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Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance
Cytokine xxx (2017) xxx–xxx
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Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance Ersilia Nigro a,b, Olga Scudiero a,c, Maria Ludovica Monaco a, Rita Polito a,b, Pietro Schettino d, Anna Grandone e, Laura Perrone e, Emanuele Miraglia Del Giudice e,1, Aurora Daniele a,b,⇑,1 a
CEINGE-Biotecnologie Avanzate Scarl, Via G. Salvatore 486, 80145 Napoli, Italy Dipartimento di Scienze e Tecnologie Ambientali Biologiche Farmaceutiche, Seconda Università degli Studi di Napoli, Via A. Vivaldi 42, 81100 Caserta, Italy Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy d Dipartimento di Medicina Clinica e Chirurgia, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy e Dipartimento della Donna del Bambino e di Chirurgia Generale e Specialistica, Università della Campania "Luigi Vanvitelli", Via L. De Crecchio 4, 80138 Napoli, Italy b c
a r t i c l e
i n f o
Article history: Received 2 August 2016 Received in revised form 10 November 2016 Accepted 21 December 2016 Available online xxxx Keywords: Adiponectin HMW oligomers Irisin Childhood obesity Insulin resistance
a b s t r a c t Adiponectin (Acrp30), its high molecular weight (HMW) oligomers, and Irisin are molecules involved in several metabolic processes. To investigate if these cytokines could represent new metabolic markers, we evaluated the expression of Acrp30 and Irisin in serum of obese children from South Italy affected by different degrees of insulin resistance (IR). The anthropometric and metabolic features were evaluated in 27 obese children versus 13 age-matched controls. The expression of Acrp30, its pattern and Irisin were investigated by ELISA, western blotting and fast protein liquid chromatography. The HOMA index was significantly higher in obese children versus controls, and metabolic syndrome was more prevalent in obese children with elevated IR versus those with normal HOMA (38% vs 16%). Total Acrp30 and HMW oligomers were significantly lower in obese than in control children, and the difference was more pronounced in children with HOMA >3.4. In control and obese children, total Acrp30 and HMW oligomers were inversely related to HOMA (r-0.38, p 0.02; r-0.35, p 0.03). Irisin was significantly higher in obese than in control children, and was inversely correlated with Acrp30 and HMW (r-0.32, p 0.04; r-0.39, p 0.01). The inverse correlation of Acpr30 and HMW oligomers with HOMA indicates that Acpr30 is directly involved in IR status. Moreover, the inverse correlation between Irisin and Acrp30 and, more significantly, between Irisin and HMW oligomers suggests that the two cytokines are closely connected. The use of Acrp30, HMW oligomers and Irisin as predictive factors of IR in obese children remains to be further elucidated. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is a complex multifactorial disease resulting from the interaction of lifestyle, the environment and genetic variants. The prevalence of this disorder has increased worldwide and affects all age ranges including the pediatric population [1]. Overweight and obesity in children is a major public health problem because it often leads to adult obesity, which is associated with an enhanced risk of type 2 diabetes (T2DM), metabolic syndrome (MS), cardiovascular diseases (CVD), and different cancers [2–4]. In addition, high insulin resistance (IR) is a harbinger of T2DM in obese children. ⇑ Corresponding author at: CEINGE-Biotecnologie Avanzate Scarl, Via G. Salvatore 486, 80145 Napoli, Italy. E-mail address: [email protected] (A. Daniele). 1 The authors equally contributed to the work.
Adipose tissue and skeletal muscle are endocrine organs that secrete adipokine and myokine hormones, respectively and are thus critical for the regulation of energy metabolism and inflammation. Dysregulation of adipokine and myokine secretion plays a pivotal role in the development and progression of both obesity and IR in adults as well as in children [5]. Being involved in the regulation of energy expenditure, insulin sensitivity and inflammation, adipokines are promising molecular targets for the treatment of obesity and its related diseases [6]. Adiponectin (Acrp30) is widely recognized for its anti-diabetic, anti-inflammatory, and cardio-protective effects [7]. Acrp30 is a protein hormone of 244 amino acids that circulates at high concentrations (5–30 lg/ mL), and accounts for 0.01% of total serum proteins. It is synthesized as a monomer of 28–30 kDa that assembles in oligomers of various molecular weights: low molecular weight (LMW), medium molecular weight (MMW), and high molecular weight (HMW). The
http://dx.doi.org/10.1016/j.cyto.2016.12.018 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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latter are the most active oligomers in terms of insulin sensitivity modulation and anti-inflammatory activities [6]. Indeed, total Acrp30 and HMW levels are reduced in adult obesity and in its associated metabolic complications [8,9]. In obese children, Acrp30 has been correlated to such metabolic factors as IR [10,11]. The myokine Irisin is a protein hormone that plays a role, together with adipokines, in the maintenance of energy balance [12]. In fact, Irisin increases energy expenditure and glucose tolerance, which implicates it in the pathogenesis of various complications of obesity including IR and T2DM. Accordingly, high circulating concentrations of Irisin have been reported in patients affected by IR [12]. Although Acrp30 and Irisin have been closely associated to obesity, IR and diabetes [6,7,12], they have not yet been simultaneously analyzed in either children or adults affected by obesity, and/or IR. In the attempt to identify early markers of IR in obese children, we analyzed serum levels of total Acrp30, its oligomeric distribution and Irisin levels in obese children from South Italy affected or not by IR with those of sex- and age-matched lean children. We also investigated correlations between total Acrp30, and/ or Acrp30 HMW oligomers with Irisin. Finally, we investigated whether the two cytokines were correlated with anthropometric/ metabolic parameters, and/or IR and/or metabolic syndrome. 2. Methods 2.1. Subjects and anthropometric and biochemical measurements We enrolled 27 obese children from South Italy (age ranging from 4 to 13 years) and 13 sex- and age-matched lean controls attending the Department of Woman, Child and General and Specialized Surgery of the Second University of Naples (Italy). All procedures were in accordance with the Helsinki Declaration of Principles and approved by the local ethics committee. Written informed consent was obtained from all parents or guardians. Physical examination included weight and height evaluation and waist/height ratio and Z-scores to calculate the body mass index (BMI) [13]. We assessed pubertal stage according to Tanner’s criteria [13]. Systolic and diastolic blood pressure was measured and standard deviation scores calculated (SBP-SDS, and DBP-SDS). A blood sample was drawn at 8 a.m. and total cholesterol, HDL, LDL, triglyceride, glucose, aspartate transaminase (AST), and alanine transaminase (ALT) levels were measured. Obesity, MS and IR were defined as previously described [14]. Patients were deemed to have increased IR when the HOMA index exceeded 3.4 [15]. Total Acrp30 was measured by ELISA using houseproduced polyclonal antibodies [16], and with a commercial kit (Millipore, MA, USA). HMW oligomers in serum were detected using a commercial kit (Millipore, Billerica, MA, USA). Serum Irisin concentrations were measured also using a commercial ELISA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The lowest detectable concentration of Irisin was 2.06 ng/ml and highest was 36.12 ng/mL. Each serum sample was tested three times in triplicate. 2.2. Western blotting analysis Ten micrograms of serum proteins were treated as previously described (8, 9). All samples were tested twice in duplicate. Incubation with Acrp30 and Irisin antibodies (Novus Biologicals, Littleton, CO, USA) was performed according to the manufacturer’s instructions. Blots were developed by enhanced chemiluminescence (ECL) with Kodak BioMax Light film, (GE Healthcare Bio-Sciences Pittsburgh, PA, USA), digitalized with a scanner (1200 dpi) and analyzed by densitometry with ImageJ Software (http://rsbweb.nih.gov.ij/).
2.3. Gel filtration analysis The Acrp30 oligomeric pattern was analyzed with a Superdex 200 10/300 GL column connected to a fast protein liquid chromatography system (GE Healthcare Bio-Sciences Pittsburgh, PA, USA) as reported elsewhere [8,9]. In detail, 1,875 mg of total proteins were fractionated at 0.5 ml/min using PBS elution buffer. Fractions (500 ll) were collected and Acrp30 was tested using ELISA (100 ll) and western blotting (20 ll). The column was calibrated using ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (44 kDa) (GE Healthcare Bio-Sciences Pittsburgh, PA, USA). This analysis was performed on 4 controls, 4 obese subjects with HOMA <3.4 and 4 obese subjects with HOMA >3.4. 2.4. Statistical analysis Data were analyzed using the SPSS (v 10.0) Software Package (SPSS, Inc., Chicago, IL, USA). The significances of anthropometrical and biochemical parameter differences were determined using the t-test for normally distributed variables and the Mann–Whitney U test for non parametric variables. The chi square test was used to compare categorical factors. A multiple logistic regression analysis and general linear model were performed to correct the significant p values obtained by the univariate analysis. The correlations between Acrp30 levels or HMW oligomers and both HOMA and Irisin levels were determined using the Spearman’s test. Statistical significance was established at p < 0.05. 3. Results 3.1. Anthropometrical and biochemical features of children Anthropometrical and biochemical features of the children enrolled in the study are reported in Table 1. Obese children had significantly higher BMI Z-scores, waist-to-height ratios, and SBP-SDS, DBP-SDS, Chol-LDL, triglycerides, glucose and insulin levels than controls (p < 0.01); the HOMA values indicated that the obese children had significantly high IR (p < 0.02). In addition, both total Acrp30 levels and HMW isomer levels were significantly lower in obese children than in controls (total Acrp30: 14.6 ± 4 lg/mL vs 18.9 ± 7.8 lg/mL; p 0.02; HMW isomers: 8.3 ± 4 vs 6 ± 2.5 ± 4.1 lg/mL, p 0.03) (Fig. 1A). In obese children but not in controls, there was an inverse relationship between total Acrp30
Table 1 Comparison of anthropometric and biochemical features between controls and obese children. Data are expressed as mean ± S.D.
Number (boys) Age (years) BMI Z-score Waist/height ratio SBP SDS (mmHg) DBP SDS (mmHg) Total cholesterol (mg/dL) LDL (mg/dL) HDL (mg/dL) Triglycerides (mg/dL) ALT (U/L) AST (U/L) Glucose (mg/dL) Insulin (lU/L) HOMA Metabolic syndrome (%)
Controls
Obeses
p-value
13 (4) 8 ± 2.4 0.46 ± 1.2 0.45 ± 0.02 0.1 ± 0.9 0.5 ± 0.5 150 ± 17 73 ± 12 60 ± 4 55 ± 3.5 18 ± 13 23 ± 4.5 73 ± 7 6±4 1.62 ± 0.3 0
27 (19) 9.7 ± 2.7 2.7 ± 0.5 0.62 ± 0.04 0.7 ± 1.3 0.8 ± 0.9 152 ± 27 83 ± 23 47 ± 9.5 104 ± 48 29 ± 13 23 ± 45.9 80 ± 6 15 ± 7 3 ± 1.4 30%
0.8 0.8 0.00001 0.01 0.01 0.01 0.8 0.05 0.01 0.007 0.3 0.9 0.03 0.01 0.02 0.01
SBP SDS, systolic pressure; DBP SDS, diastolic pressure; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Acrp30, adiponectin; HMW, high molecular weight oligomers.
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 1. Adiponectin and its HMW oligomer levels were lower d while irisin levels were higher in obese than in control children. (A) Acrp30 total and HMW levels and (B) Irisin serum concentration were determined by ELISA assay. Asterisks indicate significant differences between obese and control children. Differences between obese children with HOMA <3.4 and HOMA >3.4 are indicated as P value.
or HMW and HOMA (r-0.38, p 0.02 and r-0.35, p 0.03, respectively) (Spearman’s test). At ELISA, Irisin levels were significantly higher in obese children than in controls 16.4 ± 9.12 vs 5.62 ± 2.04 ng/mL; p 0.003) (Fig. 1B). In addition, Irisin levels were inversely related to both total Acrp30 levels and HMW oligomers (r-0.32 p 0.04 and r-0.39 p 0.01, respectively). Notably, the significance of the inverse correlation was greater between Irisin and HMW oligomer levels. No other significant correlations among Acrp30, HMW oligomers or Irisin and the main clinical features of the investigated children were found. We divided the obese children into two groups on the basis of IR levels (HOMA below or above 3.4). As shown in Table 2, the BMI Z-score, waist-to-height ratio, DBP-SDS, insulin, total Acrp30 and HMW levels were significantly higher in patients with HOMA >3.4 than in patients with HOMA <3.4. Irisin concentrations did not differ between the two groups of obese children. Lastly, MS was more prevalent in children with IR than in children without IR (38% vs 16%, p 0.01). Subsequent experiments were performed in obese children with IR and in obese children without IR. 3.2. Adiponectin analysis by Western blotting As shown in the Western blots in Fig. 2A and B, we analyzed the distribution of serum Acrp30 in the control group and in the obese children with low or without IR. Three bands corresponding to
Table 2 Comparison of anthropometric and biochemical features in obese children with HOMA <3.4 versus those with HOMA >3.4. Data are expressed as mean ± S.D.
Number (boys) Age BMI Z-score Waist/height ratio SBP SDS (mmHg) DBP SDS (mmHg) Total cholesterol (mg/dL) LDL (mg/dL) HDL (mg/dL) Triglycerides (mg/dL) ALT (U/L) AST (U/L) Glucose (mg/dL) Insulin (lU/L) HOMA Metabolic syndrome (%)
(HOMA < 3.4)
(HOMA > 3.4)
P value
15 (10) 9.4 ± 2.1 2.8 ± 0.5 0.59 ± 0.04 0.04 ± 1.5 0.16 ± 1 151 ± 32 80.5 ± 24 48 ± 12 102 ± 61 28 ± 12 23 ± 4.3 80 ± 7 10 ± 3 2 ± 0.5 16%
12 (9) 9.9 ± 2.8 3.7 ± 1.8 0.64 ± 0.03 1 ± 0.7 1.5 ± 0.6 154 ± 23 87 ± 22 46 ± 6 106 ± 35 29 ± 15 23 ± 7 81 ± 5 20 ± 6 4.2 ± 1.2 38%
0.3 0.6 0.04 0.02 0.6 0.03 0.8 0.5 0.6 0.8 0.3 0.9 0.7 0.0001 0.0001 0.01
SBP SDS, systolic pressure; DBP SDS, diastolic pressure; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Acrp30, adiponectin; HMW, high molecular weight oligomers.
HMW (250 kDa), MMW (180 kDa), and LMW (70 kDa) oligomers were evident in both control and obese children with and without IR (Fig. 2A). The densitometric evaluation of oligomeric distribution showed that the expression of HMW, MMW and LMW oligomers was higher in controls than in obese children, and that the expression of HMW oligomers, which are the most biologically relevant oligomers were significantly lower in obese children (p < 0.05). In addition, obese children with increased IR had a more pronounced reduction of Acrp30 oligomers (Fig. 2B). 3.3. Adiponectin oligomerization status by FPLC analysis To assess the distribution of Acrp30 oligomers in greater detail, we performed Fast Protein Liquid Chromatography under native conditions (FPLC). In each FPLC fraction, Acrp30 was detected by both ELISA (Fig. 3A) and Western blotting (Fig. 3B). Both analyses confirmed that Acrp30 levels were higher in controls than in obese children, and revealed a more pronounced reduction of HMW oligomers in obese children with elevated IR (HOMA >3.4) This high performance technique coupled to ELISA and Western blotting assays provides a detailed breakdown of Acrp30 HMW species and may serve as a tool with which to identify alterations of the Acrp30 profile. 3.4. Irisin expression by Western blotting We analyzed the expression of Irisin in the control group and in the subgroups of obese children with or without IR using Western blotting (Fig. 2C and D). A band of 25 kDa was obtained for both control and obese children (Fig. 2C). Densitometry showed that Irisin expression was significantly higher in obese children than in controls, (p < 0.05) (Fig. 2D); Irisin expression did not differ between children with HOMA >3.4 and children with HOMA <3.4. 4. Discussion Childhood obesity is increasing at a shocking rate worldwide [17], and it strongly predisposes to such metabolic disorders as IR. In a recent study of 208 obese children and adolescents, the rate of elevated IR was 37% in boys and 27.8% in girls in the prepubertal period, and 61.7% and 66.7%, respectively in the pubertal period [18]. The concomitant presence of obesity and increased IR is difficult to revert and frequently leads to T2DM later in life. Therefore, early markers of IR in obese children are required in order to initiate personalized therapeutic and monitoring strategies [1]. Both skeletal muscle and adipose tissues produce and secrete cytokines that could represent new markers and or new therapeu-
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 2. Adiponectin HMW oligomers and Irisin, analyzed by western blotting, were lower in sera from obese children with and without IR than in controls. (A) A representative blot of adiponectin different oligomers (HMW, MMW, and LMW) in serum of two controls, two obese patients with HOMA <3.4 and two obese patients with HOMA >3.4. (B) Graphical representation of pixel quantization of all analyzed controls and obese children. (C) A representative blot of Irisin in the serum of two controls, two obese patients with HOMA <3.4 and two obese patients with HOMA >3.4. (B) Graphical representation of pixel quantization of all analyzed controls and obese children. Values are reported as percentage compared to the control.
tic targets of metabolic disorders such as IR. In this study, we focused on Acrp30, HMW oligomers and Irisin that play key roles in energy metabolism and are involved in obesity associated metabolic disorders [6]. In this scenario, we analyzed the expression of total Acrp30, HMW oligomers and Irisin in a small group of obese children from Southern Italy. Our data demonstrate that Acpr30 decreases in obese children and that the reduction of Acpr30 is more pronounced in obese children with increased IR (HOMA >3.4). Here we show that Acpr30 HMW oligomers, which are the most biologically relevant Acpr30 form, are significantly lower in obese children than in controls, and that this reduction was more pronounced in obese children with elevated IR. Both total Acrp30 and its HMW oligomers were inversely correlated to IR. These data confirm that both Acrp30 and its HMW oligomers are closely correlated to childhood obesity as well as to IR. In addition, we found that Irisin levels were increased in obese children but they were not correlated to IR status. In our cohort of obese children, Irisin levels appeared to be inversely correlated to both total Acrp30 and HWM oligomers. Serum Acrp30 levels are reduced in obese and diabetic subjects and are thus a marker of various metabolic diseases, and also of improvement of metabolic activity after weight loss [8,9]. This far, contrasting results have been reported concerning a correlation between total Acrp30 and obesity/IR in childhood obesity [11,19–21]. In fact, some authors reported decreased Acrp30 levels in obese children and adolescents versus age-matched lean peers, especially in those with IR, and hyperinsulinemia [2,19,22,23], whereas others did not find a correlation between Acrp30 levels and IR [19–24]. Nevertheless, to our knowledge, there are no studies in children addressing the correlation between Acrp30 and obesity together with evaluation of the association between Acrp30 and IR values. Irisin, beyond its browning effect on white adipocytes, improves glucose tolerance in diet-induced obese and insulin resistant mice. It has been suggested that the insulin-sensitizing effects of Irisin are mediated by increased AMPK phosphorylation and
glucose/fatty acid uptake [25–27]. In our study, Irisin expression was significantly higher in obese children than in controls, (p < 0.05), but did not differ between children with low or high IR, although this result may be due to the small cohort size. In one study, Irisin levels were correlated with BMI [28], but not in another study [29]. Contrasting results have been reported also regarding the association of Irisin with IR. Two studies, conducted in adults, reported that Irisin is independently and positively associated with IR [30,31], whereas in another study, Irisin was reported to be inversely correlated to IR, as in our study [32]. In agreement with our data, Reinher et al. reported a direct correlation between Irisin and BMI, but not with impaired glucose tolerance in adolescents [24]. Although Irisin was reported to be inversely correlated with total Acrp30 in adults with IR and MS, to our knowledge no data have been published on this correlation in obese children [31,33,34]. Here we report an inverse correlation between Irisin levels and both total Acrp30 and its HMW oligomers. In detail, Irisin levels were specifically related to Acrp30 HMW oligomers, which are those with the most relevant insulin-sensitizing activity [6]. This novel observation highlights the connection between muscle and adipose tissues, and suggests the presence of a specific molecular mechanism that regulates the expression of both Acrp30 and Irisin [35]. Alternatively, this inverse relationship may reflect the fact that elevation of Irisin could result from the decrease of Acrp30 levels thereby creating a feedback mechanism to increase energy expenditure. In conclusion, this study demonstrates that Irisin, Acrp30, and particularly its HMW oligomers, are involved in childhood obesity. Importantly, both total Acrp30 and HMW levels were inversely correlated to IR in obese children suggesting that this adipokine plays a functional role in metabolic alterations and obesity exacerbation in children. On the contrary, although not directly associated to IR status, Irisin was inversely related to total Acrp30, particularly to its HMW oligomers, which suggests there is a connection between Irisin and Acrp30. Although produced by different
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 3. Gel filtration analysis of adiponectin in sera from controls and obese children with and without IR. (A) An aliquot of each fraction obtained from FPLC analysis was subjected to ELISA. Absorbance at 405 nm of each aliquot subjected to ELISA. The values are reported as mean of the absorbance ± S.D. (B) An image of each fraction of the most representative sample subjected to Western blotting analysis.
tissues via different molecular pathways, both proteins exert an insulin-sensitizing action and their inverse relationship suggests that they are bi-directionally regulated. Our results confirm that Acrp30 levels are modulated in children with IR, while Irisin levels are unmodified. Our data suggest that early dysregulation of Acrp30 expression could be a response to obesity-related metabolic disturbances in children. Further studies are needed to elucidate the molecular mechanisms underlying the effects of Acrp30 and Irisin on IR, and to determine whether Acrp30 and Irisin could serve as biomarkers to predict IR in obese children. Declaration of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions AD, EMDG and LP conceived and directed the study, and discussed the comprehensive assembly of data acquisition. EN, OS, MLM, RP and PS designed and performed the experiments and evaluated the results. AD, EMDG and EN wrote the manuscript, with revisions from EN, OS, MLM, RP, PS, AG, and LP. Acknowledgements Grant POR Campania FSE 2007/2013 (CAMPUS-Bioframe) (to F. S.), project DIAINTECH from the Regione Campania, Italy. We thank
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Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance Ersilia Nigro a,b, Olga Scudiero a,c, Maria Ludovica Monaco a, Rita Polito a,b, Pietro Schettino d, Anna Grandone e, Laura Perrone e, Emanuele Miraglia Del Giudice e,1, Aurora Daniele a,b,⇑,1 a
CEINGE-Biotecnologie Avanzate Scarl, Via G. Salvatore 486, 80145 Napoli, Italy Dipartimento di Scienze e Tecnologie Ambientali Biologiche Farmaceutiche, Seconda Università degli Studi di Napoli, Via A. Vivaldi 42, 81100 Caserta, Italy Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy d Dipartimento di Medicina Clinica e Chirurgia, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy e Dipartimento della Donna del Bambino e di Chirurgia Generale e Specialistica, Università della Campania "Luigi Vanvitelli", Via L. De Crecchio 4, 80138 Napoli, Italy b c
a r t i c l e
i n f o
Article history: Received 2 August 2016 Received in revised form 10 November 2016 Accepted 21 December 2016 Available online xxxx Keywords: Adiponectin HMW oligomers Irisin Childhood obesity Insulin resistance
a b s t r a c t Adiponectin (Acrp30), its high molecular weight (HMW) oligomers, and Irisin are molecules involved in several metabolic processes. To investigate if these cytokines could represent new metabolic markers, we evaluated the expression of Acrp30 and Irisin in serum of obese children from South Italy affected by different degrees of insulin resistance (IR). The anthropometric and metabolic features were evaluated in 27 obese children versus 13 age-matched controls. The expression of Acrp30, its pattern and Irisin were investigated by ELISA, western blotting and fast protein liquid chromatography. The HOMA index was significantly higher in obese children versus controls, and metabolic syndrome was more prevalent in obese children with elevated IR versus those with normal HOMA (38% vs 16%). Total Acrp30 and HMW oligomers were significantly lower in obese than in control children, and the difference was more pronounced in children with HOMA >3.4. In control and obese children, total Acrp30 and HMW oligomers were inversely related to HOMA (r-0.38, p 0.02; r-0.35, p 0.03). Irisin was significantly higher in obese than in control children, and was inversely correlated with Acrp30 and HMW (r-0.32, p 0.04; r-0.39, p 0.01). The inverse correlation of Acpr30 and HMW oligomers with HOMA indicates that Acpr30 is directly involved in IR status. Moreover, the inverse correlation between Irisin and Acrp30 and, more significantly, between Irisin and HMW oligomers suggests that the two cytokines are closely connected. The use of Acrp30, HMW oligomers and Irisin as predictive factors of IR in obese children remains to be further elucidated. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is a complex multifactorial disease resulting from the interaction of lifestyle, the environment and genetic variants. The prevalence of this disorder has increased worldwide and affects all age ranges including the pediatric population [1]. Overweight and obesity in children is a major public health problem because it often leads to adult obesity, which is associated with an enhanced risk of type 2 diabetes (T2DM), metabolic syndrome (MS), cardiovascular diseases (CVD), and different cancers [2–4]. In addition, high insulin resistance (IR) is a harbinger of T2DM in obese children. ⇑ Corresponding author at: CEINGE-Biotecnologie Avanzate Scarl, Via G. Salvatore 486, 80145 Napoli, Italy. E-mail address: [email protected] (A. Daniele). 1 The authors equally contributed to the work.
Adipose tissue and skeletal muscle are endocrine organs that secrete adipokine and myokine hormones, respectively and are thus critical for the regulation of energy metabolism and inflammation. Dysregulation of adipokine and myokine secretion plays a pivotal role in the development and progression of both obesity and IR in adults as well as in children [5]. Being involved in the regulation of energy expenditure, insulin sensitivity and inflammation, adipokines are promising molecular targets for the treatment of obesity and its related diseases [6]. Adiponectin (Acrp30) is widely recognized for its anti-diabetic, anti-inflammatory, and cardio-protective effects [7]. Acrp30 is a protein hormone of 244 amino acids that circulates at high concentrations (5–30 lg/ mL), and accounts for 0.01% of total serum proteins. It is synthesized as a monomer of 28–30 kDa that assembles in oligomers of various molecular weights: low molecular weight (LMW), medium molecular weight (MMW), and high molecular weight (HMW). The
http://dx.doi.org/10.1016/j.cyto.2016.12.018 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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latter are the most active oligomers in terms of insulin sensitivity modulation and anti-inflammatory activities [6]. Indeed, total Acrp30 and HMW levels are reduced in adult obesity and in its associated metabolic complications [8,9]. In obese children, Acrp30 has been correlated to such metabolic factors as IR [10,11]. The myokine Irisin is a protein hormone that plays a role, together with adipokines, in the maintenance of energy balance [12]. In fact, Irisin increases energy expenditure and glucose tolerance, which implicates it in the pathogenesis of various complications of obesity including IR and T2DM. Accordingly, high circulating concentrations of Irisin have been reported in patients affected by IR [12]. Although Acrp30 and Irisin have been closely associated to obesity, IR and diabetes [6,7,12], they have not yet been simultaneously analyzed in either children or adults affected by obesity, and/or IR. In the attempt to identify early markers of IR in obese children, we analyzed serum levels of total Acrp30, its oligomeric distribution and Irisin levels in obese children from South Italy affected or not by IR with those of sex- and age-matched lean children. We also investigated correlations between total Acrp30, and/ or Acrp30 HMW oligomers with Irisin. Finally, we investigated whether the two cytokines were correlated with anthropometric/ metabolic parameters, and/or IR and/or metabolic syndrome. 2. Methods 2.1. Subjects and anthropometric and biochemical measurements We enrolled 27 obese children from South Italy (age ranging from 4 to 13 years) and 13 sex- and age-matched lean controls attending the Department of Woman, Child and General and Specialized Surgery of the Second University of Naples (Italy). All procedures were in accordance with the Helsinki Declaration of Principles and approved by the local ethics committee. Written informed consent was obtained from all parents or guardians. Physical examination included weight and height evaluation and waist/height ratio and Z-scores to calculate the body mass index (BMI) [13]. We assessed pubertal stage according to Tanner’s criteria [13]. Systolic and diastolic blood pressure was measured and standard deviation scores calculated (SBP-SDS, and DBP-SDS). A blood sample was drawn at 8 a.m. and total cholesterol, HDL, LDL, triglyceride, glucose, aspartate transaminase (AST), and alanine transaminase (ALT) levels were measured. Obesity, MS and IR were defined as previously described [14]. Patients were deemed to have increased IR when the HOMA index exceeded 3.4 [15]. Total Acrp30 was measured by ELISA using houseproduced polyclonal antibodies [16], and with a commercial kit (Millipore, MA, USA). HMW oligomers in serum were detected using a commercial kit (Millipore, Billerica, MA, USA). Serum Irisin concentrations were measured also using a commercial ELISA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The lowest detectable concentration of Irisin was 2.06 ng/ml and highest was 36.12 ng/mL. Each serum sample was tested three times in triplicate. 2.2. Western blotting analysis Ten micrograms of serum proteins were treated as previously described (8, 9). All samples were tested twice in duplicate. Incubation with Acrp30 and Irisin antibodies (Novus Biologicals, Littleton, CO, USA) was performed according to the manufacturer’s instructions. Blots were developed by enhanced chemiluminescence (ECL) with Kodak BioMax Light film, (GE Healthcare Bio-Sciences Pittsburgh, PA, USA), digitalized with a scanner (1200 dpi) and analyzed by densitometry with ImageJ Software (http://rsbweb.nih.gov.ij/).
2.3. Gel filtration analysis The Acrp30 oligomeric pattern was analyzed with a Superdex 200 10/300 GL column connected to a fast protein liquid chromatography system (GE Healthcare Bio-Sciences Pittsburgh, PA, USA) as reported elsewhere [8,9]. In detail, 1,875 mg of total proteins were fractionated at 0.5 ml/min using PBS elution buffer. Fractions (500 ll) were collected and Acrp30 was tested using ELISA (100 ll) and western blotting (20 ll). The column was calibrated using ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (44 kDa) (GE Healthcare Bio-Sciences Pittsburgh, PA, USA). This analysis was performed on 4 controls, 4 obese subjects with HOMA <3.4 and 4 obese subjects with HOMA >3.4. 2.4. Statistical analysis Data were analyzed using the SPSS (v 10.0) Software Package (SPSS, Inc., Chicago, IL, USA). The significances of anthropometrical and biochemical parameter differences were determined using the t-test for normally distributed variables and the Mann–Whitney U test for non parametric variables. The chi square test was used to compare categorical factors. A multiple logistic regression analysis and general linear model were performed to correct the significant p values obtained by the univariate analysis. The correlations between Acrp30 levels or HMW oligomers and both HOMA and Irisin levels were determined using the Spearman’s test. Statistical significance was established at p < 0.05. 3. Results 3.1. Anthropometrical and biochemical features of children Anthropometrical and biochemical features of the children enrolled in the study are reported in Table 1. Obese children had significantly higher BMI Z-scores, waist-to-height ratios, and SBP-SDS, DBP-SDS, Chol-LDL, triglycerides, glucose and insulin levels than controls (p < 0.01); the HOMA values indicated that the obese children had significantly high IR (p < 0.02). In addition, both total Acrp30 levels and HMW isomer levels were significantly lower in obese children than in controls (total Acrp30: 14.6 ± 4 lg/mL vs 18.9 ± 7.8 lg/mL; p 0.02; HMW isomers: 8.3 ± 4 vs 6 ± 2.5 ± 4.1 lg/mL, p 0.03) (Fig. 1A). In obese children but not in controls, there was an inverse relationship between total Acrp30
Table 1 Comparison of anthropometric and biochemical features between controls and obese children. Data are expressed as mean ± S.D.
Number (boys) Age (years) BMI Z-score Waist/height ratio SBP SDS (mmHg) DBP SDS (mmHg) Total cholesterol (mg/dL) LDL (mg/dL) HDL (mg/dL) Triglycerides (mg/dL) ALT (U/L) AST (U/L) Glucose (mg/dL) Insulin (lU/L) HOMA Metabolic syndrome (%)
Controls
Obeses
p-value
13 (4) 8 ± 2.4 0.46 ± 1.2 0.45 ± 0.02 0.1 ± 0.9 0.5 ± 0.5 150 ± 17 73 ± 12 60 ± 4 55 ± 3.5 18 ± 13 23 ± 4.5 73 ± 7 6±4 1.62 ± 0.3 0
27 (19) 9.7 ± 2.7 2.7 ± 0.5 0.62 ± 0.04 0.7 ± 1.3 0.8 ± 0.9 152 ± 27 83 ± 23 47 ± 9.5 104 ± 48 29 ± 13 23 ± 45.9 80 ± 6 15 ± 7 3 ± 1.4 30%
0.8 0.8 0.00001 0.01 0.01 0.01 0.8 0.05 0.01 0.007 0.3 0.9 0.03 0.01 0.02 0.01
SBP SDS, systolic pressure; DBP SDS, diastolic pressure; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Acrp30, adiponectin; HMW, high molecular weight oligomers.
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 1. Adiponectin and its HMW oligomer levels were lower d while irisin levels were higher in obese than in control children. (A) Acrp30 total and HMW levels and (B) Irisin serum concentration were determined by ELISA assay. Asterisks indicate significant differences between obese and control children. Differences between obese children with HOMA <3.4 and HOMA >3.4 are indicated as P value.
or HMW and HOMA (r-0.38, p 0.02 and r-0.35, p 0.03, respectively) (Spearman’s test). At ELISA, Irisin levels were significantly higher in obese children than in controls 16.4 ± 9.12 vs 5.62 ± 2.04 ng/mL; p 0.003) (Fig. 1B). In addition, Irisin levels were inversely related to both total Acrp30 levels and HMW oligomers (r-0.32 p 0.04 and r-0.39 p 0.01, respectively). Notably, the significance of the inverse correlation was greater between Irisin and HMW oligomer levels. No other significant correlations among Acrp30, HMW oligomers or Irisin and the main clinical features of the investigated children were found. We divided the obese children into two groups on the basis of IR levels (HOMA below or above 3.4). As shown in Table 2, the BMI Z-score, waist-to-height ratio, DBP-SDS, insulin, total Acrp30 and HMW levels were significantly higher in patients with HOMA >3.4 than in patients with HOMA <3.4. Irisin concentrations did not differ between the two groups of obese children. Lastly, MS was more prevalent in children with IR than in children without IR (38% vs 16%, p 0.01). Subsequent experiments were performed in obese children with IR and in obese children without IR. 3.2. Adiponectin analysis by Western blotting As shown in the Western blots in Fig. 2A and B, we analyzed the distribution of serum Acrp30 in the control group and in the obese children with low or without IR. Three bands corresponding to
Table 2 Comparison of anthropometric and biochemical features in obese children with HOMA <3.4 versus those with HOMA >3.4. Data are expressed as mean ± S.D.
Number (boys) Age BMI Z-score Waist/height ratio SBP SDS (mmHg) DBP SDS (mmHg) Total cholesterol (mg/dL) LDL (mg/dL) HDL (mg/dL) Triglycerides (mg/dL) ALT (U/L) AST (U/L) Glucose (mg/dL) Insulin (lU/L) HOMA Metabolic syndrome (%)
(HOMA < 3.4)
(HOMA > 3.4)
P value
15 (10) 9.4 ± 2.1 2.8 ± 0.5 0.59 ± 0.04 0.04 ± 1.5 0.16 ± 1 151 ± 32 80.5 ± 24 48 ± 12 102 ± 61 28 ± 12 23 ± 4.3 80 ± 7 10 ± 3 2 ± 0.5 16%
12 (9) 9.9 ± 2.8 3.7 ± 1.8 0.64 ± 0.03 1 ± 0.7 1.5 ± 0.6 154 ± 23 87 ± 22 46 ± 6 106 ± 35 29 ± 15 23 ± 7 81 ± 5 20 ± 6 4.2 ± 1.2 38%
0.3 0.6 0.04 0.02 0.6 0.03 0.8 0.5 0.6 0.8 0.3 0.9 0.7 0.0001 0.0001 0.01
SBP SDS, systolic pressure; DBP SDS, diastolic pressure; AST, aspartate aminotransferase; ALT, alanine aminotransferase; Acrp30, adiponectin; HMW, high molecular weight oligomers.
HMW (250 kDa), MMW (180 kDa), and LMW (70 kDa) oligomers were evident in both control and obese children with and without IR (Fig. 2A). The densitometric evaluation of oligomeric distribution showed that the expression of HMW, MMW and LMW oligomers was higher in controls than in obese children, and that the expression of HMW oligomers, which are the most biologically relevant oligomers were significantly lower in obese children (p < 0.05). In addition, obese children with increased IR had a more pronounced reduction of Acrp30 oligomers (Fig. 2B). 3.3. Adiponectin oligomerization status by FPLC analysis To assess the distribution of Acrp30 oligomers in greater detail, we performed Fast Protein Liquid Chromatography under native conditions (FPLC). In each FPLC fraction, Acrp30 was detected by both ELISA (Fig. 3A) and Western blotting (Fig. 3B). Both analyses confirmed that Acrp30 levels were higher in controls than in obese children, and revealed a more pronounced reduction of HMW oligomers in obese children with elevated IR (HOMA >3.4) This high performance technique coupled to ELISA and Western blotting assays provides a detailed breakdown of Acrp30 HMW species and may serve as a tool with which to identify alterations of the Acrp30 profile. 3.4. Irisin expression by Western blotting We analyzed the expression of Irisin in the control group and in the subgroups of obese children with or without IR using Western blotting (Fig. 2C and D). A band of 25 kDa was obtained for both control and obese children (Fig. 2C). Densitometry showed that Irisin expression was significantly higher in obese children than in controls, (p < 0.05) (Fig. 2D); Irisin expression did not differ between children with HOMA >3.4 and children with HOMA <3.4. 4. Discussion Childhood obesity is increasing at a shocking rate worldwide [17], and it strongly predisposes to such metabolic disorders as IR. In a recent study of 208 obese children and adolescents, the rate of elevated IR was 37% in boys and 27.8% in girls in the prepubertal period, and 61.7% and 66.7%, respectively in the pubertal period [18]. The concomitant presence of obesity and increased IR is difficult to revert and frequently leads to T2DM later in life. Therefore, early markers of IR in obese children are required in order to initiate personalized therapeutic and monitoring strategies [1]. Both skeletal muscle and adipose tissues produce and secrete cytokines that could represent new markers and or new therapeu-
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 2. Adiponectin HMW oligomers and Irisin, analyzed by western blotting, were lower in sera from obese children with and without IR than in controls. (A) A representative blot of adiponectin different oligomers (HMW, MMW, and LMW) in serum of two controls, two obese patients with HOMA <3.4 and two obese patients with HOMA >3.4. (B) Graphical representation of pixel quantization of all analyzed controls and obese children. (C) A representative blot of Irisin in the serum of two controls, two obese patients with HOMA <3.4 and two obese patients with HOMA >3.4. (B) Graphical representation of pixel quantization of all analyzed controls and obese children. Values are reported as percentage compared to the control.
tic targets of metabolic disorders such as IR. In this study, we focused on Acrp30, HMW oligomers and Irisin that play key roles in energy metabolism and are involved in obesity associated metabolic disorders [6]. In this scenario, we analyzed the expression of total Acrp30, HMW oligomers and Irisin in a small group of obese children from Southern Italy. Our data demonstrate that Acpr30 decreases in obese children and that the reduction of Acpr30 is more pronounced in obese children with increased IR (HOMA >3.4). Here we show that Acpr30 HMW oligomers, which are the most biologically relevant Acpr30 form, are significantly lower in obese children than in controls, and that this reduction was more pronounced in obese children with elevated IR. Both total Acrp30 and its HMW oligomers were inversely correlated to IR. These data confirm that both Acrp30 and its HMW oligomers are closely correlated to childhood obesity as well as to IR. In addition, we found that Irisin levels were increased in obese children but they were not correlated to IR status. In our cohort of obese children, Irisin levels appeared to be inversely correlated to both total Acrp30 and HWM oligomers. Serum Acrp30 levels are reduced in obese and diabetic subjects and are thus a marker of various metabolic diseases, and also of improvement of metabolic activity after weight loss [8,9]. This far, contrasting results have been reported concerning a correlation between total Acrp30 and obesity/IR in childhood obesity [11,19–21]. In fact, some authors reported decreased Acrp30 levels in obese children and adolescents versus age-matched lean peers, especially in those with IR, and hyperinsulinemia [2,19,22,23], whereas others did not find a correlation between Acrp30 levels and IR [19–24]. Nevertheless, to our knowledge, there are no studies in children addressing the correlation between Acrp30 and obesity together with evaluation of the association between Acrp30 and IR values. Irisin, beyond its browning effect on white adipocytes, improves glucose tolerance in diet-induced obese and insulin resistant mice. It has been suggested that the insulin-sensitizing effects of Irisin are mediated by increased AMPK phosphorylation and
glucose/fatty acid uptake [25–27]. In our study, Irisin expression was significantly higher in obese children than in controls, (p < 0.05), but did not differ between children with low or high IR, although this result may be due to the small cohort size. In one study, Irisin levels were correlated with BMI [28], but not in another study [29]. Contrasting results have been reported also regarding the association of Irisin with IR. Two studies, conducted in adults, reported that Irisin is independently and positively associated with IR [30,31], whereas in another study, Irisin was reported to be inversely correlated to IR, as in our study [32]. In agreement with our data, Reinher et al. reported a direct correlation between Irisin and BMI, but not with impaired glucose tolerance in adolescents [24]. Although Irisin was reported to be inversely correlated with total Acrp30 in adults with IR and MS, to our knowledge no data have been published on this correlation in obese children [31,33,34]. Here we report an inverse correlation between Irisin levels and both total Acrp30 and its HMW oligomers. In detail, Irisin levels were specifically related to Acrp30 HMW oligomers, which are those with the most relevant insulin-sensitizing activity [6]. This novel observation highlights the connection between muscle and adipose tissues, and suggests the presence of a specific molecular mechanism that regulates the expression of both Acrp30 and Irisin [35]. Alternatively, this inverse relationship may reflect the fact that elevation of Irisin could result from the decrease of Acrp30 levels thereby creating a feedback mechanism to increase energy expenditure. In conclusion, this study demonstrates that Irisin, Acrp30, and particularly its HMW oligomers, are involved in childhood obesity. Importantly, both total Acrp30 and HMW levels were inversely correlated to IR in obese children suggesting that this adipokine plays a functional role in metabolic alterations and obesity exacerbation in children. On the contrary, although not directly associated to IR status, Irisin was inversely related to total Acrp30, particularly to its HMW oligomers, which suggests there is a connection between Irisin and Acrp30. Although produced by different
Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018
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Fig. 3. Gel filtration analysis of adiponectin in sera from controls and obese children with and without IR. (A) An aliquot of each fraction obtained from FPLC analysis was subjected to ELISA. Absorbance at 405 nm of each aliquot subjected to ELISA. The values are reported as mean of the absorbance ± S.D. (B) An image of each fraction of the most representative sample subjected to Western blotting analysis.
tissues via different molecular pathways, both proteins exert an insulin-sensitizing action and their inverse relationship suggests that they are bi-directionally regulated. Our results confirm that Acrp30 levels are modulated in children with IR, while Irisin levels are unmodified. Our data suggest that early dysregulation of Acrp30 expression could be a response to obesity-related metabolic disturbances in children. Further studies are needed to elucidate the molecular mechanisms underlying the effects of Acrp30 and Irisin on IR, and to determine whether Acrp30 and Irisin could serve as biomarkers to predict IR in obese children. Declaration of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions AD, EMDG and LP conceived and directed the study, and discussed the comprehensive assembly of data acquisition. EN, OS, MLM, RP and PS designed and performed the experiments and evaluated the results. AD, EMDG and EN wrote the manuscript, with revisions from EN, OS, MLM, RP, PS, AG, and LP. Acknowledgements Grant POR Campania FSE 2007/2013 (CAMPUS-Bioframe) (to F. S.), project DIAINTECH from the Regione Campania, Italy. We thank
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Please cite this article in press as: E. Nigro et al., Adiponectin profile and Irisin expression in Italian obese children: Association with insulin-resistance, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2016.12.018