The association between pentraxin 3 and insulin resistance in obese children at baseline and after physical activity intervention

Clinica Chimica Acta 413 (2012) 1430–1437

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The association between pentraxin 3 and insulin resistance in obese children at baseline and after physical activity intervention Sang Hui Chu b, 1, Ji-Hye Park a, 1, Mi Kyung Lee a, Yoonsuk Jekal a, Ki Yong Ahn a, Jae Youn Chung a, Dong Hoon Lee a, Eun Sung Kim a, Masayo Naruse a, Jee-Aee Im g, Deok Kong d, Choon Hee Chung d, Ji Won Lee e, Kyong-Mee Chung c, Young-Bum Kim f,⁎, Justin Y. Jeon a,⁎⁎ a

Department of Sport and Leisure Studies, Yonsei University, Seoul, Korea Department of Clinical Nursing Science, Yonsei University College of Nursing, Nursing policy and Research Institute, Biobehavioral Research Center, Seoul, Korea c Department of Psychology, Yonsei University, Seoul, Korea d Department of Internal Medicine, Yonsei University Wonju College of Medicine, Seoul, Korea e Department of Family Medicine, Yonsei University College of Medicine, Seoul, Korea f Department of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA g Sport and Medicine Research Center, INTOTO Inc. Seoul, Korea b

a r t i c l e

i n f o

Article history: Received 13 March 2012 Received in revised form 29 May 2012 Accepted 1 June 2012 Available online 9 June 2012 Keywords: Obesity HOMA-IR PTX3 Physical activity Exercise

a b s t r a c t Background: The role of pentraxin-3 (PTX3) in the development of insulin resistance is still not clear. We aimed to test 1) whether circulating PTX3 levels are associated with insulin resistance and 2) whether changes in PTX3 levels after the physical activity are associated with changes in insulin resistance. Methods: Fifty-seven overweight or obese children (39 boys, 18 girls; age: 12.04 ± 0.82 y, BMI: 26.5± 1.2 kg/m2) participated in the study. All participants were housed together and their amount of physical activity (1823.5 ± 1.34 kcal/day) and food intake (1882 ± 68.8 kcal/day) were tightly controlled. Results: Circulating PTX3 levels at baseline were negatively associated with fasting insulin (r = −.336, p = 0.012) and homeostasis model assessment of insulin resistance (HOMA-IR) (r = −.334, p = 0.014) even after adjustment for BMI and Tanner stage. The degree of change in PTX3 levels notably associated with changes in fasting insulin (r=−.280, p=0.035) and HOMA-IR (r=−.281, p=.034) in response to the physical activity intervention. Subgroup analysis further indicates that HOMA-IR was improved more in subjects whose PTX3 levels were increased compared with subjects who PTX3 levels were decreased (HOMA-IR delta: −2.33± 1.3 vs −1.46±0.70, p=0.004). Conclusion: PTX3 is negatively associated with insulin resistance and associated with changes in insulin resistance induced by physical activity in overweight and obese children. © 2012 Published by Elsevier B.V.

1. Introduction Childhood obesity has increased significantly during the last decade and obesity associated with metabolic diseases such as cardiovascular disease and type 2 diabetes have become more prevalent among children [1,2]. Inflammatory markers have been identified as one of

Abbreviations: PTX3, pentraxin 3; HOMA-IR, homeostasis model assessment of insulin resistance; OGTT, oral glucose tolerance test; WC, waist circumference; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; PI3K, phosphatidylinositol 3-kinase. ⁎ Correspondence to: Y.-B. Kim, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA. Tel.: +1 617 735 3216; fax: +1 617 735 3323. ⁎⁎ Correspondence to: J.Y. Jeon, Department of Sport and Leisure Studies, 134 Shinchon-dong, Seodaemoon-ku, Seoul, 120‐749, Korea. Tel.: + 82 2 2123 6197; fax: + 82 2 2123 3198. E-mail addresses: [email protected] (Y-B. Kim), [email protected] (J.Y. Jeon). 1 Both authors contributed equally to this work. 0009-8981/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.cca.2012.06.002

main contributors for the development of obesity associated insulin resistance [3–5]. Exercise and physical activity are important to treat childhood obesity and obesity associated insulin resistance [6]. However, the exercise induced improvement of insulin resistance and its association with adipocytokines and/or inflammatory markers in children remained to be fully elucidated. Pentraxin 3 (PTX3), a novel inflammatory marker, is a member of the long pentraxin family, a component of the humoral arm of innate immunity [7,8]. Unlike the classic short pentraxin C-reactive protein (CRP), which is synthesized in the liver as a systemic response to local inflammation [9], PTX3 is produced rapidly in damaged tissue and may reflect more of a tissue-specific inflammatory response that includes smooth and skeletal muscle and adipocytes [10–12]. Although the role of PTX3 in the pathogenesis of atherosclerosis [13,14] and heart failure [15] has been identified, studies still report conflicting data on the association of PTX3 with adiposity and insulin resistance in human among many different health conditions [14,16,17]. These conflicting reports on the relationship among PTX3, adiposity and

S.H. Chu et al. / Clinica Chimica Acta 413 (2012) 1430–1437

insulin resistance could be due to the ages of the participants. Considering that PTX3 increases with the development of atherosclerosis, it might be more appropriate to investigate the relationship between PTX3, adiposity and insulin resistance in subjects without atherosclerosis but still have some degree of obesity [11,13,16]. Therefore, studying the relationship between PTX3 and obese children without atherosclerosis would be logical. In addition, most studies that reported an association between adiposity and circulating PTX3 levels have used body mass index (BMI) and waist circumference (WC) as an adiposity index. To completely understand the role of PTX3 in the development of obesity associated insulin resistance, the association of PTX3 with adiposity and insulin resistance should be investigated using more accurate adiposity measurements such as computed tomography (CT). Physical activity decreases insulin resistance and also alters various adipocytokines including adiponectin, tumor necrosis factor-alpha and interleukin-6 [18,19]. Fukuda et al. [20] and Nakajima et al. [21] recently reported changes in PTX3 levels after cardiac rehabilitation exercise and after high intensity aerobic and resistance exercise. However, the effects of exercise or short-term physical activity on PTX3 levels and its relationship to anthropometric, insulin resistance and metabolic parameters have not been studied. Therefore, the purpose of the study is to test whether 1) circulating PTX3 levels are associated with insulin resistance, anthropometric measurements and metabolic parameters and 2) whether 7 days of intense physical activity would alter PTX3 levels in association with changes in insulin resistance, anthropometric measurements and metabolic parameters. 2. Methods 2.1. Subject Fifty‐seven overweight or obese elementary school students in the fourth, fifth or sixth grade participated in the study. All subjects were classified as either overweight or obese according to the criteria based on Korean Society for the Study of Obesity guidelines for children [22]. Subject characteristics are summarized in Table 1. This study

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was approved by the Institutional Ethics Review Board at Yonsei University College of Medicine, Wonju Campus. Written informed consent was obtained from the parents of all participants. 2.2. The study design and short term physical activity program The baseline anthropometric measurements as well as blood samples were taken 3 days prior to the start of the program. The baseline CT measurements were taken between 2:00–4:00 p.m. of Day 1. All participants were housed together at Yonsei University Wonju Campus dormitory and followed strict nutrition and physical activity programs for 7 days (Supplementary Table 1). The physical activity program started at 2:00 p.m. on day 1 and ended at 5:00 p.m. of day 7. The postintervention anthropometric measurements and blood samples were taken between 7:30 a.m. and 8:30 a.m. of day 8. The post-intervention CT measurements were taken between 2:00–4:00 p.m. on day 8. All subjects participated in daily physical activities of an average energy expenditure of 1823±134 kcal per day. Daily physical activities included soccer, basketball, swimming, golf, line dance, jazz dance, hip hop dance, dodge-ball, structured-game-oriented physical activity, rope skipping and circuit training. While participating in the study, subjects consumed an average of 1882 ± 68 kcal (carbohydrate 63.5%, protein 21.5%, fat 15%) with strict inhibition of additional food intake (Supplementary Table 2). Participants went to bed at 10:00 p.m. and woke up at 7:00 a.m., which allowed them for 9 h of sleep per day during the period of the study. The short-term physical activity was organized and administered by a multi-disciplinary team composed of an exercise physiologist, clinical psychologist, clinical nutritionist, clinical nurse, and medical doctors from Yonsei University. 2.3. Anthropometric measurements Anthropometric measurements are described in detail in a previous publication [22]. Pubertal status was assessed by a physician using Tanner staging [23]. Of the children examined, 68.4% showed no evidence of

Table 1 Characteristics of participants.

Age Pubertal status Anthropometric measures BMI (kg/m2) WC (cm) WHR Body fat (%) VAT (cm2) SAT (cm2) Muscle mass (kg) MT muscle mass (cm2) Blood Pressure SBP (mm Hg) DBP (mm Hg) Glucose metabolism Glucose (mg/dl) Insulin (μU/ml) HOMA-IR Lipid profile TC (mg/dl) HDL-C (mg/dl) 40–86 (Girls) TG (mg/dl) Other hs-CRP (mg/dl) PTX3 (ng/dl)

Boys (n = 39)

Girls (n = 18)

Total (n = 57)

12.03 ± 0.84 1.18 ± 0.46

12.06 ± 0.80 1.89 ± 1.02

12.04 ± 0.82 1.41 ± 0.76

Reference range$

26.93 ± 2.89 86.84 ± 7.66 .95 ± .05 26.59 ± 4.06 77.96 ± 27.68 235.66 ± 64.52 40.80 ± 4.50 111.41 ± 17.12

25.56 ± 3.70 78.97 ± 10.02* .86 ± .07* 29.70 ± 4.11* 64.86 ± 27.23 185.39 ± 78.86* 36.8 ± 4.30* 102.41 ± 10.73*

26.50 ± 3.20 84.35 ± 9.16 .92 ± .07 27.57 ± 2.30 73.83 ± 27.98 219.78 ± 72.58 39.54 ± 5.11 108.67 ± 15.92

– – – – – – – –

111.67 ± 11.83 73.33 ± 9.82

110.28 ± 12.66 73.61 ± 10.26

111.23 ± 12 73.42 ± 9.87

– –

76.15 ± 6.92 12.35 ± 5.94 2.34 ± 1.19

77.39 ± 6.84 16.55 ± 7.58* 3.15 ± 1.41*

76.54 ± 6.86 13.67 ± 6.73 2.59 ± 1.31

172.44 ± 29.71 48.68 ± 7.60

171.67 ± 35.18 50.22 ± 9.22

172.19 ± 31.22 49.16 ± 8.10

100–220 40–80 (Boys)

118.90 ± 38.87

142.33 ± 69.45

126.30 ± 51.09

44–166

.17 ± .15 1.10 ± .74

.15 ± .21 .84 ± .48

.16 ± .17 1.02 ± .68

60–100 2.6–24.9 0.39–6.15

0.1–0.3 0.23–1.98^

Data are mean ± SD*p b 0.05 between genders, WC: waist circumference, WHR: waist-hip ratio, VAT: Visceral adipose tissue, SAT: Subcutaneous adipose tissue, MT muscle mass: Mid-thigh muscle mass, HOMA-IR: homeostasis model assessment-insulin resistance, QUICKI: quantitative insulin sensitivity check index , PTX3: pentraxin 3, $Reference range from Severance Hospital age category similar to participating subjects, Seoul, Korea. ^Reference range for PTX3 was calculated from the value of the participants in the current study since there was no reference range available and conducting the reference range study was the beyond the scope of the current study.

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−50 HU. The coefficients of variation for inter- and intra-observer reproducibility were 1.4% and 0.5%, respectively.

pubertal development (Tanner stage 1), 23.2% showed very early signs of pubertal development (Tanner stage 2), and 7.2% of the children were determined to be Tanner stage 3.

2.5. Insulin sensitivity measurements 2.4. Whole body composition and visceral and subcutaneous adipose tissue measurements

Since post-intervention insulin sensitivity needed to be measured in 57 individuals on the same day at the same time while subjects were still fasting, it was impossible to utilize euglycemic insulin clamps or an oral glucose tolerance test (OGTT). Although there is controversy over using the homeostasis model assessment of insulin resistance (HOMA-IR) as a measure of insulin sensitivity among children [24,25], the use of fasting insulin and HOMA-IR are the best available options when euglycemic clamps or OGTT are not feasible and QUICKI was not used because of reports that QUICKI may not accurately reflect changes insulin sensitivity with exercise training [26,27].

Percent body fat and total body fat mass were measured using bioelectric impedance analysis equipment (Inbody 4.0 Biospace, Seoul Korea). An abdominal adiposity and the mid-thigh muscle were quantified by CT (Tomoscan 350; Philips, Mahwah, NJ). The visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured with a 10-mm CT slice scan at the L4–L5 level with the subject in a supine position. Skeletal muscle attenuation was determined by measuring the mean value of all pixels within the range of 0–100 Hounsfield units (HU); adipose tissue areas fell in the range −150 to

PTX3 (ng/dl)

A

5

B5

R=-.001, p=.993

4

4

3

3

2

2

1

1

R=-.050, p=.713

0

0 15

20

25

30

35

40

0

50

30 20 10 0

Pentraxin-3 tertile

PTX3 (ng/dl)

5

F5

R=-.319, p=. 015

4

4

3

3

2

2

1

1

R=-.323, p=.014

0

0 0

10

20

30

40

-1

1

Insulin (micro unit per ml)

3

5

7

HOMA-IR

H

20

4 15

HOMA-IR

Insulin (micro U/ ml)

200

80 60 40 20 0

Pentraxin-3 tertile

G

150

D 100 40

VAT (cm2)

Body fat (%)

C

E

100

Visceral fat area

BMI

*

10 5

3

*

2 1 0

0

Pentraxin-3 tertile

Pentraxin-3 tertile

1stTertile

2ndTertile

3rdTertile

Fig. 1. Association between circulating levels of pentraxin 3 with adiposity, insulin and HOMA-IR. Data represent mean ± S.E., HOMA-IR: Homeostasis of insulin resistance, VFA: visceral fat area *Statistically different compared with the variable of the first tertile of PTX3 A, Relationship between baseline fasting PTX3 and baseline BMI, Correlation coefficient (r) and p values are included in the respective panels. B, Relationship between baseline fasting PTX3 and baseline visceral fat area, Correlation coefficient (r) and p values are included in the respective panels. C, Baseline percent body fat of participants according to the tertiles of PTX3 measured at baseline. D, Baseline visceral fat of participants according to the tertiles of PTX3 measured at baseline. E, Relationship between baseline fasting PTX3 and baseline fasting insulin levels, Correlation coefficient (r) and p values are included in the respective panels. F, Relationship between baseline fasting PTX3 and baseline HOMA-IR levels, Correlation coefficient (r) and p values are included in the respective panels. G, Baseline fasting insulin levels according to the tertiles of PTX3 measured at baseline. H, Baseline HOMA-IR of participants according to the tertiles of PTX3 measured at baseline.

S.H. Chu et al. / Clinica Chimica Acta 413 (2012) 1430–1437

2.6. Blood collection and biochemical analyses Blood samples were collected between 7:30 a.m. to 8:30 a.m. after 12 hours of overnight fasting. Venous blood was collected, and after centrifugation, the serum was frozen immediately at − 80 °C. Serum levels of fasting glucose, total cholesterol (TC), HDL-cholesterol (HDL-C) and triglyceride (TG) were assayed using an ADVIA 1650 Chemistry system (Siemens, Tarrytown, NY). Fasting insulin was assayed via an electrochemiluminescence immunoassay using Elecsys 2010 (Roche, Indianapolis, IN). Insulin resistance was estimated according to the HOMA-IR, (insulin (μIU/ml) × fasting blood glucose (mg/dl)/ 22.5). hs-CRP was measured using an ADVIA 1650 Chemistry system (Siemens). Plasma PTX3 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA, R&D system, Minneapolis, MN) and inter- and intra-assay CV were 5.1 ± 1.0 and 4.0 ± 0.4%. 2.7. Statistical analysis SPSS 17.0 (SPSS Inc., Chicago, IL) was used for statistical analyses. A t-test was used to compare baseline characteristics and PTX3 levels between boys and girls. For cross-sectional part of the study, Pearson's correlation coefficients and partial correlations were used to evaluate the relationships between plasma PTX3 level and other variables. Multiple regression analysis was performed with PTX3 as a dependent variable and variables that significantly correlated with PTX3 as independent variables. In the case of variables that were not normally distributed, a log transformation was performed. To further investigate the relationship between PTX3 and anthropometric measurements, glucose metabolism, participants were divided into three groups based on plasma PTX3 level (tertiles). One-way ANOVA was used to compare the three groups. A paired sample t-test was used to determine the difference before and after the intervention. To determine which variables are related to changes in PTX3 values, delta values were calculated (post-intervention value − pre-intervention value) and then correlation coefficient analyses were analyzed. The statistical significance was set at p b 0.05. 3. Results 3.1. PTX3 was not associated with adiposity but was significantly associated with whole body muscle mass and mid-thigh muscle mass (Fig. 1, Table 2) There was no correlation between circulating PTX3 levels and any of the adiposity measurements including WC, percent body fat, VAT and SAT. However, there was a significant association between circulating PTX3 levels and whole body muscle mass and mid-thigh muscle mass. The association between circulating PTX3 and measurements of muscle mass remained statistically significant after controlling for gender, BMI and Tanner stage. 3.2. PTX3 was associated with plasma insulin levels, HOMA-IR and TG levels Correlation coefficient analyses demonstrated that PTX3 levels were negatively associated with fasting insulin levels, HOMA-IR, but not with fasting glucose levels. These negative associations remained significant even after controlling for BMI and Tanner stages. To further analyze to relationship between PTX3 and insulin resistance, subjects were stratified into three groups (tertiles) according to their baseline PTX3 levels, and their insulin and HOMA-IR levels were compared. Compared with subjects in the lowest PTX3 tertile, subjects in the highest PTX3 tertile have significantly lower insulin and HOMA-IR. When multiple stepwise regression analyses were performed with PTX3 as the dependent variable and age, TG, HOMA-IR, and mid-thigh muscle mass as independent variables, HOMA-IR and

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Table 2 Relationships between pentraxin-3 with anthropometric and metabolic parameters at baseline. Pentraxin-3

Anthropometric BMI WC Body fat VAT SAT Muscle mass MT muscle mass Glucose metabolism Glucose Insulin HOMA-IR Lipid profile TC HDL-C TG Other hs-CRP

Model 1

Model 2

Model 3

− 0.056 − 0.08 − 0.235 0.059 − 0.106 .282* .352*

.360* .375*

.342* .370*

− 0.054 − .319⁎ − .323⁎

− 0.094 − .335* − .332*

− 0.101 − .339* − .334*

0.129 0.187 − .302⁎

0.175 0.172 − .288*

0.138 0.167 − .320*

0.093

0.102

0.123

Coefficients are calculated using the Pearson correlation model. *p b 0.05, ^p = 0.053 Model 1. Correlation between pentraxin-3 and other parameters, Model 2. Partial correlation between pentraxin-3 and other parameters after adjusting for gender and BMI, Model 3. Partial correlation between pentraxin-3 and other parameters after adjusting for BMI and Tanner stage. WC: waist circumference, VAT: visceral adipose tissue, SAT: subcutaneous adipose tissue, MT muscle mass: mid thigh muscle mass, HOMA-IR: homeostasis model assessment-insulin resistance, QUICKI: quantitative insulin sensitivity check index.

mid-thigh muscle mass remained significant predictors for PTX3 levels (Fig. 1, Table 2, Table 3). 3.3. Seven days of intense physical activity significantly reduced HOMA-IR, TC, TG, hs-CRP and abdominal adiposity All participants engaged in 7 days of intense short-term physical activity and healthy balanced diet to investigate the effects of short-term intense short-term physical activity induced body weight loss on body adiposity, HOMA-IR, lipid profiles and PTX3. The intervention program significantly reduced body adiposity including VAT, and SAT. The program also significantly reduced fasting insulin, HOMA-IR, TC, TG, and hs-CRP. It was noteworthy that about 4.1% of weight reduction was accompanied by 4.2% reduction in SAT and 13.2% reduction of VAT after the intervention. More interestingly, HOMA-IR, TG, and hs-CRP levels were reduced by 74.5%, 72.7%, and 40.6%, respectively (Table 4). 3.4. PTX3 levels increased after 7 days of intense physical activity in a subgroup of subjects with low baseline PTX3 levels As mentioned earlier, PTX3 levels were negatively correlated with insulin, and HOMA-IR. When subjects were stratified into three groups, it became clear that subjects who were in the lowest tertile of PTX3

Table 3 Multiple linear regression adjusted for confounding variables with pentraxin 3 as a dependent variable at baseline. Predictors

Standardized effect estimate

t

p Value

Age Tanner stage Triglyceride HOMA-IR MT muscle mass

0.049 − 0.105 − 0.215 − 0.261 0.362

0.343 0.829 − 1.721 − 2.063 2.699

NS NS NS 0.047 0.009

MT muscle mass: mid-thigh muscle mass, Multiple linear regression was performed with age, Tanner stage, triglyceride, HOMA, MT muscle mass as independent variables and PTX3 as a dependent variable. All independent variables used in the model correlated with PTX3.

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Table 4 The effects of 7 days of intense physical activity on anthropometric and metabolic parameters.

Anthropometric Weight (kg) BMI (kg/m2) WC (cm) $ Fat mass (kg) VAT (cm2) SAT (cm2) Muscle mass (kg) MT muscle mass (cm)$ Blood pressure SBP (mm Hg) DBP (mm Hg) Glucose metabolism Glucose (mg/dl) Insulin (μU/ml) HOMA-IR Lipid metabolism TC (mg/dl) HDL-C (mg/dl) TG (mg/dl) PTX3 (ng/dl) hs-CRP (mg/dl)

Before

After

Change

% change

61.01 ± 9.81 26.50 ± 3.20 84.31 ± 9.24 16.71 ± 5.05 73.83 ± 27.98 219.78 ± 72.58 39.54 ± 5.11 107.72 ± 15.39

58.48 ± 9.48 25.33 ± 3.09 81.07 ± 8.67 15.65 ± 4.90 64.09 ± 31.03 210.56 ± 65.78 38.83 ± 5.07 105.60 ± 14.79

− 2.53 ± 0.85* − 1.17 ± 0.38* − 3.24 ± 2.31* − 1.06 ± 0.09* − 9.73 ± 1.9* + 9.22 ±2.77 − .71 ± 0.63* − 1.72 ± 4.3*

− 4.1 − 4.5 − 3.8 − 6.3 − 13.2 − 4.2 − 1.3 −2

111.23 ± 12.0 73.42 ± 9.87

110.4 ± 11.38 67.79 ± 7.55

− .83 ± 12.64 − 5.63 ± 10.23*

− 6.8

76.54 ± 6.86 13.67 ± 6.73 2.59 ± 1.31

75.75 ± 5.33 3.49 ± 1.98 .65 ± .38

−.79 ± .7.57 − 10.18 ± .5.80* − 1.93 ± 1.14*

− 74.4 − 74.5

172.19 ± 31.22 49.16 ± 8.10 126.30 ± 51.09 1.02 ± .68 .16 ± .17

142.44 ± 25.79 50.16 ± 7.79 34.58 ± 11.83 1.11 ± .69 .09 ± .09

− 29.75 ± 38.83* + 1.0 ± 6.82 − 91.72 ± 47.83* + 0.085 ± 0.99 −.066 ± .16*

− 17.3 − 72.7 − 40.6

Data are mean ± SD, *p b 0.05, $: Pre-intervention values do not agree with baseline value in Table 1 due to missing post-intervention value. BMI: body mass index, WC: waist circumference, VAT: Visceral adipose tissue, SAT, Subcutaneous adipose tissue MT muscle mass: Mid thigh muscle mass, HOMA-IR: homeostasis model assessment-insulin resistance, QUICKI: quantitative insulin sensitivity check index.

levels had 32.7% higher HOMA-IR compared to patients in the highest tertile of PTX3 levels (Fig. 2). Since the negative association between PTX3 and HOMA-IR remained even after controlling for gender and BMI, this association is probably independent of adiposity and suggests that lower PTX3 levels may predict higher insulin resistance in study participants. Therefore, HOMA-IR and insulin levels were analyzed according to PTX3 sub-groups (low, mid and high levels of PTX3). Interestingly, significant increases in PTX3 were only observed in subjects who had low baseline levels of PTX3. 3.5. Delta PTX3 levels were negatively associated with delta HOMA-IR and delta insulin levels To determine the relationship between intervention-induced changes in PTX3 with anthropometric and metabolic parameters, coefficient correlation analyses were employed using the delta values of each variable (post-intervention value−pre-intervention value). These analyses revealed a significant negative association between delta PTX3 with delta insulin (r=−.280, p=0.035) and HOMA-IR (r=−.281, p=0.034); and a positive association between delta PTX3 and delta TC. In order to further investigate the effects of post-intervention changes in PTX3 levels on insulin and HOMA-IR levels, subjects were divided into 2 groups: a group with increased post-intervention PTX3 levels and a group with decreased post-intervention PTX3 levels. Analysis showed that subjects with increased PTX3 levels had significantly greater improvements in post-intervention HOMA-IR (subjects with increased PTX3: −2.33 ± 1.3 vs. subjects with decreased PTX3: −1.46 ± .70, p = 0.004), and insulin levels (subjects with increased PTX3: −11.95± 6.68 vs. subjects with decreased PTX3: −8.08± 3.67 μU/ml, p = 0.035) (Fig. 2). 4. Discussion This reports the association between PTX3 and insulin resistance and the effects of short-term physical activity on circulating PTX3 levels and its association with insulin resistance in children. In the crosssectional analyses, HOMA-IR was associated with PTX3 even after adjusting for age, gender, BMI and Tanner stage. Furthermore, the degree of changes in PTX3 levels were negatively associated with fasting and insulin and HOMA-IR, which provide evidence of the link between

PTX3 and insulin resistance at baseline and after the physical activity intervention. When sub-analyses were performed in subjects with increased post-intervention PTX3 levels, this sub-group had significantly greater improvement in HOMA-IR compared to subjects whose post-intervention PTX3 levels were decreased. These results may suggest that PTX3 may play an important physiological role in regulation of insulin resistance in obese children. However, the link between physical activity-associated changes in PTX3 levels and physical activity-associated reduction in HOMA-IR should be done with caution since circulating PTX3 levels did not change while HOMA-IR significantly changed after intervention. Although several studies have reported an association of PTX3 with atherosclerosis [13,28–31] and metabolic syndrome [32], the relationship between PTX3 and insulin resistance is not yet fully understood. Previous studies reported no association of PTX3 with glucose [33] and insulin resistance [14,31]. However, the current study found a negative association of PTX3 with insulin and HOMA-IR, even after controlling for gender and BMI. It is unclear why the results of this study are not consistent with those of previous studies. Previous studies have reported significantly higher PTX3 levels with increased age. The average age of the participants in other studies is approximately 60 y [31,33] while the average ages of participants in this study is 12 y. This difference in age may explain why the present study found a relationship between PTX3 and the measures of insulin resistance, while previous studies did not find a relationship between PTX3 and insulin resistance. Another explanation for the inconsistent study findings may be the presence of atherosclerosis with an aged population. Atherosclerosis is known to be associated with PTX3 levels. Although subjects in this study are overweight and obese, increase in intima media thickness were not observed in our participants, indicating that they have not developed atherosclerosis. Therefore, the source of PTX3 from endothelial cells due to atherosclerosis might have not been a factor among the study subjects. Lack of atherosclerosis among the participants in the study due to their relatively young age might explain the differences between this study and previously published studies. Pubertal stage influences insulin resistance, so the relationship between pubertal stage and PTX3 levels was also analyzed but no significant relationship between PTX3 levels and pubertal stage was observed. The fact that increased PTX3 levels were negatively associated

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A

2.50

5.00 4.00 3.00

2.00 1.50 1.00

2.00

0.50

2.00 1.00

0.00

0.00

2.00

2.00

1.50

1.50

1.00

1.00

1.00

0.50

0.50

0.50

0.00

0.00

0.00

*

1.50

Pre

B delta HOMA-IR

-6

-4

-2

R=-.281, p=.034

Post

Pre

1 0 -1 0 -2 -3 -4 -5 -6 -7

C 2

delta pentraxin 3

delta HOMA-IR

D 0

Post

-6

4

delta Insulin

Pentraxin-3 (ng/dl)

Highest Third (N=19)

Middle Third (N=19)

Lowest Third (N=19) 3.00 2.50 2.00 1.50 1.00 0.50 0.00

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Post

Pre

-4

-2

R=-.280, p=.035

0 -5 0 -10 -15 -20 -25 -30 -35

2

4

delta pentraxin 3

PTX3 increased PTX decreased

-1 -2 -3

*

Fig. 2. Effects of short-term intense short-term physical activity on circulating PTX3 levels. Data represents mean ± S.E., PTX3: pentraxin-3, *Statistically significant difference compared with the baseline value. A, Baseline and post intervention fasting value of PTX3 levels according to the tertile of baseline PTX3 levels B. Coefficient correlation between delta PTX3 and delta HOMA-IR, C. Coefficient correlation between delta PTX3 and delta Insulin, D. Comparison of delta HOMA-IR between subjects whose PTX3 was increased vs decreased.

with insulin resistance suggests that PTX3 may play a role in the prevention of insulin resistance; however, this hypothesis needs to be examined further. In addition, it is still unclear if exercise (which is known to decrease insulin resistance) regulates PTX3 levels and if there is a relationship between exercise-induced PTX3 levels and insulin resistance. In this tightly controlled intervention study, significantly reduced insulin resistance and improved lipid profiles were observed after short-term physical activity, but there were no changes in PTX3 levels. However, it PTX3 levels increased significantly in response to the intervention among subjects in the lowest tertile of PTX3 who had the highest level of insulin resistance. Further correlation analysis also showed a negative association of delta PTX3 with delta insulin and delta HOMA-IR, which suggest that a post-intervention increase in PTX3 levels may mediate the reduction in insulin resistance. To further analyze the effects of changes in PTX3 in response to the intervention on the changes in insulin resistance, subjects were divided into two groups (PTX3 increment and PTX3 decrement) and their insulin and HOMA-IR changes were compared. This analysis showed that subjects whose PTX3 levels increased after training had almost 60% better insulin resistance compared to subjects whose PTX3 levels

decreased after the training. Insulin resistance significantly improved in both groups after the intervention, so an increase in PTX3 was not the sole reason for improvement in insulin sensitivity. This study suggests that PTX3 is closely related to intervention-associated improvements in insulin resistance. There have been only 2 studies which investigated the effects of exercise on circulating PTX3 levels [20,21]. One study reported decreased PTX3 levels during six months of cardiac rehabilitation exercise [20]. Another study reported increased PTX3 levels after an acute bout of maximal aerobic and resistance exercise, which may have been the result of exercise induced muscle damage [21]. PTX3 expression in cardiac, smooth and skeletal muscle and circulating levels of PTX3 acutely increase in response to various stimuli such as lipopolysaccharides [12]. Therefore, increased PTX3 release from the potentially damaged muscle due to acute maximal exercise would not be surprising. In addition, cardiac rehabilitation exercise is known to improve atherosclerosis likely also decreases circulating PTX3 levels. Because of this, it is difficult to investigate the effects of exerciseassociated changes in PTX3 levels on insulin resistance with acute bouts of maximal intensity exercise in subjects who have already had myocardial infarction. Since subjects in the current study did

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not likely to have atherosclerosis, and also were not involved with maximal levels of exercise which may influence PTX3 levels, the significant association in the current study between interventioninduced changes in PTX3 levels and changes in insulin resistance is important evidence that may improve the understanding of the role of PTX3 in insulin resistance. The findings of the current study are also supported by a recent study conducted by Miyaki et al. [34] which reported that endurance-trained individuals had higher levels of circulating PTX3 than sedentary controls and suggested that PTX3 may play a partial role in endurance exercise training-induced cardioprotection. Based on previous studies, several mechanisms may be involved in the negative association of PTX3 with insulin resistance and the potential contribution of PTX3 to short-term physical activity induced improvement in insulin resistance. First, it has been proposed that an accumulation of macrophages that secrete pro-inflammatory cytokines as well as altered functioning in insulin target cells might be the cause of insulin resistance [35]. PTX3 inhibits the classical complement pathway by enhancing binding between apoptotic cells and C1q, the recognition subunit of the classical complement pathway [36]. Thus, increased plasma PTX3 levels in obese children might decelerate chronic inflammation, which is the cause of insulin resistance, by inhibiting the complement pathway. Another potential mechanism linking PTX3 levels and insulin resistance could be the phosphatidylinositol 3-kinase (PI3K) and downstream signaling pathways, including protein kinase B [37]. Recent studies have found PI3K/Akt activation-dependent expression of PTX3 in endothelial cells [38], suggesting a possible link between PTX3 and insulin signaling. Further investigation is required to elucidate this important issue. The degree of changes in insulin, HOMA-IR and TG after the intervention in this study deserve some attention. Both HOMA-IR and TG levels were reduced by more than 70% after only 7 days of short-term physical activity. The reduction in fat mass due to significantly increased physical activity (11751 kcal expended over 7 days) and controlled caloric intake (1882 ± 68 kcal per day), may contribute to reductions in insulin, HOMA-IR and TG. Indeed, visceral fat was reduced by 13.2% while body weight and subcutaneous fat were reduced by 4.1% and 4.2%, respectively. However, it is unclear whether this reduction in fat mass was solely responsible for greater than 70 percent reduction in HOMA-IR. Previous studies have tried to identify the relationship between PTX3 and adiposity; however, it is still not clear whether PTX3 increases with obesity since conflicting results on the association between adiposity and PTX3 concentration have been reported [14,32,33,39,40]. In this study, there was no association between PTX3 levels and any of the adiposity measurements including percent body fat, WC, VAT and SAT. On the other hand, PTX3 was associated with whole body muscle mass and mid-thigh muscle mass, after controlling for gender and BMI. Furthermore, multiple linear regression analysis showed that mid-thigh muscles mass is a significant predictor for PTX3 levels. The association between PTX3 and skeletal muscle mass has not been previously reported. Considering that skeletal muscle is a tissue that strongly expresses PTX3 [12], the significant association between skeletal muscle and PTX3 in this study is not surprising. Limitations of the current study include the use of a surrogate marker of insulin resistance. Fasting insulin and HOMA-IR may not accurately reflect insulin resistance in children [24,27], but were used in this study because they were only options since insulin sensitivity of all 57 participants had to be measured during a two-hour window while the participants were fasting [26]. Another limitation is the relatively small sample size for cross-sectional analysis. Although the number of participants was small, the participants were very homogenous in terms of population origin, the level of adiposity and age. While the correlation noted between insulin resistance measures and PTX3 levels may still be meaningful, the study data should be interpreted cautiously.

In conclusion, a significant association between PTX3 and insulin resistance was observed at baseline and also after the physical activity intervention, which may suggest the possible role of PTX3 in regulating insulin resistance. This was the first study to report a negative association between PTX3 and insulin resistance as well as to provide the information on the impact of short term physical activity on PTX3 levels. Disclosure statements No author has anything to declare. Acknowledgments We appreciate Dr. Young Hee Lee and Mr. Sung Soo Jeon for their technical expertise and advice on measuring abdominal adiposity. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009‐0070217) and Sports ToTo. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.cca.2012.06.002. References [1] Ogden CL, Carroll MD, Curtin LR, et al. Prevalence of overweight and obesity in the United States, 1999–2004. JAMA 2006;295:1549–55. [2] Sinha R, Fisch G, Teague B, et al. Prevalence of impaired glucose tolerance among children and adolescents with marked obesity. N Engl J Med 2002;346:802–10. [3] Cai D, Yuan M, Frantz DF, et al. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005;11:183–90. [4] Alikasifoglu A, Gonc EN, Ozon ZA, et al. The relationship between serum adiponectin, tumor necrosis factor-alpha, leptin levels and insulin sensitivity in childhood and adolescent obesity: adiponectin is a marker of metabolic syndrome. J Clin Res Pediatr Endocrinol 2009;1:233–9. [5] Goldfine AB, Fonseca V, Shoelson SE. Therapeutic approaches to target inflammation in type 2 diabetes. Clin Chem 2011;57:162–7. [6] Kim ES, Im JA, Kim KC, et al. Improved insulin sensitivity and adiponectin level after exercise training in obese Korean youth. Obesity 2007;15:3023–30. [7] Garlanda C, Bottazzi B, Bastone A, et al. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol 2005;23:337–66. [8] Cotena A, Maina V, Sironi M, et al. Complement dependent amplification of the innate response to a cognate microbial ligand by the long pentraxin PTX3. J Immunol 2007;179:6311–7. [9] Castell JV, Gomez-Lechon MJ, David M, et al. Acute-phase response of human hepatocytes: regulation of acute-phase protein synthesis by interleukin-6. Hepatology 1990;12:1179–86. [10] Abderrahim-Ferkoune A, Bezy O, Chiellini C, et al. Characterization of the long pentraxin PTX3 as a TNFalpha-induced secreted protein of adipose cells. J Lipid Res 2003;44:994–1000. [11] Klouche M, Peri G, Knabbe C, et al. Modified atherogenic lipoproteins induce expression of pentraxin-3 by human vascular smooth muscle cells. Atherosclerosis 2004;175:221–8. [12] Introna M, Alles VV, Castellano M, et al. Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood 1996;87: 1862–72. [13] Norata GD, Marchesi P, Pulakazhi Venu VK, et al. Deficiency of the long pentraxin PTX3 promotes vascular inflammation and atherosclerosis. Circulation 2009;120: 699–708. [14] Zanetti M, Bosutti A, Ferreira C, et al. Circulating pentraxin 3 levels are higher in metabolic syndrome with subclinical atherosclerosis: evidence for association with atherogenic lipid profile. Clin Exp Med 2009;9:243–8. [15] Suzuki S, Takeishi Y, Niizeki T, et al. Pentraxin 3, a new marker for vascular inflammation, predicts adverse clinical outcomes in patients with heart failure. Am Heart J 2008;155:75–81. [16] Kasai T, Inoue K, Kumagai T, et al. Plasma pentraxin3 and arterial stiffness in men with obstructive sleep apnea. Am J Hypertens 2011;24:401–7. [17] Hollan I, Bottazzi B, Cuccovillo I, et al. Increased levels of serum pentraxin 3, a novel cardiovascular biomarker, in patients with inflammatory rheumatic disease. Arthritis Care Res (Hoboken) 2010;62:378–85. [18] Balagopal P, George D, Patton N, et al. Lifestyle-only intervention attenuates the inflammatory state associated with obesity: a randomized controlled study in adolescents. J Pediatr 2005;146:342–8.

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