulating irisin levels. Given the role of irisin to promote improvements in glucose tolerance and insulin resistance in mice [10], the present and prior findings [13, 18] showing an inverse correlation between irisin levels and insulin sensitivity are surprising. It is tempting to speculate that increased irisin release from skeletal muscle and adipose tissue may represent a physiological compensatory mechanism in response to deterioration of insulin sensitivity. Alternatively, elevated circulating irisin levels may reflect a condition of tissue irisin resistance similar to the conditions of insulin and leptin resistance associated with elevated circulating insulin and leptin levels. Further mechanistic studies in humans aimed at investigating the molecular mechanisms regulating irisin release from tissues, the site(s) of action, and the signaling pathway are absolutely necessary to allow a better understanding of the pathophysiological role of irisin. It would also be interesting to investigate whether the activation of peroxisome proliferator-activated receptor-c (PPARc) by thiazolidinediones, which has been shown to induce an increase in mitochondrial content, and expression of UCP-1 and PGC-1a [34], is also able to modulate irisin expression. In fact, it is well established that the activation of PPARc by thiazolidinediones improves glucose metabolism by several mechanisms including enhancing insulin sensitivity, promoting differentiation of adipocytes, and reducing the inflammatory state [35, 36]; the modulation of irisin expression and secretion may represent an additional mechanism underlying the action of these drugs. The present study has several strengths including the relatively large sample comprising men and women, the homogeneity of the sample comprising White adults of European ancestry, the exclusion of confounding conditions such as previous cardiovascular disease, history of malignant disease, liver or kidney failure, the assessment of glucose tolerance by OGTT, the centralized measurements of biochemical variables, and the strict quality control of ultrasound studies performed by experienced examiners who were blinded to the subjects’ clinical and laboratory findings. The present results are also strengthened by the use of the gold standard euglycemic–hyperinsulinemic clamp for insulin sensitivity and insulin clearance assessment. Nevertheless, some limitations should be acknowledged in the interpretation of our results. First, the cross-sectional nature of the study precludes us to draw any conclusion on the role of irisin in the development of metabolic disorders and cardiovascular disease,
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and therefore, no conclusion regarding cause–effect relationships can be made. Second, all anthropometric and metabolic variables including OGTT euglycemic–hyperinsulinemic clamp were measured once. Although such an approach is a common limitation to large epidemiological studies, intra-individual variation cannot be taken into account, and some individuals might have been misclassified. Additionally, circulating irisin levels were measured at resting condition, and the possibility of obtaining different results after exercise cannot be excluded. Moreover, body composition was assessed by bioimpedance analysis. Although this method is well established, inexpensive, and simple to perform in the clinical routine, the analysis of body composition by bioimpedance provides a less accurate measure of various components of the body as compared with computed tomography or magnetic resonance imaging, which are not suitable for mass screening. Finally, all participants to the present study were Whites, and whether these observations can also be extended to nonwhite ethnic groups with different body composition remains to be determined. Conflict of interest G. Sesti, F. Andreozzi, T. V. Fiorentino, G. C. Mannino, A. Sciacqua, M. A. Marini and F. Perticone declare they have no conflict of interest. Human and Animal Rights disclosure All procedures followed were approved by approved Institutional Ethics Committee of the University of Rome Tor Vergata and Catanzaro and in accordance with the ethical of the Helsinki declaration of 1975, as revised in 2008. Informed consent disclosure Informed consent was obtained from all patients for being included in the study.
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ORIGINAL ARTICLE
High circulating irisin levels are associated with insulin resistance and vascular atherosclerosis in a cohort of nondiabetic adult subjects G. Sesti • F. Andreozzi • T. V. Fiorentino • G. C. Mannino • A. Sciacqua • M. A. Marini F. Perticone
•
Received: 6 February 2014 / Accepted: 20 February 2014 Ó Springer-Verlag Italia 2014
Abstract Irisin, a novel myokine, was proposed to be able to regulate glucose homeostasis and obesity in mice. Whether irisin levels are associated with cardio-metabolic variables, insulin sensitivity, and vascular atherosclerosis in humans remain unsettled. To determine the associations between circulating irisin levels, cardio-metabolic variables, insulin sensitivity, and common carotid intimamedia thickness (IMT), an indicator of vascular atherosclerosis, a cross-sectional evaluation of circulating irisin levels and cardio-metabolic variables in 192 White adults was conducted. Insulin sensitivity and insulin clearance were assessed by euglycemic–hyperinsulinemic clamp. Common carotid IMT was measured by ultrasound. After adjusting for age and gender, irisin levels were positively correlated with body fat mass (r = 0.12, P \ 0.05), fasting (r = 0.17, P \ 0.01), 2 h post-load insulin (r = 0.15, P \ 0.02) levels, and IMT (r = 0.29, P \ 0.0001) and were negatively correlated with insulin-stimulated glucose disposal (r = -0.18, P = 0.007), Matsuda index (r = -0.13, P \ 0.04), disposition index (r = -0.278, P \ 0.0001), and insulin clearance (r = -0.26, P \ 0.0001). After adjusting for age, gender, and BMI, individuals in the highest tertile of irisin levels exhibited
Managed by Antonio Secchi. G. Sesti (&) F. Andreozzi T. V. Fiorentino G. C. Mannino A. Sciacqua F. Perticone Dipartimento Scienze Mediche e Chirurgiche, Universita` ‘‘Magna-Græcia’’ di Catanzaro, Viale Europa, 88100 Catanzaro, Italy e-mail: [email protected] M. A. Marini Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy
higher body fat mass (P \ 0.01), fasting (P \ 0.05), 2 h post-load (P \ 0.01) insulin levels, carotid IMT (P \ 0.001), lower insulin-stimulated glucose disposal (P \ 0.001), Matsuda index (P \ 0.01), disposition index (P \ 0.01), and insulin clearance (P \ 0.001) as compared with subjects in the lowest tertile of circulating irisin levels. Irisin is inversely associated with insulin sensitivity and positively associated with carotid IMT in humans, suggesting either increased release by adipose/ muscle tissue in response to deterioration of insulin sensitivity or a compensatory increase in irisin to overcome an underlying irisin resistance. Keywords Irisin Insulin sensitivity Intima-media thickness Vascular atherosclerosis
Introduction Obesity is a serious threat to public health and is reaching epidemic proportions worldwide [1]. It is a component of the metabolic syndrome, a condition characterized by a clustering of metabolic and atherosclerotic risk factors including visceral adiposity, dyslipidemia, hypertension, and hyperglycemia [2] and is associated with the development of type 2 diabetes mellitus and cardiovascular disease [3–6]. Insulin resistance in skeletal muscle, the major site of insulin-stimulated glucose disposal, is a key feature of obesity, metabolic syndrome, and type 2 diabetes [7]. Sedentary lifestyle is considered a central contributory factor for obesity and obesity-induced metabolic disorders including insulin resistance, metabolic syndrome, type 2 diabetes, and lifestyle interventions that include increased physical activity lead to improvement in insulin sensitivity [8]. Increasing evidence suggests that skeletal muscle acts
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as a secretory organ, releasing a variety of cytokines, termed myokines, especially during or immediately after the physical activity, which mediate some of the beneficial effects of exercise on metabolism [9]. Irisin is a novel myokine released upon cleavage of the plasma membrane protein fibronectin type III domain-containing protein 5 (FNDC5) [10]. In mice, muscle FNDC5 gene is induced by transgenic expression of peroxisome proliferator-activated receptor-a coactivator-1 (PGC1)-a and, more physiologically, by 3 weeks of free wheel running [10]. Irisin induces the browning of subcutaneous white adipocytes and stimulates thermogenic genes including uncoupling protein 1 (UCP1) both in stromal vascular fraction (SVF) cells and in mouse models [10]. In addition, mice injected with adenoviral vectors containing full-length FNDC5 exhibited a threefold–fourfold increase in plasma irisin levels, which led to lower weight gain, improvement in glucose tolerance, and insulin resistance induced by high-fat diet [10]. Although these results have provided evidence that irisin may mediate some of the positive effects of training on body weight, insulin sensitivity, and glucose homeostasis, studies in humans have led to mixed results. Thus, some studies have reported a positive correlation between body mass index (BMI) and circulating irisin levels [12–15] or muscle FNDC5 mRNA expression [11, 16], while other studies have reported a negative correlation between BMI and circulating irisin levels [16]. In addition, weight loss induced by bariatric surgery decreased both circulating irisin and muscle FNDC5 expression [12]. Likewise, conflicting results have been reported in studies investigating the correlation between glucose levels and FNDC5 mRNA expression in skeletal muscle or circulating irisin levels. Although three studies have reported that individuals with type 2 diabetes have lower levels of irisin as compared with nondiabetic controls [15–17], others have reported a positive association between circulating irisin and fasting plasma glucose [12, 13] or muscle FNDC5 mRNA expression and 2 h glucose during an oral glucose tolerance test (OGTT) [18]. In addition, whether circulating irisin levels are associated with insulin sensitivity in humans is an object of debate with divergent results reported by previous studies [11, 13, 16–18]. So far, no prior investigation has assessed the relationship between irisin and insulin sensitivity, cardio-metabolic risk factors, and subclinical atherosclerosis after adjusting for potential confounders. Exploring these relationships is important in order to gain insight into potential pathophysiological roles of irisin in the development of cardio-metabolic disease. To examine this, we have measured, by ultrasonography, intima-media thickness (IMT) of common carotid artery, a well-recognized index of subclinical vascular
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atherosclerosis, in a cohort of 192 nondiabetic individuals in whom insulin sensitivity was assessed by the euglycemic–hyperinsulinemic clamp technique.
Subjects, materials and methods The study group included 192 nondiabetic offspring of European ancestry participating in the EUGENE2 project [19, 20] who had only one of the parents with type 2 diabetes. Participants were consecutively recruited at the Department of Systems Medicine of the University of Rome Tor Vergata and at the Department of Medical and Surgical Sciences of the University ‘‘Magna Graecia’’ of Catanzaro [21]. Subjects were excluded if they had history of cardiovascular disease including myocardial infarction, stoke, peripheral atherosclerosis, chronic gastrointestinal diseases associated with malabsorption, chronic pancreatitis, history of any malignant disease, history of alcohol or drug abuse, positivity for antibodies to hepatitis C virus (HCV) or hepatitis B surface antigen (HBsAg), and liver or kidney failure. All individuals were studied according to a previously described protocol [22]. All anthropometric and biochemical measurements were taken in the morning after a 12 h fast using standardized methods. Weight was measured with subjects in undergarments, height was measured by stadiometer, and BMI was calculated as body weight (kg) divided by the square of height (m). Waist circumference was measured as the narrowest circumference between the lower rib margin and the anterior superior iliac spine, and body composition evaluated by bioelectrical impedance. Brachial blood pressure was measured in the left arm of the supine subjects, after 5 min of quiet rest, with a digital electronic tensiometer (regular or large adult cuffs were used according to arm circumference). Values were calculated as the average of the last two of three consecutive measurements obtained at 3-min intervals. A 75-g OGTT was performed with 0-, 30-, 60-, 90-, and 120-min sampling for plasma glucose and insulin assay. Insulin sensitivity was assessed by euglycemic–hyperinsulinemic clamp study, as previously described [23]. Briefly, a continuous insulin infusion was initiated at the rate of 40 mU/m2 of body surface area per min, after a priming dose, in order to reach and maintain steady-state plasma insulin of 80 ± 5 lU/ml. Plasma glucose was assessed at 5-min intervals during the 2 h clamp study by a glucose analyzer. In the study subjects, mean plasma glucose concentration during the last hour of the clamp was 93 ± 5 mg/dl. Plasma insulin concentrations were measured every 20 min during the insulin infusion. The study was approved by Institutional Ethics Committees of the
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University of Rome Tor Vergata and Catanzaro, and written informed consent was obtained from each participant in accordance with principles of the Declaration of Helsinki.
Insulin secretion was estimated by insulinogenic index I30I0/G30-G0 (DI30/DG30). To evaluate b-cell function, the disposition index was calculated as DI30/DG30 9 MFFM/ SSPI.
Ultrasound measurement of IMT of the common carotid artery
Definitions of metabolic status
High-resolution B-mode ultrasound was used to measure IMT of the common carotid artery using an ATL HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothell, WA) equipped with a 5-MHz linear array transducer. The thickness of the intima-media complex was assessed as previously described [23]. Manual measurements were taken in plaque-free portions of the 10-mm linear segment proximal to the carotid bulb. For each individual, two measurements were taken bilaterally and the values were averaged. Ultrasound measurement of IMT was taken by a trained examiner who was unaware of the subjects’ anthropometric and metabolic findings. Analytical determinations Glucose, triglyceride, and total and HDL-C concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). Circulating irisin concentrations were measured using commercial enzyme-linked immunosorbent assays (ELISA, Catalog No. EK-067-52, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). Serum insulin concentration was determined by a chemiluminescencebased assay (ImmuliteÒ, Siemens, Italy). Calculation Glucose disposal (M) was calculated as the mean rate of glucose infusion measured during the last 60 min of the clamp examination (steady-state) and is expressed as milligrams per minute per kilogram fat-free mass (MFFM) measured with the use of electrical bioimpedance. MFFM value was corrected for steady-state plasma insulin concentration during the last hour of the clamp (MFFM/SSPI). Insulin clearance was calculated by dividing the rate of insulin infusion by the mean steady-state plasma insulin concentration during the euglycemic clamp [24]. The Matsuda index was calculated as follows [25]:
10; 000=
Subjects were classified into glucose tolerance status according to the American Diabetes Association (ADA) criteria [26]: NGT with fasting plasma glucose (FPG) \100 mg/dl and 2 h post-load \140 mg/dl, isolated impaired fasting glucose (IFG) with FPG ranging from 100 to 125 mg/dl and 2 h post-load \140 mg/dl, isolated impaired glucose tolerance (IGT) with FPG \ 100 mg/dl and 2 h post-load [140 and \200 mg/dl, and combined IFG/IGT with FPG ranging from 100 to 125 mg/dl and 2 h post-load [140 and \200 mg/dl. Subjects were divided into three groups on the basis of their BMI: normal weight (BMI \ 25.0 kg/m2), overweight (BMI 25.0–29.9 kg/m2), and obese (BMI [ 30 kg/m2). Statistical analyses Variables with skewed distribution including triglyceride, fasting, and 2 h post-load insulin were natural log-transformed for statistical analyses. Continuous variables are expressed as mean ± SD. Categorical variables were compared by v2 test. Unpaired Student’s t test was used to compare differences of continuous variables between two groups. Partial correlation coefficients adjusted for age, gender, and BMI were computed between variables. Subjects were stratified into tertile according to their circulating irisin values. Anthropometric and metabolic differences between groups were tested after adjusting for gender, age, and BMI using a general linear model with post hoc Bonferroni correction for multiple comparisons. For all analyses, a P value B0.05 was considered to be statistically significant. All analyses were performed using SPSS software version 16.0 for Windows. Results Clinical characteristics and laboratory findings of the study group are shown in Table 1. Of the 192 subjects examined, 149 (77.6 %) had NGT, 17 had IFG (8.9 %), and 26
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Acta Diabetol Table 1 Clinical characteristics of the study cohort Overall cohort
Men
Women
P value
Number
192
101
91
–
Age (years)
39 ± 10
40 ± 10
38 ± 9
0.06
Body weight (kg)
83 ± 18
89 ± 16
77 ± 19
\0.0001
2
BMI (kg/m )
29.6 ± 6.1
29.5 ± 4.7
29.7 ± 7.4
0.76
Waist circumference (cm)
97 ± 13
100 ± 11
93 ± 15
\0.0001
Fat body mass (kg)
28 ± 15
25 ± 11
30 ± 16
0.01
Fat-free mass (kg)
55 ± 12
63 ± 10
46 ± 9
\0.0001
SBP (mmHg)
127 ± 15
131 ± 13
122 ± 13
\0.0001
DBP (mmHg)
82 ± 10
85 ± 10
78 ± 10
\0.0001
Total cholesterol (mg/dl)
198 ± 37
199 ± 36
1977 ± 38
0.63
HDL cholesterol (mg/dl) Triglyceride (mg/dl)
50 ± 14 120 ± 71
45 ± 11 137 ± 77
57 ± 14 102 ± 58
\0.0001 \0.0001
Fasting glucose (mg/dl)
89 ± 9
91 ± 9
86 ± 9
\0.0001
2 h Glucose (mg/dl)
112 ± 27
112 ± 24
113 ± 30
0.95 0.42
Fasting insulin (lU/ml)
12 ± 8
12 ± 8
11 ± 8
2 h Insulin (lU/ml)
83 ± 80
82 ± 74
85 ± 84
0.74
Irisin (ng/ml)
156 ± 81
153 ± 72
156 ± 87
0.81
Matsuda index
78 ± 47
74 ± 46
83 ± 47
0.21
DI30/DG30
26 ± 19
27 ± 22
25 ± 15
0.44
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
11.3 ± 7.2
9.0 ± 4.9
13.8 ± 8.3
\0.0001
Disposition index
261 ± 206
222 ± 193
304 ± 234
0.01
Insulin clearance (ml/min 9 m2)
718 ± 476
643 ± 274
764 ± 513
0.04
Intima-media thickness (mm)
0.68 ± 0.16
0.70 ± 0.17
0.66 ± 0.15
0.13
NGT/IFG/IGT (n)
149/17/26
74/4/13
75/13/13
0.11
Normal weight/overweight/obese
47/64/81
19/39/43
28/25/38
0.10
Data are mean ± SD. Triglyceride, fasting, and 2 h insulin levels were log-transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Differences between means were compared using unpaired Student’s t test. Categorical variables were compared by v2 test SBP systolic blood pressure, DBP diastolic blood pressure, NGT normal glucose tolerance, IGT impaired glucose tolerance, and IFG impaired fasting glucose
(13.5 %) had isolated IGT or combined IFG/IGT. In addition, 81 (42.2 %) subjects were obese and 64 (33.3 %) were overweight with no gender-specific differences in the distribution of BMI categories. Men were slightly older and exhibited a worse cardiometabolic risk profile including higher fat body mass, blood pressure, triglyceride, and fasting plasma glucose levels, and lower HDL cholesterol, insulin sensitivity, assessed by euglycemic–hyperinsulinemic clamp, and disposition index. No gender-specific differences were observed in circulating irisin levels between men and women. Univariate correlations between circulating irisin levels and anthropometric and metabolic variables are shown in Table 2. Circulating irisin levels were significantly correlated with age. After adjusting for age and gender, circulating irisin levels were positively correlated with body weight, body fat mass, fasting, 2 h post-load insulin levels,
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and carotid IMT and were negatively correlated with insulin-stimulated glucose disposal, Matsuda index of insulin sensitivity, disposition index, and insulin clearance. These correlations remained significant after further adjusting for BMI in addition to gender and age (Table 2). When univariate correlations between circulating irisin levels and anthropometric measures were analyzed in the obese subgroup (n = 81), irisin was positively correlated with body weight (r = 0.29, P = 0.005), BMI (r = 0.21, P = 0.02), waist circumference (r = 0.24, P = 0.01), and body fat mass (r = 0.28, P = 0.006) after adjusting for age and gender. Additionally, the correlations between circulating irisin levels, insulin sensitivity, disposition index, insulin clearance, and carotid IMT remained statistically significant when analysis was performed separately in men and women (Table 3). Anthropometric and metabolic characteristics of the study group stratified according to tertile of circulating
Acta Diabetol Table 2 Univariate correlations between circulating irisin levels and anthropometric and biochemical variables
Age (years)
Age- and gender-adjusted correlations between circulating irisin levels and metabolic variables
Age-, gender-, and BMI-adjusted correlations between circulating irisin levels and metabolic variables
Pearson’s correlation coefficient (r)
Pearson’s correlation coefficient (r)
-0.19
P 0.003*
-0.19
P 0.003*
Body weight (kg)
0.12
0.05
–
–
BMI (kg/m2)
0.04
0.28
–
–
Waist circumference (cm)
0.05
0.23
–
–
Fat body mass (kg)
0.12
0.05
–
–
Fat-free mass (kg)
0.06
0.47
–
–
SBP (mmHg)
0.05
0.27
0.03
0.31
DBP (mmHg)
0.10
0.08
0.05
0.24
Total cholesterol (mg/dl)
0.06
0.21
0.06
0.22
HDL cholesterol (mg/dl) Triglycerides (mg/dl)
-0.09 0.01
0.09 0.46
-0.11 0.02
0.06 0.38
Fasting glucose (mg/dl)
0.06
0.18
0.07
0.17
2 h Glucose (mg/dl)
0.11
0.07
0.10
0.08
Fasting insulin (lU/ml)
0.17
0.01
0.18
0.007
2 h Insulin (lU/ml)
0.15
0.02
0.14
0.03
Matsuda index
-0.13
0.04
-0.12
0.05
DI30/DG30
-0.11
0.09
-0.12
0.06
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
-0.18
0.007
-0.17
0.007
Disposition index
-0.27
\0.0001
-0.26
\0.0001
-0.26
\0.0001
-0.25
\0.0001
0.29
\0.0001
0.28
\0.0001
2
Insulin clearance (ml/min 9 m ) Intima-media thickness (mm)
BMI body mass index, SBP systolic blood pressure, and DBP diastolic blood pressure * P values refer to results after analyses with adjustment for gender
irisin levels are shown in Table 4. After adjusting for age, gender, and BMI, individuals in the highest tertile of irisin levels exhibited higher body fat mass, fasting, 2 h post-load insulin levels, carotid IMT, lower insulin-stimulated glucose disposal, Matsuda index of insulin sensitivity, disposition index, and insulin clearance as compared with subjects in the lowest tertile of circulating irisin levels.
Discussion Irisin is a novel myokine whose expression in skeletal muscle is stimulated by exercise; it exerts beneficial effects on metabolism by inducing the browning of subcutaneous white adipocytes [10]. Mildly, overexpression of irisin in mice fed a high-fat diet resulted in an improvement of obesity, glucose tolerance, and insulin resistance [10]. Although these initial promising results point to a potential beneficial role of irisin in regulating body weight, insulin sensitivity, and glucose homeostasis, studies in humans have led to divergent results [11–18]. Furthermore, the role
of irisin in cardiovascular disease remains still unsettled. These observations coupled with the accessibility of a carefully characterized cohort of nondiabetic adult individuals have provided the rationale for examining the relationship between circulating irisin levels, glucose homeostasis, insulin sensitivity, cardio-metabolic risk factors, and subclinical atherosclerosis. In the present crosssectional study, we report an inverse relationship between circulating irisin levels and insulin sensitivity, measured either by the euglycemic–hyperinsulinemic clamp or by the Matsuda index, a validated OGTT-derived insulin sensitivity index [25]. These findings obtained using gold standard techniques are consistent with previous studies showing both a positive association between circulating irisin levels and insulin resistance measured by the homeostasis model assessment for insulin resistance (HOMA-IR) index [13] and an inverse relationship between human myotube FNDC5 expression and Matsuda index of insulin sensitivity [18]. Accordingly, an association between insulin sensitivity and a single nucleotide polymorphism (SNP rs726344) in the FNDC5 locus,
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Acta Diabetol Table 3 Univariate correlations between circulating irisin levels and anthropometric and biochemical variables
Age (years)
Age-adjusted correlations between circulating irisin levels and metabolic variables in men
Age-adjusted correlations between circulating irisin levels and metabolic variables in women
Pearson’s correlation coefficient (r)
P
Pearson’s correlation coefficient (r)
-0.10
0.08*
-0.31
P 0.001*
Body weight (kg)
0.12
0.05
0.13
0.10
BMI (kg/m2)
0.04
0.36
0.08
0.23
Waist circumference (cm)
0.08
0.22
0.07
0.26
Fat body mass (kg)
0.12
0.06
0.11
0.07
Fat-free mass (kg)
0.07
0.25
0.04
0.46
SBP (mmHg)
0.10
0.14
0.03
0.49
DBP (mmHg)
0.06
0.28
0.04
0.37
Total cholesterol (mg/dl)
0.01
0.48
0.22
0.02
HDL cholesterol (mg/dl) Triglycerides (mg/dl)
-0.02 0.02
0.43 0.44
0.02 0.03
0.39 0.37
Fasting glucose (mg/dl)
0.09
0.19
0.07
0.24
2 h Glucose (mg/dl)
0.10
0.17
0.14
0.09
Fasting insulin (lU/ml)
0.10
0.10
0.24
0.01
2 h Insulin (lU/ml)
0.24
0.01
0.08
0.25
Matsuda index
-0.27
0.004
-0.11
0.19
DI30/DG30
-0.07
0.52
-0.18
0.06
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
-0.16
0.05
-0.24
0.01
Disposition index
-0.23
0.01
-0.34
0.001
-0.18
0.04
-0.31
0.18
0.04
0.47
2
Insulin clearance (ml/min 9 m ) Intima-media thickness (mm)
0.001 \0.0001
BMI body mass index, SBP systolic blood pressure, and DBP diastolic blood pressure * P values refer to results after analyses without adjustment
encoding the irisin precursor, has been reported. Interestingly, it was observed a trend for an association of the insulin-desensitizing minor A allele of SNP rs726344 with higher FNDC5 mRNA contents (P = 0.19) in human myotubes [18]. We also observed a positive association between circulating irisin levels, fasting, and 2 h post-load insulin levels, in agreement with most [12, 14, 18], but not all prior studies [16]. It has been reported that impaired insulin clearance is a major determinant of hyperinsulinemia after an oral glucose load [27, 28], and therefore, it is possible that the increases in fasting and 2 h post-load insulin levels observed in individuals with higher circulating irisin levels may be due to differences in insulin clearance. Accordingly, we observed an inverse relationship between insulin clearance and circulating irisin levels as well as a lower insulin clearance in the individuals in the highest tertile of irisin levels. A decrease in insulin clearance may represent a compensatory mechanism to reduce the demand on b-cells as insulin resistance worsens [29]. Interestingly, we found that
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subjects in the highest tertile of irisin levels exhibit both a decrease in disposition index, which reflects b-cell adequacy to adjust to prevailing insulin resistance, and diminished insulin clearance. Thus, it is possible to hypothesize that the reduced insulin clearance observed in subjects with elevated levels of irisin represents an adaptive mechanism to preserve b-cells from the overburden to compensate for impaired insulin sensitivity. Importantly, the inverse relationship between circulating irisin levels, fasting, 2 h post-load insulin levels, insulin sensitivity, disposition index, and insulin clearance remains significant after adjustment for confounders potentially affecting circulating irisin including age, gender, and BMI. In fact, there is evidence that circulating irisin levels [12] or muscle FNDC5 gene expression [16] is inversely associated with age, and accordingly, an inverse association has been also observed in the present study (Table 2). In addition, a positive correlation between BMI or body fat mass and circulating irisin levels [12–15] or muscle FNDC5 mRNA expression [11, 16] has been reported in
Acta Diabetol Table 4 Anthropometric and metabolic characteristics of the study subjects stratified according to tertile of circulating irisin levels
Gender (male/female)
Tertile 1 (n = 64)
Tertile 2 (n = 64)
Tertile 3 (n = 64)
34/30
34/30
33/31
P
0.97 b
0.03*
Age (years)
41 ± 9
38 ± 10
37 ± 9
Body weight (kg)
79 ± 15
85 ± 20a
86 ± 19b
0.03§
BMI (kg/m )
28.8 ± 5.1
30.3 ± 6.7
29.8 ± 6.4
0.17§
Waist circumference (cm)
95 ± 11
98 ± 13
98 ± 16
0.20§
2
b
b
0.02§ 0.40§ 0.31
Fat body mass (kg)
24 ± 9
30 ± 17
29 ± 16
Fat-free mass (kg) SBP (mmHg)
55 ± 11 126 ± 13
54 ± 14 125 ± 15
56 ± 13 128 ± 17
DBP (mmHg)
82 ± 11
81 ± 9
82 ± 10
0.30
Total cholesterol (mg/dl)
202 ± 33
191 ± 34
200 ± 39
0.07
HDL cholesterol (mg/dl)
52 ± 14
48 ± 13
49 ± 12
0.40
Triglyceride (mg/dl)
124 ± 67
115 ± 56
125 ± 86
0.63
Fasting glucose (mg/dl)
90 ± 9
90 ± 8
89 ± 11
0.40
2 h Glucose (mg/dl)
112 ± 25
112 ± 26
114 ± 30
0.76
Fasting insulin (lU/ml)
9±6
13 ± 9a
14 ± 8a
0.05
2 h Insulin (lU/ml)
66 ± 65
92 ± 90
90 ± 69b
0.02
Irisin (ng/ml)
78 ± 27
141 ± 15
246 ± 63
\0.0001
Matsuda index
91 ± 54
75 ± 44a
70 ± 40b
0.02
DI30/DG30
26 ± 10
28 ± 16
23 ± 16
0.50
Insulin-stimulated glucose disposal (mg min kg FFM lU ml) 9 100
13.0 ± 7.5
11.3 ± 8.1
9.5 ± 5.2c
0.01
Disposition index
304 ± 211
288 ± 268
193 ± 130b
0.01
579 ± 223c
0.001
0.74 ± 0.13c
0.005
2
b
Insulin clearance (ml/min 9 m )
906 ± 583
669 ± 281
Intima-media thickness (mm)
0.64 ± 0.16
0.69 ± 0.17
Data are mean ± SD. Triglyceride, fasting, and 2 h insulin levels were log-transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Categorical variables were compared by v2 test. Comparisons between the three groups were made using a general linear model SBP systolic blood pressure, DBP diastolic blood pressure P values refer to results after analyses with adjustment for age, gender, and BMI. * P values refer to results after analyses with adjustment for gender. § P values refer to results after analyses with adjustment for age and gender a
P \ 0.05 versus tertile 1 group
b
P \ 0.01 versus tertile 1 group
c
P \ 0.001 versus tertile 1 group
most, but not all studies [16]. Accordingly, we found that circulating irisin levels were positively associated with body fat mass in the whole cohort and with BMI in the subgroup of obese individuals. These findings may explain some of the divergent results observed in prior studies in relation to insulin sensitivity and related traits and highlight the importance to consider confounders potentially affecting circulating irisin including age, and adiposity when comparing different groups of individuals. Next, we report a positive relationship between circulating irisin levels and carotid IMT, a validated measure of vascular atherosclerosis [30]. To the best of our knowledge, this is the first evidence linking higher circulating irisin levels with signs of vascular atherosclerosis. These data are consistent with the previous observation that circulating
irisin levels were associated with an increased 10-year risk of cardiovascular diseases, assessed by the general Framingham risk profile for cardiovascular diseases risk assessment tool [13]. It is well established that impaired insulin action and sustained hyperinsulinemia due to decreased insulin clearance may have adverse effects on target tissues of insulin action including vasculature [22– 24, 31]. Thus, it has been reported that exposure of vascular cells to high insulin concentrations induces proatherogenic effects. In cultured endothelial cells, elevated insulin concentrations induce the secretion of endothelin-1 (ET-1), a potent vasoconstrictor protein [32], the expression of vascular cell adhesion molecule (VCAM)-1, and monocytes adhesion to cultured endothelial cells [32, 33]. It is conceivable that impaired insulin sensitivity, elevated fasting,
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and postprandial insulinemia due to decreased insulin clearance may have adverse effects on vasculature in individuals with elevated circulating irisin levels. Given the role of irisin to promote improvements in glucose tolerance and insulin resistance in mice [10], the present and prior findings [13, 18] showing an inverse correlation between irisin levels and insulin sensitivity are surprising. It is tempting to speculate that increased irisin release from skeletal muscle and adipose tissue may represent a physiological compensatory mechanism in response to deterioration of insulin sensitivity. Alternatively, elevated circulating irisin levels may reflect a condition of tissue irisin resistance similar to the conditions of insulin and leptin resistance associated with elevated circulating insulin and leptin levels. Further mechanistic studies in humans aimed at investigating the molecular mechanisms regulating irisin release from tissues, the site(s) of action, and the signaling pathway are absolutely necessary to allow a better understanding of the pathophysiological role of irisin. It would also be interesting to investigate whether the activation of peroxisome proliferator-activated receptor-c (PPARc) by thiazolidinediones, which has been shown to induce an increase in mitochondrial content, and expression of UCP-1 and PGC-1a [34], is also able to modulate irisin expression. In fact, it is well established that the activation of PPARc by thiazolidinediones improves glucose metabolism by several mechanisms including enhancing insulin sensitivity, promoting differentiation of adipocytes, and reducing the inflammatory state [35, 36]; the modulation of irisin expression and secretion may represent an additional mechanism underlying the action of these drugs. The present study has several strengths including the relatively large sample comprising men and women, the homogeneity of the sample comprising White adults of European ancestry, the exclusion of confounding conditions such as previous cardiovascular disease, history of malignant disease, liver or kidney failure, the assessment of glucose tolerance by OGTT, the centralized measurements of biochemical variables, and the strict quality control of ultrasound studies performed by experienced examiners who were blinded to the subjects’ clinical and laboratory findings. The present results are also strengthened by the use of the gold standard euglycemic–hyperinsulinemic clamp for insulin sensitivity and insulin clearance assessment. Nevertheless, some limitations should be acknowledged in the interpretation of our results. First, the cross-sectional nature of the study precludes us to draw any conclusion on the role of irisin in the development of metabolic disorders and cardiovascular disease,
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and therefore, no conclusion regarding cause–effect relationships can be made. Second, all anthropometric and metabolic variables including OGTT euglycemic–hyperinsulinemic clamp were measured once. Although such an approach is a common limitation to large epidemiological studies, intra-individual variation cannot be taken into account, and some individuals might have been misclassified. Additionally, circulating irisin levels were measured at resting condition, and the possibility of obtaining different results after exercise cannot be excluded. Moreover, body composition was assessed by bioimpedance analysis. Although this method is well established, inexpensive, and simple to perform in the clinical routine, the analysis of body composition by bioimpedance provides a less accurate measure of various components of the body as compared with computed tomography or magnetic resonance imaging, which are not suitable for mass screening. Finally, all participants to the present study were Whites, and whether these observations can also be extended to nonwhite ethnic groups with different body composition remains to be determined. Conflict of interest G. Sesti, F. Andreozzi, T. V. Fiorentino, G. C. Mannino, A. Sciacqua, M. A. Marini and F. Perticone declare they have no conflict of interest. Human and Animal Rights disclosure All procedures followed were approved by approved Institutional Ethics Committee of the University of Rome Tor Vergata and Catanzaro and in accordance with the ethical of the Helsinki declaration of 1975, as revised in 2008. Informed consent disclosure Informed consent was obtained from all patients for being included in the study.
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