Pediatric Exercise Science, 2015, 27, 525 -534 http://dx.doi.org/10.1123/pes.2015-0050 © 2015 Human Kinetics, Inc.
ORIGINAL RESEARCH
Cytokine Responses to Acute Intermittent Aerobic Exercise in Children With Prader-Willi Syndrome and Nonsyndromic Obesity Andrea T. Duran
Erik Gertz
California State University Fullerton
United States Department of Agriculture
Daniel A. Judelson
Andrea M. Haqq
California State University Fullerton
University of Alberta
Susan J. Clark
Kavin W. Tsang and Daniela Rubin
Children’s Hospital of Orange County
California State University Fullerton
Prader-Willi Syndrome (PWS), the best characterized form of syndromic obesity, presents with abnormally high fat mass. In children, obesity presents with low-grade systemic inflammation. This study evaluated if PWS and/or nonsyndromic obesity affected cytokine responses to intermittent aerobic exercise in children. Eleven children with PWS (11 ± 2 y, 45.4 ± 9.5% body fat), 12 children with obesity (OB) (9 ± 1 y, 39.9 ± 6.8% body fat), and 12 lean (LN) children (9 ± 1 y, 17.5 ± 4.6% body fat) participated. Children completed 10 2-min cycling bouts of vigorous intensity, separated by 1-min rest. Blood samples were collected preexercise (PRE), immediately postexercise (IP), and 15, 30, and 60 min into recovery to analyze possible changes in cytokines. In all groups, IL-6 and IL-8 concentrations were greater during recovery compared with PRE. PWS and OB exhibited higher IL-6 area under the curve (AUC) than LN (p < .01 for both). PWS demonstrated higher IL-8 AUC than LN (p < .04). IL-10, TNF-α, and IFN-γ did not change with exercise (p > .05 for all). Results indicate that children with PWS respond with increased Il-6 and IL-8 concentrations to acute exercise similarly to controls. Excess adiposity and epigenetic modifications may explain the greater integrated IL-6 and IL-8 responses in PWS compared with controls. In children and adults, obesity is associated with chronic low-grade systemic inflammation (12,44). At rest, proinflammatory cytokines can be released into circulation from adipocytes embedded in the connective tissue matrix and macrophages located within the subcutaneous and visceral adipose tissue (4,24,25). Understanding these cytokines is important because in children with obesity, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6), and interferon-gamma (IFN-γ) have been linked to
Duran, Tsang, and Rubin are with the Dept. of Kinesiology, California State University Fullerton, Fullerton, CA. Gertz is with the Obesity and Metabolism Research Unit, United States Department of Agriculture, Davis, CA. Judelson is with the Dept. of Kinesiology, California State University Fullerton, Fullerton, CA. Haqq is with the Dept. of Pediatrics, University of Alberta, Edmonton, Alberta, Canada. Clark is with the Dept. of Endocrinology, Children’s Hospital of Orange County, Orange, CA. Address author correspondence to Daniela Rubin at [email protected].
cardiovascular disease, hypertension, insulin resistance and atherosclerosis (12,19,33,36). Prader-Willi Syndrome (PWS) is the best-characterized form and most common genetic cause of childhood morbid obesity (41). Children with PWS present hyperphagia, hypotonia, high fat mass, low lean mass and endocrine abnormalities such as growth hormone deficiency and hypogonadism (5,8). Haqq and colleagues (2011) reported that children with PWS exhibited lower concentrations of IL-6 at rest when compared with obese controls. However, Cadoudal and colleagues (2014) recently demonstrated similar levels of circulating IL-6 and greater interleukin-8 (IL-8) and TNF-α at rest in children with PWS compared with those with nonsyndromic obesity. The paucity of information suggests that the cytokine profile in children with PWS needs further investigation. Exercise is vital to the management of PWS and prevention of pediatric obesity (39,40). Cytokines released in response to exercise, such as IL-6, IL-8 and interleukin-10 (IL-10), help regulate metabolism, muscle growth, and general pro- and anti-inflammatory processes
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(7,11,14,38). Previous research in healthy children demonstrated that aerobic exercise, at least 30 min in duration and of moderate to vigorous intensity, elicited elevated systemic IL-6 and IL-8 concentrations during exercise and recovery compared with concentrations at rest (31,38). In contrast, IL-10 and TNF-α concentrations, in healthy children, did not change in response to aerobic exercise (31,35). The mentioned cytokine responses to exercise vary in children with nonysndromic obesity (23,30,32) and have yet to be explored in children with PWS. In adults, obese individuals exhibit increased proinflammatory markers compared with lean individuals in response to aerobic exercise (9). In children with obesity, studies have demonstrated an increase in some cytokines in response to aerobic exercise compared with children with normal weight (23,30,32). Given the beneficial role of exercise on the management of obesity, the involvement exercise induced cytokines have in metabolic and inflammatory processes, and the possibility that the cytokine profile at rest may differ between children with PWS and nonsyndromic obesity (6,17), it is relevant to determine if the cytokine responses to exercise are altered in children with PWS. Therefore, this study characterized the cytokine responses to acute intermittent aerobic exercise in children with PWS and compared it to obese and lean controls.
Methods Participants Eleven children with PWS (age range 8–13 years) participated. Participants with PWS provided documentation of molecular and cytogenetic testing (i.e., chromosomes, FISH 15, DNA methylation and/or DNA polymorphism studies) to verify diagnoses. Twelve healthy lean children (LN) and 12 obese children (OB) (age range 8–11 years) were recruited through fliers, emails, and word of mouth. Nonsyndromic children were categorized as obese if they had a body fat percentage higher than the 95th percentile and as lean if their body fat percentage was lower than the 85th percentile for age and sex (22). Written assent and consent were obtained by all participants and their parents before participation. The assent and consent forms were approved by the California State University, Fullerton and the Human Research Protection Office at the United States Army Medical Research and Materiel Command Institutional Review Boards. Participant’s parents completed a medical and exercise history form to screen participants for potential health risks and contraindications to exercise. Exclusion criteria for participants included confirmed pregnancy or inability to perform vigorous exercise. Tanner stage established by breast, genital and pubic hair development for children with PWS was determined by a study physician. Tanner stage for children without PWS was determined using the Pubertal Development Scale filled out by the child and his or her parents (28).
Study Protocol All participants completed 2 visits. Each visit was separated by a minimum of 2 days and a maximum of two weeks. Visit one involved the following measurements: anthropometrics, body composition, resting heart rate, and resting blood pressure. Participants also completed an incremental, graded exercise test (McMaster protocol) to determine each child’s relative peak power output. At the second visit, participants consumed a standardized breakfast (260 kcal, 7 g of fat [21.5%], 37 g of carbohydrate [57%], and 14 g of protein [21.5%]) two hours before arrival to the laboratory or the hospital. The breakfast included one breakfast bar (strawberry or chocolate) and an apple sauce, which contained no caffeine. Participants consumed unlimited amounts of water, but were not required to record the consumed volume. Participants refrained from any strenuous physical activity 24 hr before exercise testing. Upon arrival, a trained phlebotomist inserted an indwelling catheter into an antecubital vein or the dorsal vein of the hand of the participant. After 30 min of rest, a resting blood sample (PRE) was obtained and participants completed an intermittent aerobic exercise protocol based on a resistance setting from the McMaster protocol. Blood samples were obtained immediate postexercise (IP), +15, +30, and +60 min during recovery from the exercise challenge. Blood samples were analyzed for IL-6, IL-8, IL-10, TNF-α, and IFN-γ.
Anthropometric and Physiologic Measurements Body mass was obtained using a digital scale (ES2001; Ohaus, Pinewood, N.J.) with the participant only wearing a t-shirt, shorts and no shoes. Stature was measured at the end of inhalation (10) against a wall-mounted stadiometer (Seca, Ontario, Calif., USA). BMI was computed by dividing body mass in kilograms by the stature in meters, squared. After 5 min of seated rest, resting heart rate was measured via telemetry (Polar USA, Lake, Success, NY), and resting blood pressure was measured with an aneroid sphygmomanometer using appropriate diameter cuffs (Diagnostix 752, American Diagnostic Corporation, Hauppage, NY).
Body Composition Body composition was determined with whole body dual x-ray absorptiometry (DXA) scan (Lunar Prodigy Advance; GE Healthcare, Madison, Wisc., USA) to obtain total body, android, gynoid and trunk fat percentage and total lean mass. Participants were positioned following manufacturer’s indications (GE Healthcare, GE Lunar Corp., Madison WI). All scans were done and analyzed using the Pediatric Encore software version 12.30.008, which automatically demarcates the regional boundaries for all regions.
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McMaster Protocol This protocol is a multistage, incremental protocol, requiring subjects to cycle in 2-min stages at progressively increasing workloads (3). Work rate at each stage was based on participant sex and height. Exercise intensity increased every 2 min until the subject reached volitional exhaustion, failed to sustain the desired workload, requested to stop, stood up on the bike pedals, or experienced fatigue-related symptoms. Participants were required to cycle at a minimum pedaling rate of 50 rpm (3), monitored by an EM900 pocket metronome (Franz Manufacturing Company, Incorporated, East Haven, CT). Workload, heart rate, and rating of perceived exertion using the Pediatric OMNI scale (29) were recorded for each stage and relative peak power output (Watts per kg lean body mass) was computed by dividing the test termination load by the participant’s lean body mass obtained from the DXA scan.
Experimental Exercise Protocol The intermittent aerobic exercise protocol consisted of 10 cycling bouts (2 min each), separated by 1-min rest intervals. The workload for this discontinuous protocol was set based on the first resistance from the McMaster protocol that successfully elicited a heart rate ≥160 bpm. Previous studies have shown acute hormonal and inflammatory responses at a target heart rate ≥160 bpm in children (13,16). As 8 children with PWS were not able to attain peak heart rates >160 bpm, they exercised at the second to last workload completed in the McMaster protocol. Heart rate was measured continuously and ratings of perceived exertion were measured at the end of every 2-min interval.
Blood Processing and Analysis Blood samples obtained at PRE, IP, +15, +30, and +60 min during recovery from the experimental exercise protocol were collected into ethylene-diamine-tetra-acetic acid (EDTA) containing tubes (BD Diagnostics, Franklin Lakes, NJ) and centrifuged at 4 °C and 3,000 rpm for 15 min. Plasma was then placed in Eppendorf tubes for storage in -70 °C freezer until analyses. Proinflammatory Panel 1 (human) V-PLEX Kit (Catolog # K15049D) from Meso Scale Discovery (MSD) was used for cytokine analysis. The mean intra-assay CV of IL-6, IL-8, IL-10, TNF-α and IFN-γ were 24.9%, 6.9%, 29.9%, 11.1% and 26.6%; respectively.
Statistical Analyses All cytokines were nonnormally distributed. Log transformations were used to obtain normal distributions for all cytokines. TNF-α data for two participants were omitted from analyses because concentrations were below the detection limit of the assay. Three (group) by five (time) repeated measures mixed model ANOVAs were used to
identify differences in cytokine concentrations between children with PWS, obese and lean controls over time. In the case of significant interaction, group or time effect, Tukey’s HSD assessed pairwise differences. In addition, total areas under the curve (AUC; cytokine concentration x time) were calculated for each cytokine using the standardized trapezoidal methods (21) to determine group differences. Group comparisons for AUC were performed using the nonparametric Kruskal-Wallis test. In case of significant findings, two group comparisons for distribution and medians of AUC were done using Mann-Whitney U test and Median test, respectively. Significance level for all analyses was set at p < .05. Statistical analyses were performed using SPSS, Version 20.0 for Windows (SPSS, Inc., Chicago, IL).
Results Participant Characteristics Uniparental deletion (n = 9) and methylation (n = 2) diagnoses were confirmed for participants with PWS. PWS participants were classified as Tanner Stage I (Breast/ genital (BG): n = 5, pubic hair (PH): n = 2), Tanner Stage II (BG: n = 3, PH: n = 1), Tanner Stage III (BG: n = 1, PH: n = 2), Tanner Stage IV (BG: n = 2, PH: n = 5,) or Tanner Stage V (BG: n = 0, PH: n = 1). PWS participants also displayed type II diabetes mellitus (n = 1), asthma (n = 2), apnea (n = 3), pneumonia (n = 2), hypothyroidism (n = 3) and hyperthyroidism (n = 1). PWS participants currently received (n = 9), previously received (n = 1), or never received (n = 1) GH replacement therapy. The obese and lean controls were classified as Tanner Stage I (OB: n = 6, LN: n = 8), Tanner Stage II (OB: n = 4, LN: n = 3) or Tanner Stage III (OB: n = 2, LN: n = 1) based on the Pubertal Development Scale. Participant characteristics for each group are presented in Table 1. Children with PWS were older than obese and lean controls (p = .01, p = .03, respectively), while obese and lean had a similar age (p = .89). All children exhibited a similar height (p > .10 for all), with PWS presenting higher body mass than lean (p < .01). There were no significant differences in lean body mass among groups (p > .20 for all). Children with PWS exhibited similar android, gynoid, trunk and total body fat percentage as obese (p = .95, p = .70, p = .62 and p = .17; respectively). Lean controls had a significantly lower android, gynoid, trunk and total body fat percentage than children with PWS or obese (p < .01 for all).
Physiological Responses to Exercise Exercise responses to the intermittent aerobic protocol for each group are presented in Table 2. In response to the intermittent aerobic protocol, children with PWS had a significantly lower submaximal exercise heart rate, peak heart rate and relative submaximal workload compared
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Table 1 Participant Characteristics for Children with PWS, Obese, and Lean Controls Participant Characteristics Age (yrs)
11 ±
Males/Females
Obese (n = 12)
Lean (n = 12)
9±1
10 ± 1
8/4
8/4
2a,b
7/4
Height (cm)
148.9 ± 13.6
140.6 ± 8.1
140.1 ± 7.6
Body Mass (kg)
62.7 ± 30.8b
50.5 ± 13.3
32.6 ± 6.0
10.5b
4.4c
16.5 ± 1.4
39.9 ± 6.8c
17.5 ± 4.6
Body Mass Index
(kg/m2)
Body Fat Mass (%)
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PWS (n = 11)
27.2 ±
45.4 ± 9.5b
25.2 ±
Lean Body Mass (kg)
31.1 ± 11.6
28.5 ± 4.9
25.7 ± 4.1
Android Fat (%)
49.9 ± 12.1b
48.7 ± 8.1c
17.1 ± 6.8
Gynoid Fat (%)
52.3 ± 6.2b
47.1 ± 4.9c
29.5 ± 5.0
Trunk Fat (%)
45.0 ± 12.1b
41.60 ± 7.9c
15.3 ± 5.0
Resting heart rate (bpm)
80 ± 13
79 ± 12
79 ± 8
Resting SBP (mm Hg)
103 ± 15
105 ± 12
95 ± 15
Resting DBP (mm Hg)
72 ± 12
65 ± 9
62 ± 14
Note. Data reported as mean ± SD or frequency. ap
< .05, PWS different than OB.
bp
< .05, PWS different than LN.
cp
< .05, OB different than LN.
Table 2 Group Exercise Responses to the Intermittent Aerobic Exercise protocols Intermittent Aerobic Exercise Responses Absolute submaximal workload (W)
OB (n = 12)
LN (n = 12)
55 ± 26
70 ± 17
70 ± 13
2.5 ± 0.3
2.7 ± 0.4
66.5 ± 11.6
75.7 ± 13.4
129 ± 30a,b
163 ± 6
168 ± 10
Peak heart rate (bpm)a
151 ± 27a,b
193 ± 13
186 ± 13
Percentage of peak heart rate (%)
85.4 ± 15.9
84.9 ± 5.9
90.3 ± 6.7
123 ± 24
133 ± 10
123 ± 13
64 ± 11
55 ± 15
Relative submaximal workload (W·LBM
kg-1)
PWS (n = 11) 1.8 ±
0.7a,b
Percentage of peak relative workload (%)
75.3 ± 5.9
Submaximal exercise heart rate (bpm)
Ending systolic blood pressure (mm Hg) Ending diastolic blood pressure (mm Hg)
72 ±
6b
3c
7±2 9±2
Mean RPE (1–10)
5±1
4±
Peak RPE (1–10)
8±2
6±3c
Note. Data reported as mean ± SD. Peak heart rate was obtained during McMaster Protocol. ap
< .05, PWS different than OB.
bp
< .05, PWS different than LN.
cp
< .05, OB different than LN.
with obese and lean controls (OB: p < .01, p < .01, p = .01, respectively; LN: p < .01 for all). Obese and lean controls had a similar submaximal exercise heart rate, peak heart rate and relative submaximal workload (p > .99, p = .68, p = .32, respectively). Children with PWS did not show any difference in mean or peak RPE when compared with obese and lean controls (PWS vs. OB:
p = .57 and p = .22, respectively; PWS vs. LN: p = .24 and p = .75, respectively). Lean controls had significantly greater mean RPE and peak RPE values compared with obese (p = .01 and p = .02, respectively). There were no differences among groups in percentage of peak relative workload or percentage of peak heart rate (p = .38 and p = .08, respectively).
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Figure 1 — IL-6 and IL-8 concentrations at rest and in response to the intermittent aerobic exercise protocol in children with PWS, obese and lean controls. a = significant time effect: time point different than PRE (p < .05). b = significant time effect: time point different than IP (p < .05). # = overall group differences (p < .05).
Cytokine Responses to Exercise IL-6. IL-6 concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 1. There was no significant group by time interaction in circulating IL-6 concentrations in response to the exercise challenge (p = .73). Children with PWS showed no significant differences in overall mean IL-6 concentrations compared with the obese and lean controls (p = .68 and p = .60, respectively). Obese had
a greater overall mean IL-6 concentration than the lean controls (p = .02). In all groups, IL-6 concentrations were significantly greater at +15, +30, and +60 compared with PRE (p < .01 for all). Total IL-6 AUC distributions and medians (presented in Table 3) were significantly greater in PWS and obese groups compared with the lean controls (PWS: p = .03, p < .01; respectively, OB: p = .01, p < .01; respectively). Total IL-6 AUC distributions and medians were the same between the PWS and obese groups (p = .64, p = .70, respectively).
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IL-8. IL-8 concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 1. There was no significant group by time interaction or group effect in circulating IL-8 concentrations in response to the exercise challenge (p = .63 and p = .14; respectively). In all groups, IL-8 concentrations were significantly greater at IP compared with baseline, and at +15, and +30 (p < .01 for all) and significantly lower at +60 than IP (p = .02). Total IL-8 AUC distributions and medians were significantly greater in children with PWS compared with the lean controls (p = .01, p = .04, respectively). Total IL-8 AUC distributions and medians (see Table 3) were the same between the PWS and obese groups (p = .26, p = .68, respectively) and the obese and lean controls (p = .32, p = .68, respectively).
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IL-10, TNF-α, & IFN-γ. IL-10, TNF-α, and IFN-γ concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 2. There were no significant group by time interactions, group or time effects in circulating IL-10, TNF-α, or IFN- γ concentrations in response to the exercise challenge (IL-10: p = .98, p = .66 and p = .18, respectively; TNF-α : p = .29, p = .29 and p = .50, respectively; IFN- γ: p = .63, p = .31 and p = .19, respectively). Total IL-10 AUC, TNF-α AUC, and IFN- γ AUC distributions and medians (presented in Table 3) were also the same among all groups (IL-10: p = .33 and p = .75, respectively; TNF-α: p = .316 and p = .213, respectively; IFN-γ: p = .112 and p = .111, respectively).
Discussion In all children, 30 min of vigorous, intermittent, aerobic exercise elicited elevations of systemic IL-6 and IL-8 concentrations immediately after and during the recovery period, with no changes in IL-10, TNF-α, or IFN-γ. Those with PWS and nonsyndromic obesity demonstrated a greater integrated response measured by the AUC for IL-6. Those with PWS had a greater integrated response
measured by the AUC for IL-8 when compared with lean controls. No differences were observed for IL-10, TNF-α, or IFN- γ AUC among groups. All groups presented increases in IL-6 during recovery from exercise, which is consistent with results by previous studies (23,32). This increase in IL-6 was probably due to IL-6 released by skeletal muscle as groups presented comparable lean mass (27). Children with PWS and nonsyndromic obesity had a greater integrated IL-6 response to exercise compared with lean controls, possibly due to similar fat distribution (total body, android, gynoid, and trunk fat) and fat content in these groups. Likely, excess body fat led to higher circulating proinflammatory IL-6 concentrations at rest in these groups relative to the lean controls as shown before by others (34,44). We could not demonstrate significant IL-6 differences at baseline among groups; perhaps because of the low statistical power (observed power: 0.371). However, these group differences probably became larger when evaluating IL-6 concentrations over several time points. A greater tissue exposure to absolute systemic concentrations of IL-6, as presented in the obese children, is relevant because IL-6 has been linked with cardiometabolic complications at rest (33), as well as with improved hepatic glucose production, fatty acid oxidation and insulin sensitivity in response to exercise (7,14). As for PWS, our results support a normal IL-6 response over time, with increased concentrations in response to exercise. Perhaps a greater IL-6 response in PWS when considering the lower absolute workload that the majority of these children exercised at when compared with controls. All children presented increased exercise-induced systemic IL-8 concentrations as early as 15 min into recovery, with IL-8 concentrations returning to baseline within one hour. When children exercise longer than 30 min, concentrations of circulating IL-8 may take more recovery time to return to baseline (38). In children, the exercise-induced elevations of IL-8 during recovery may be from mobilized white blood cells or active skeletal muscle (2,15,23). Since all children presented similar lean mass,
Table 3 Total Area Under the Curve for Cytokine Concentrations in Response to the Intermittent Aerobic Exercise Protocol in Children with PWS, Obese, and Lean Controls PWS (n = 11) IL-6 (pg*min/mL) IL-8 (pg*min/mL)
67.2 (37.5, 315.8 (249.0,
IL-10 (pg*min/mL)
78.2)a 369.8)a
36.8 (24.5, 47.3)
OB (n = 12) 68.3 (43.7,
93.4)b
Lean (n = 12) 29.3 (27.1, 35.4)
284.6 (165.8, 320.1)
247.1 (192.2, 269.1)
40.5 (36.2, 57.0)
40.1 (24.2, 48.7)
TNF-α (pg*min/mL)
174.0 (130.5–265.5)
225.0 (156.8–273.8)
159.0 (144.8–222.0)
IFN-γ (pg*min/mL)
504.0 (357.8–808.5)
771.8 (411.0–1106.1)
378.0 (378.0–685.7)
Note: Data reported as median (interquartile range). ap
< .05, PWS different than LN
bp
< .05, OB different than LN
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Figure 2 — IL-10, TNF-α, IFN-γ concentrations at rest and in response to the intermittent aerobic exercise protocol in children with PWS, obese and lean controls.
the primary source of exercise-induced IL-8 observed in the current study may have been the skeletal muscle (2,15). Children with PWS presented a greater integrated IL-8 response to exercise and recovery, possibly due to the epigenetic modifications associated with the syndrome (41,45). Epigenetic modulations, such as genomic imprinting or allelic silencing, are recognized regulators of the immune and inflammatory responses (45). These epigenetic modifications in PWS may cause an overactive innate immune system, possibly leading to higher IL-8 concentrations at rest and during recovery from exercise
(41,45). In support of this speculation, Cadoudal and colleagues (2014) reported that children with PWS, who were not on GH treatment, exhibited higher circulating IL-8 at rest when compared with nonsyndromic obese and lean controls, suggesting that IL-8 may be a proinflammatory cytokine differentiating PWS-associated obesity from simple obesity in children. Furthermore, the greater absolute concentrations of IL-8 in children with PWS compared with controls may be relevant because IL-8 has been linked to angiogenesis, muscle adaptation and growth (1,38).
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This is the first study to evaluate the recovery period IL-10 response to exercise in children with and without PWS. We originally expected to see increased IL-10 concentrations during recovery from exercise because IL-10 production helps control the magnitude and duration of the proinflammatory cytokine response to exercise (11,26). However, the duration and/or intensity of the current study’s exercise protocol may not have been enough to trigger IL-10 release from CD8+ T-cells (20,26). Further investigation is needed to determine the duration and intensity of exercise that elicits an increase in circulating IL-10 concentrations in children. Similar to the above IL-10 findings, TNF-α and IFN-γ levels were unchanged immediately post exercise and during recovery compared with baseline values. At rest, a lack of TNF-α and IFN-γ differences between PWS and controls were expected based on findings from previous researchers (17,18,37). However, a lack of TNF-α group differences in response to exercise were unexpected, as Rosa and colleagues (2011) reported that children with obesity had significantly elevated TNF-α concentrations throughout exercise compared with lean (30). With regards to IFN-γ, previous researchers reported either an increase or decrease in IFN-γ concentration in response to acute aerobic exercise in healthy young adults and adults (20,42). The incongruity between our TNF-α and IFN-γ data and those previously published could be due to pubertal maturation differences (adults vs. children), experimental differences (mode, intensity, or duration of the exercise bout), or the training status of the participants (38,43). To our knowledge, this is the first study to evaluate cytokine responses to exercise in children with PWS. Studying exercise responses in children with PWS presents with numerous challenges because of syndrome related characteristics, such as challenging behaviors, intellectual disability, poor stamina and poor cardiovascular fitness (8). Thus, obtaining maximal or peak oxygen consumption measurements in this study was not feasible. Moreover, our relatively small sample size resulted in the large cytokine concentration variability exhibited by all groups; suggesting the need for a larger sample size when evaluating cytokines. However, recruitment of child participants with PWS, a rare condition, who were also able to complete this exercise protocol was extremely challenging. Lastly, it would have been beneficial to evaluate cytokine responses at the mRNA and protein level to better understand the elevated overall integrated exercise-induced systemic IL-6 and IL-8 concentrations in children with PWS and nonsyndromic obesity. In conclusion, children with PWS had a similar pattern in the circulating cytokines’ response to acute intermittent aerobic exercise as children without the syndrome. The integrated IL-6 response to exercise was greater in children with obesity compared with lean controls; supporting the link between excess body fat, syndromic or nonsyndromic in origin, and high IL-6 in pediatric obesity. Furthermore, the larger integrated IL-8
response to exercise in PWS than controls may be due to epigenetic modifications associated with the syndrome; possibly differentiating PWS-associated obesity from simple obesity in children. Acknowledgments Thanks to participating children, Joane Less, RN, Cheryl Wilkinson, RN and Nancy Varni, RN (Children’s Hospital of Orange County). Special thanks to Dr. Robert Kersey from the Department of Kinesiology at California State University Fullerton and to Dr. Marta D. Van Loan for conducting our cytokine analyses in her laboratory, USDA ARS Western Human Nutrition Research Center (Address: 430 West Health Sciences Drive, University of California Davis, CA 95616). This project was funded by US Army Medical Research and Materiel Command Contract W81XWH-08-1-0025. Andrea M. Haqq was supported by the Canadian Institutes of Health Research and the Alberta Diabetes Institute.
References 1. Adair TH, Montani JP. Angiogenesis. San Rafael, CA: Morgan & Claypool Life Sciences; 2010. 2. Akerstrom T, Steensberg A, Keller P, Keller C, Penkowa M, Pedersen BK. Exercise induces interleukin-8 expression in human skeletal muscle. J Physiol. 2005; 563(Pt 2):507–516. PubMed doi:10.1113/jphysiol.2004.077610 3. Bar-Or O, Rowland TW. Procedures for exercise testing in children. In: O Bar-Or and TW Rowland, editors. Pediatric Exercise Medicine: From Physiologic Principles to Health Care Application. Champaign: Human Kinetics; 2004. p. 343–365. 4. Berg AH, Scherer PE. Adipose tissue, inflammation, and cardiovascular disease. Circ Res. 2005; 96(9):939–949. PubMed doi:10.1161/01.RES.0000163635.62927.34 5. Butler MG. Management of obesity in Prader-Willi syndrome. Nat Clin Pract Endocrinol Metab. 2006; 2(11):592–593. PubMed doi:10.1038/ncpendmet0320 6. Cadoudal T, Buleon M, Sengenes C, et al. Impairment of adipose tissue in Prader-Willi syndrome rescued by growth hormone treatment. Int J Obes. 2014; 38(9):1234–1240. PubMed doi:10.1038/ijo.2014.3 7. Carey AL, Steinberg GR, Macaulay SL, et al. Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes. 2006; 55(10):2688–2697. PubMed doi:10.2337/db05-1404 8. Cassidy SB, Schwartz S, Miller JL, Driscoll DJ. Prader-Willi syndrome. Genet Med. 2012; 14(1):10–26. PubMed doi:10.1038/gim.0b013e31822bead0 9. Christiansen T, Bruun JM, Paulsen SK, et al. Acute exercise increases circulating inflammatory markers in overweight and obese compared with lean subjects. Eur J Appl Physiol. 2013; 113(6):1635–1642. PubMed doi:10.1007/s00421013-2592-0 10. Centers for Disease Control. National Health and Nutrition Examination Survey (NHANES). Atlanta, Ga: Anthropometry Procedures Manual. NCFH Statistics; 2007.
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11. Das UN. Anti-inflammatory nature of exercise. Nutrition. 2004; 20(3):323–326. PubMed doi:10.1016/j. nut.2003.11.017 12. de Heredia FP, Gomez-Martinez S, Marcos A. Obesity, inflammation and the immune system. Proc Nutr Soc. 2012; 71(2):332–338. PubMed doi:10.1017/ S0029665112000092 13. Eliakim A, Nemet D, Zaldivar F, et al. Reduced exercise-associated response of the GH-IGF-I axis and catecholamines in obese children and adolescents. J Appl Physiol. 2006; 100(5):1630–1637. PubMed doi:10.1152/ japplphysiol.01072.2005 14. Febbraio MA, Hiscock N, Sacchetti M, Fischer CP, Pedersen BK. Interleukin-6 is a novel factor mediating glucose homeostasis during skeletal muscle contraction. Diabetes. 2004; 53(7):1643–1648. PubMed doi:10.2337/ diabetes.53.7.1643 15. Frydelund-Larsen L, Penkowa M, Akerstrom T, Zankari A, Nielsen S, Pedersen BK. Exercise induces interleukin-8 receptor (CXCR2) expression in human skeletal muscle. Exp Physiol. 2007; 92(1):233–240. PubMed doi:10.1113/ expphysiol.2006.034769 16. Galassetti P, Larson J, Iwanaga K, Salsberg SL, Eliakim A, Pontello A. Effect of a high-fat meal on the growth hormone response to exercise in children. J Pediatr Endocrinol Metab. 2006; 19(6):777–786. PubMed doi:10.1515/ JPEM.2006.19.6.777 17. Haqq AM, Muehlbauer MJ, Newgard CB, Grambow S, Freemark M. The metabolic phenotype of Prader-Willi syndrome (PWS) in childhood: heightened insulin sensitivity relative to body mass index. J Clin Endocrinol Metab. 2011; 96(1):E225–E232. PubMed doi:10.1210/jc.2010-1733 18. Hoybye C. Inflammatory markers in adults with Prader-Willi syndrome before and during 12 months growth hormone treatment. Horm Res. 2006; 66(1):27–32. PubMed doi:10.1159/000093229 19. Inzaugarat ME, Billordo LA, Vodanovich F, et al. Alterations in innate and adaptive immune leukocytes are involved in paediatric obesity. Pediatr Obes. 2013; 9(5):381–390. PubMed doi:10.1111/j.2047-6310.2013.00179.x 20. LaVoy EC, Bosch JA, Lowder TW, Simpson RJ. Acute aerobic exercise in humans increases cytokine expression in CD27(-) but not CD27(+) CD8(+) T-cells. Brain Behav Immun. 2012; 27(1):54–62. PubMed 21. Matthews JN, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in medical research. BMJ. 1990; 300(6719):230–235. PubMed doi:10.1136/ bmj.300.6719.230 22. McCarthy HD, Cole TJ, Fry T, Jebb SA, Prentice AM. Body fat reference curves for children. Int J Obes. 2006; 30(4):598–602. PubMed doi:10.1038/sj.ijo.0803232 23. McMurray RG, Zaldivar F, Galassetti P, et al. Cellular immunity and inflammatory mediator responses to intense exercise in overweight children and adolescents. J Investig Med. 2007; 55(3):120–129. PubMed doi:10.2310/6650.2007.06031 24. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014; 41(1):36–48. PubMed doi:10.1016/j.immuni.2014.05.010
25. Neels JG, Olefsky JM. Inflamed fat: what starts the fire? J Clin Invest. 2006; 116(1):33–35. PubMed doi:10.1172/ JCI27280 26. Ostrowski K, Rohde T, Asp S, Schjerling P, Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol. 1999; 515(Pt 1):287–291. PubMed doi:10.1111/j.1469-7793.1999.287ad.x 27. Pedersen BK, Febbraio MA. Muscle as an endocrine organ: focus on muscle-derived interleukin-6. Physiol Rev. 2008; 88(4):1379–1406. PubMed doi:10.1152/physrev.90100.2007 28. Petersen AC, Crockett L, Richards M, Boxer A. A self-report measure of pubertal status: Reliability, validity, and initial norms. J Youth Adolesc. 1988; 17(2):117–133. PubMed doi:10.1007/BF01537962 29. Robertson RJ, Goss FL, Bell JA, et al. Self-regulated cycling using the Children’s OMNI Scale of Perceived Exertion. Med Sci Sports Exerc. 2002; 34(7):1168–1175. PubMed doi:10.1097/00005768-200207000-00018 30. Rosa JS, Heydari S, Oliver SR, et al. Inflammatory cytokine profiles during exercise in obese, diabetic, and healthy children. J Clin Res Pediatr Endocrinol. 2011; 3(3):115–121. PubMed doi:10.4274/jcrpe.v3i3.23 31. Rosa JS, Oliver SR, Flores RL, et al. Kinetic profiles of 18 systemic pro- and anti-inflammatory mediators during and following exercise in children. J Pediatr Endocrinol Metab. 2007; 20(12):1293–1305. PubMed doi:10.1515/ JPEM.2007.20.12.1293 32. Rosa JS, Oliver SR, Flores RL, et al. Altered inflammatory, oxidative, and metabolic responses to exercise in pediatric obesity and type 1 diabetes. Pediatr Diabetes. 2011; 12(5):464–472. PubMed doi:10.1111/j.13995448.2010.00724.x 33. Rubin DA, Hackney AC. Inflammatory cytokines and metabolic risk factors during growth and maturation: influence of physical activity. Med Sport Sci. 2010; 55:43–55. PubMed doi:10.1159/000321971 34. Rubin DA, McMurray RG, Harrell JS, Hackney AC, Haqq AM. Do surrogate markers for adiposity relate to cytokines in adolescents? J Investig Med. 2008; 56(5):786–792. PubMed 35. Scheett TP, Mills PJ, Ziegler MG, Stoppani J, Cooper DM. Effect of exercise on cytokines and growth mediators in prepubertal children. Pediatr Res. 1999; 46(4):429–434. PubMed doi:10.1203/00006450-199910000-00011 36. Schwarzenberg SJ, Sinaiko AR. Obesity and inflammation in children. Paediatr Respir Rev. 2006; 7(4):239–246. PubMed doi:10.1016/j.prrv.2006.08.002 37. Sohn YB, Kwak MJ, Kim SJ, et al. Correlation of adiponectin receptor expression with cytokines and insulin sensitivity in growth hormone (GH)-treated children with Prader-Willi syndrome and in non-GH-treated obese children. J Clin Endocrinol Metab. 2010; 95(3):1371–1377. PubMed doi:10.1210/jc.2009-1489 38. Timmons BW, Tarnopolsky MA, Snider DP, Bar-Or O. Immunological changes in response to exercise: influence of age, puberty, and gender. Med Sci Sports Exerc. 2006; 38(2):293–304. PubMed doi:10.1249/01. mss.0000183479.90501.a0
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42. Vijayaraghava A. K R. Alteration of Interferon Gamma (IFN-gamma) in Human Plasma with Graded Physical Activity. J Clin Diagn Res. 2014; 8(6):BC05–BC07. PubMed 43. Walsh NP, Gleeson M, Shephard RJ, et al. Position statement. Part one: Immune function and exercise. Exerc Immunol Rev. 2011; 17:6–63. PubMed 44. Weiss R, Dziura J, Burgert TS, et al. Obesity and the metabolic syndrome in children and adolescents. N Engl J Med. 2004; 350(23):2362–2374. PubMed doi:10.1056/ NEJMoa031049 45. Wilson AG. Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. J Periodontol. 2008; 79(8, Suppl.):1514–1519. PubMed doi:10.1902/jop.2008.080172
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39. Trinh A, Campbell M, Ukoumunne OC, Gerner B, Wake M. Physical activity and 3-year BMI change in overweight and obese children. Pediatrics. 2013; 131(2):e470–e477. PubMed doi:10.1542/peds.2012-1092 40. van Mil EG, Westerterp KR, Gerver WJ, Van Marken Lichtenbelt WD, Kester AD, Saris WH. Body composition in Prader-Willi syndrome compared with nonsyndromal obesity: Relationship to physical activity and growth hormone function. J Pediatr. 2001; 139(5):708–714. PubMed doi:10.1067/mpd.2001.118399 41. Viardot A, Sze L, Purtell L, et al. Prader-Willi syndrome is associated with activation of the innate immune system independently of central adiposity and insulin resistance. J Clin Endocrinol Metab. 2010; 95(7):3392–3399. PubMed doi:10.1210/jc.2009-2492
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ORIGINAL RESEARCH
Cytokine Responses to Acute Intermittent Aerobic Exercise in Children With Prader-Willi Syndrome and Nonsyndromic Obesity Andrea T. Duran
Erik Gertz
California State University Fullerton
United States Department of Agriculture
Daniel A. Judelson
Andrea M. Haqq
California State University Fullerton
University of Alberta
Susan J. Clark
Kavin W. Tsang and Daniela Rubin
Children’s Hospital of Orange County
California State University Fullerton
Prader-Willi Syndrome (PWS), the best characterized form of syndromic obesity, presents with abnormally high fat mass. In children, obesity presents with low-grade systemic inflammation. This study evaluated if PWS and/or nonsyndromic obesity affected cytokine responses to intermittent aerobic exercise in children. Eleven children with PWS (11 ± 2 y, 45.4 ± 9.5% body fat), 12 children with obesity (OB) (9 ± 1 y, 39.9 ± 6.8% body fat), and 12 lean (LN) children (9 ± 1 y, 17.5 ± 4.6% body fat) participated. Children completed 10 2-min cycling bouts of vigorous intensity, separated by 1-min rest. Blood samples were collected preexercise (PRE), immediately postexercise (IP), and 15, 30, and 60 min into recovery to analyze possible changes in cytokines. In all groups, IL-6 and IL-8 concentrations were greater during recovery compared with PRE. PWS and OB exhibited higher IL-6 area under the curve (AUC) than LN (p < .01 for both). PWS demonstrated higher IL-8 AUC than LN (p < .04). IL-10, TNF-α, and IFN-γ did not change with exercise (p > .05 for all). Results indicate that children with PWS respond with increased Il-6 and IL-8 concentrations to acute exercise similarly to controls. Excess adiposity and epigenetic modifications may explain the greater integrated IL-6 and IL-8 responses in PWS compared with controls. In children and adults, obesity is associated with chronic low-grade systemic inflammation (12,44). At rest, proinflammatory cytokines can be released into circulation from adipocytes embedded in the connective tissue matrix and macrophages located within the subcutaneous and visceral adipose tissue (4,24,25). Understanding these cytokines is important because in children with obesity, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6), and interferon-gamma (IFN-γ) have been linked to
Duran, Tsang, and Rubin are with the Dept. of Kinesiology, California State University Fullerton, Fullerton, CA. Gertz is with the Obesity and Metabolism Research Unit, United States Department of Agriculture, Davis, CA. Judelson is with the Dept. of Kinesiology, California State University Fullerton, Fullerton, CA. Haqq is with the Dept. of Pediatrics, University of Alberta, Edmonton, Alberta, Canada. Clark is with the Dept. of Endocrinology, Children’s Hospital of Orange County, Orange, CA. Address author correspondence to Daniela Rubin at [email protected].
cardiovascular disease, hypertension, insulin resistance and atherosclerosis (12,19,33,36). Prader-Willi Syndrome (PWS) is the best-characterized form and most common genetic cause of childhood morbid obesity (41). Children with PWS present hyperphagia, hypotonia, high fat mass, low lean mass and endocrine abnormalities such as growth hormone deficiency and hypogonadism (5,8). Haqq and colleagues (2011) reported that children with PWS exhibited lower concentrations of IL-6 at rest when compared with obese controls. However, Cadoudal and colleagues (2014) recently demonstrated similar levels of circulating IL-6 and greater interleukin-8 (IL-8) and TNF-α at rest in children with PWS compared with those with nonsyndromic obesity. The paucity of information suggests that the cytokine profile in children with PWS needs further investigation. Exercise is vital to the management of PWS and prevention of pediatric obesity (39,40). Cytokines released in response to exercise, such as IL-6, IL-8 and interleukin-10 (IL-10), help regulate metabolism, muscle growth, and general pro- and anti-inflammatory processes
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(7,11,14,38). Previous research in healthy children demonstrated that aerobic exercise, at least 30 min in duration and of moderate to vigorous intensity, elicited elevated systemic IL-6 and IL-8 concentrations during exercise and recovery compared with concentrations at rest (31,38). In contrast, IL-10 and TNF-α concentrations, in healthy children, did not change in response to aerobic exercise (31,35). The mentioned cytokine responses to exercise vary in children with nonysndromic obesity (23,30,32) and have yet to be explored in children with PWS. In adults, obese individuals exhibit increased proinflammatory markers compared with lean individuals in response to aerobic exercise (9). In children with obesity, studies have demonstrated an increase in some cytokines in response to aerobic exercise compared with children with normal weight (23,30,32). Given the beneficial role of exercise on the management of obesity, the involvement exercise induced cytokines have in metabolic and inflammatory processes, and the possibility that the cytokine profile at rest may differ between children with PWS and nonsyndromic obesity (6,17), it is relevant to determine if the cytokine responses to exercise are altered in children with PWS. Therefore, this study characterized the cytokine responses to acute intermittent aerobic exercise in children with PWS and compared it to obese and lean controls.
Methods Participants Eleven children with PWS (age range 8–13 years) participated. Participants with PWS provided documentation of molecular and cytogenetic testing (i.e., chromosomes, FISH 15, DNA methylation and/or DNA polymorphism studies) to verify diagnoses. Twelve healthy lean children (LN) and 12 obese children (OB) (age range 8–11 years) were recruited through fliers, emails, and word of mouth. Nonsyndromic children were categorized as obese if they had a body fat percentage higher than the 95th percentile and as lean if their body fat percentage was lower than the 85th percentile for age and sex (22). Written assent and consent were obtained by all participants and their parents before participation. The assent and consent forms were approved by the California State University, Fullerton and the Human Research Protection Office at the United States Army Medical Research and Materiel Command Institutional Review Boards. Participant’s parents completed a medical and exercise history form to screen participants for potential health risks and contraindications to exercise. Exclusion criteria for participants included confirmed pregnancy or inability to perform vigorous exercise. Tanner stage established by breast, genital and pubic hair development for children with PWS was determined by a study physician. Tanner stage for children without PWS was determined using the Pubertal Development Scale filled out by the child and his or her parents (28).
Study Protocol All participants completed 2 visits. Each visit was separated by a minimum of 2 days and a maximum of two weeks. Visit one involved the following measurements: anthropometrics, body composition, resting heart rate, and resting blood pressure. Participants also completed an incremental, graded exercise test (McMaster protocol) to determine each child’s relative peak power output. At the second visit, participants consumed a standardized breakfast (260 kcal, 7 g of fat [21.5%], 37 g of carbohydrate [57%], and 14 g of protein [21.5%]) two hours before arrival to the laboratory or the hospital. The breakfast included one breakfast bar (strawberry or chocolate) and an apple sauce, which contained no caffeine. Participants consumed unlimited amounts of water, but were not required to record the consumed volume. Participants refrained from any strenuous physical activity 24 hr before exercise testing. Upon arrival, a trained phlebotomist inserted an indwelling catheter into an antecubital vein or the dorsal vein of the hand of the participant. After 30 min of rest, a resting blood sample (PRE) was obtained and participants completed an intermittent aerobic exercise protocol based on a resistance setting from the McMaster protocol. Blood samples were obtained immediate postexercise (IP), +15, +30, and +60 min during recovery from the exercise challenge. Blood samples were analyzed for IL-6, IL-8, IL-10, TNF-α, and IFN-γ.
Anthropometric and Physiologic Measurements Body mass was obtained using a digital scale (ES2001; Ohaus, Pinewood, N.J.) with the participant only wearing a t-shirt, shorts and no shoes. Stature was measured at the end of inhalation (10) against a wall-mounted stadiometer (Seca, Ontario, Calif., USA). BMI was computed by dividing body mass in kilograms by the stature in meters, squared. After 5 min of seated rest, resting heart rate was measured via telemetry (Polar USA, Lake, Success, NY), and resting blood pressure was measured with an aneroid sphygmomanometer using appropriate diameter cuffs (Diagnostix 752, American Diagnostic Corporation, Hauppage, NY).
Body Composition Body composition was determined with whole body dual x-ray absorptiometry (DXA) scan (Lunar Prodigy Advance; GE Healthcare, Madison, Wisc., USA) to obtain total body, android, gynoid and trunk fat percentage and total lean mass. Participants were positioned following manufacturer’s indications (GE Healthcare, GE Lunar Corp., Madison WI). All scans were done and analyzed using the Pediatric Encore software version 12.30.008, which automatically demarcates the regional boundaries for all regions.
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McMaster Protocol This protocol is a multistage, incremental protocol, requiring subjects to cycle in 2-min stages at progressively increasing workloads (3). Work rate at each stage was based on participant sex and height. Exercise intensity increased every 2 min until the subject reached volitional exhaustion, failed to sustain the desired workload, requested to stop, stood up on the bike pedals, or experienced fatigue-related symptoms. Participants were required to cycle at a minimum pedaling rate of 50 rpm (3), monitored by an EM900 pocket metronome (Franz Manufacturing Company, Incorporated, East Haven, CT). Workload, heart rate, and rating of perceived exertion using the Pediatric OMNI scale (29) were recorded for each stage and relative peak power output (Watts per kg lean body mass) was computed by dividing the test termination load by the participant’s lean body mass obtained from the DXA scan.
Experimental Exercise Protocol The intermittent aerobic exercise protocol consisted of 10 cycling bouts (2 min each), separated by 1-min rest intervals. The workload for this discontinuous protocol was set based on the first resistance from the McMaster protocol that successfully elicited a heart rate ≥160 bpm. Previous studies have shown acute hormonal and inflammatory responses at a target heart rate ≥160 bpm in children (13,16). As 8 children with PWS were not able to attain peak heart rates >160 bpm, they exercised at the second to last workload completed in the McMaster protocol. Heart rate was measured continuously and ratings of perceived exertion were measured at the end of every 2-min interval.
Blood Processing and Analysis Blood samples obtained at PRE, IP, +15, +30, and +60 min during recovery from the experimental exercise protocol were collected into ethylene-diamine-tetra-acetic acid (EDTA) containing tubes (BD Diagnostics, Franklin Lakes, NJ) and centrifuged at 4 °C and 3,000 rpm for 15 min. Plasma was then placed in Eppendorf tubes for storage in -70 °C freezer until analyses. Proinflammatory Panel 1 (human) V-PLEX Kit (Catolog # K15049D) from Meso Scale Discovery (MSD) was used for cytokine analysis. The mean intra-assay CV of IL-6, IL-8, IL-10, TNF-α and IFN-γ were 24.9%, 6.9%, 29.9%, 11.1% and 26.6%; respectively.
Statistical Analyses All cytokines were nonnormally distributed. Log transformations were used to obtain normal distributions for all cytokines. TNF-α data for two participants were omitted from analyses because concentrations were below the detection limit of the assay. Three (group) by five (time) repeated measures mixed model ANOVAs were used to
identify differences in cytokine concentrations between children with PWS, obese and lean controls over time. In the case of significant interaction, group or time effect, Tukey’s HSD assessed pairwise differences. In addition, total areas under the curve (AUC; cytokine concentration x time) were calculated for each cytokine using the standardized trapezoidal methods (21) to determine group differences. Group comparisons for AUC were performed using the nonparametric Kruskal-Wallis test. In case of significant findings, two group comparisons for distribution and medians of AUC were done using Mann-Whitney U test and Median test, respectively. Significance level for all analyses was set at p < .05. Statistical analyses were performed using SPSS, Version 20.0 for Windows (SPSS, Inc., Chicago, IL).
Results Participant Characteristics Uniparental deletion (n = 9) and methylation (n = 2) diagnoses were confirmed for participants with PWS. PWS participants were classified as Tanner Stage I (Breast/ genital (BG): n = 5, pubic hair (PH): n = 2), Tanner Stage II (BG: n = 3, PH: n = 1), Tanner Stage III (BG: n = 1, PH: n = 2), Tanner Stage IV (BG: n = 2, PH: n = 5,) or Tanner Stage V (BG: n = 0, PH: n = 1). PWS participants also displayed type II diabetes mellitus (n = 1), asthma (n = 2), apnea (n = 3), pneumonia (n = 2), hypothyroidism (n = 3) and hyperthyroidism (n = 1). PWS participants currently received (n = 9), previously received (n = 1), or never received (n = 1) GH replacement therapy. The obese and lean controls were classified as Tanner Stage I (OB: n = 6, LN: n = 8), Tanner Stage II (OB: n = 4, LN: n = 3) or Tanner Stage III (OB: n = 2, LN: n = 1) based on the Pubertal Development Scale. Participant characteristics for each group are presented in Table 1. Children with PWS were older than obese and lean controls (p = .01, p = .03, respectively), while obese and lean had a similar age (p = .89). All children exhibited a similar height (p > .10 for all), with PWS presenting higher body mass than lean (p < .01). There were no significant differences in lean body mass among groups (p > .20 for all). Children with PWS exhibited similar android, gynoid, trunk and total body fat percentage as obese (p = .95, p = .70, p = .62 and p = .17; respectively). Lean controls had a significantly lower android, gynoid, trunk and total body fat percentage than children with PWS or obese (p < .01 for all).
Physiological Responses to Exercise Exercise responses to the intermittent aerobic protocol for each group are presented in Table 2. In response to the intermittent aerobic protocol, children with PWS had a significantly lower submaximal exercise heart rate, peak heart rate and relative submaximal workload compared
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Table 1 Participant Characteristics for Children with PWS, Obese, and Lean Controls Participant Characteristics Age (yrs)
11 ±
Males/Females
Obese (n = 12)
Lean (n = 12)
9±1
10 ± 1
8/4
8/4
2a,b
7/4
Height (cm)
148.9 ± 13.6
140.6 ± 8.1
140.1 ± 7.6
Body Mass (kg)
62.7 ± 30.8b
50.5 ± 13.3
32.6 ± 6.0
10.5b
4.4c
16.5 ± 1.4
39.9 ± 6.8c
17.5 ± 4.6
Body Mass Index
(kg/m2)
Body Fat Mass (%)
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PWS (n = 11)
27.2 ±
45.4 ± 9.5b
25.2 ±
Lean Body Mass (kg)
31.1 ± 11.6
28.5 ± 4.9
25.7 ± 4.1
Android Fat (%)
49.9 ± 12.1b
48.7 ± 8.1c
17.1 ± 6.8
Gynoid Fat (%)
52.3 ± 6.2b
47.1 ± 4.9c
29.5 ± 5.0
Trunk Fat (%)
45.0 ± 12.1b
41.60 ± 7.9c
15.3 ± 5.0
Resting heart rate (bpm)
80 ± 13
79 ± 12
79 ± 8
Resting SBP (mm Hg)
103 ± 15
105 ± 12
95 ± 15
Resting DBP (mm Hg)
72 ± 12
65 ± 9
62 ± 14
Note. Data reported as mean ± SD or frequency. ap
< .05, PWS different than OB.
bp
< .05, PWS different than LN.
cp
< .05, OB different than LN.
Table 2 Group Exercise Responses to the Intermittent Aerobic Exercise protocols Intermittent Aerobic Exercise Responses Absolute submaximal workload (W)
OB (n = 12)
LN (n = 12)
55 ± 26
70 ± 17
70 ± 13
2.5 ± 0.3
2.7 ± 0.4
66.5 ± 11.6
75.7 ± 13.4
129 ± 30a,b
163 ± 6
168 ± 10
Peak heart rate (bpm)a
151 ± 27a,b
193 ± 13
186 ± 13
Percentage of peak heart rate (%)
85.4 ± 15.9
84.9 ± 5.9
90.3 ± 6.7
123 ± 24
133 ± 10
123 ± 13
64 ± 11
55 ± 15
Relative submaximal workload (W·LBM
kg-1)
PWS (n = 11) 1.8 ±
0.7a,b
Percentage of peak relative workload (%)
75.3 ± 5.9
Submaximal exercise heart rate (bpm)
Ending systolic blood pressure (mm Hg) Ending diastolic blood pressure (mm Hg)
72 ±
6b
3c
7±2 9±2
Mean RPE (1–10)
5±1
4±
Peak RPE (1–10)
8±2
6±3c
Note. Data reported as mean ± SD. Peak heart rate was obtained during McMaster Protocol. ap
< .05, PWS different than OB.
bp
< .05, PWS different than LN.
cp
< .05, OB different than LN.
with obese and lean controls (OB: p < .01, p < .01, p = .01, respectively; LN: p < .01 for all). Obese and lean controls had a similar submaximal exercise heart rate, peak heart rate and relative submaximal workload (p > .99, p = .68, p = .32, respectively). Children with PWS did not show any difference in mean or peak RPE when compared with obese and lean controls (PWS vs. OB:
p = .57 and p = .22, respectively; PWS vs. LN: p = .24 and p = .75, respectively). Lean controls had significantly greater mean RPE and peak RPE values compared with obese (p = .01 and p = .02, respectively). There were no differences among groups in percentage of peak relative workload or percentage of peak heart rate (p = .38 and p = .08, respectively).
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Figure 1 — IL-6 and IL-8 concentrations at rest and in response to the intermittent aerobic exercise protocol in children with PWS, obese and lean controls. a = significant time effect: time point different than PRE (p < .05). b = significant time effect: time point different than IP (p < .05). # = overall group differences (p < .05).
Cytokine Responses to Exercise IL-6. IL-6 concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 1. There was no significant group by time interaction in circulating IL-6 concentrations in response to the exercise challenge (p = .73). Children with PWS showed no significant differences in overall mean IL-6 concentrations compared with the obese and lean controls (p = .68 and p = .60, respectively). Obese had
a greater overall mean IL-6 concentration than the lean controls (p = .02). In all groups, IL-6 concentrations were significantly greater at +15, +30, and +60 compared with PRE (p < .01 for all). Total IL-6 AUC distributions and medians (presented in Table 3) were significantly greater in PWS and obese groups compared with the lean controls (PWS: p = .03, p < .01; respectively, OB: p = .01, p < .01; respectively). Total IL-6 AUC distributions and medians were the same between the PWS and obese groups (p = .64, p = .70, respectively).
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IL-8. IL-8 concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 1. There was no significant group by time interaction or group effect in circulating IL-8 concentrations in response to the exercise challenge (p = .63 and p = .14; respectively). In all groups, IL-8 concentrations were significantly greater at IP compared with baseline, and at +15, and +30 (p < .01 for all) and significantly lower at +60 than IP (p = .02). Total IL-8 AUC distributions and medians were significantly greater in children with PWS compared with the lean controls (p = .01, p = .04, respectively). Total IL-8 AUC distributions and medians (see Table 3) were the same between the PWS and obese groups (p = .26, p = .68, respectively) and the obese and lean controls (p = .32, p = .68, respectively).
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IL-10, TNF-α, & IFN-γ. IL-10, TNF-α, and IFN-γ concentrations in response to the intermittent aerobic exercise protocol for each group can be seen in Figure 2. There were no significant group by time interactions, group or time effects in circulating IL-10, TNF-α, or IFN- γ concentrations in response to the exercise challenge (IL-10: p = .98, p = .66 and p = .18, respectively; TNF-α : p = .29, p = .29 and p = .50, respectively; IFN- γ: p = .63, p = .31 and p = .19, respectively). Total IL-10 AUC, TNF-α AUC, and IFN- γ AUC distributions and medians (presented in Table 3) were also the same among all groups (IL-10: p = .33 and p = .75, respectively; TNF-α: p = .316 and p = .213, respectively; IFN-γ: p = .112 and p = .111, respectively).
Discussion In all children, 30 min of vigorous, intermittent, aerobic exercise elicited elevations of systemic IL-6 and IL-8 concentrations immediately after and during the recovery period, with no changes in IL-10, TNF-α, or IFN-γ. Those with PWS and nonsyndromic obesity demonstrated a greater integrated response measured by the AUC for IL-6. Those with PWS had a greater integrated response
measured by the AUC for IL-8 when compared with lean controls. No differences were observed for IL-10, TNF-α, or IFN- γ AUC among groups. All groups presented increases in IL-6 during recovery from exercise, which is consistent with results by previous studies (23,32). This increase in IL-6 was probably due to IL-6 released by skeletal muscle as groups presented comparable lean mass (27). Children with PWS and nonsyndromic obesity had a greater integrated IL-6 response to exercise compared with lean controls, possibly due to similar fat distribution (total body, android, gynoid, and trunk fat) and fat content in these groups. Likely, excess body fat led to higher circulating proinflammatory IL-6 concentrations at rest in these groups relative to the lean controls as shown before by others (34,44). We could not demonstrate significant IL-6 differences at baseline among groups; perhaps because of the low statistical power (observed power: 0.371). However, these group differences probably became larger when evaluating IL-6 concentrations over several time points. A greater tissue exposure to absolute systemic concentrations of IL-6, as presented in the obese children, is relevant because IL-6 has been linked with cardiometabolic complications at rest (33), as well as with improved hepatic glucose production, fatty acid oxidation and insulin sensitivity in response to exercise (7,14). As for PWS, our results support a normal IL-6 response over time, with increased concentrations in response to exercise. Perhaps a greater IL-6 response in PWS when considering the lower absolute workload that the majority of these children exercised at when compared with controls. All children presented increased exercise-induced systemic IL-8 concentrations as early as 15 min into recovery, with IL-8 concentrations returning to baseline within one hour. When children exercise longer than 30 min, concentrations of circulating IL-8 may take more recovery time to return to baseline (38). In children, the exercise-induced elevations of IL-8 during recovery may be from mobilized white blood cells or active skeletal muscle (2,15,23). Since all children presented similar lean mass,
Table 3 Total Area Under the Curve for Cytokine Concentrations in Response to the Intermittent Aerobic Exercise Protocol in Children with PWS, Obese, and Lean Controls PWS (n = 11) IL-6 (pg*min/mL) IL-8 (pg*min/mL)
67.2 (37.5, 315.8 (249.0,
IL-10 (pg*min/mL)
78.2)a 369.8)a
36.8 (24.5, 47.3)
OB (n = 12) 68.3 (43.7,
93.4)b
Lean (n = 12) 29.3 (27.1, 35.4)
284.6 (165.8, 320.1)
247.1 (192.2, 269.1)
40.5 (36.2, 57.0)
40.1 (24.2, 48.7)
TNF-α (pg*min/mL)
174.0 (130.5–265.5)
225.0 (156.8–273.8)
159.0 (144.8–222.0)
IFN-γ (pg*min/mL)
504.0 (357.8–808.5)
771.8 (411.0–1106.1)
378.0 (378.0–685.7)
Note: Data reported as median (interquartile range). ap
< .05, PWS different than LN
bp
< .05, OB different than LN
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Figure 2 — IL-10, TNF-α, IFN-γ concentrations at rest and in response to the intermittent aerobic exercise protocol in children with PWS, obese and lean controls.
the primary source of exercise-induced IL-8 observed in the current study may have been the skeletal muscle (2,15). Children with PWS presented a greater integrated IL-8 response to exercise and recovery, possibly due to the epigenetic modifications associated with the syndrome (41,45). Epigenetic modulations, such as genomic imprinting or allelic silencing, are recognized regulators of the immune and inflammatory responses (45). These epigenetic modifications in PWS may cause an overactive innate immune system, possibly leading to higher IL-8 concentrations at rest and during recovery from exercise
(41,45). In support of this speculation, Cadoudal and colleagues (2014) reported that children with PWS, who were not on GH treatment, exhibited higher circulating IL-8 at rest when compared with nonsyndromic obese and lean controls, suggesting that IL-8 may be a proinflammatory cytokine differentiating PWS-associated obesity from simple obesity in children. Furthermore, the greater absolute concentrations of IL-8 in children with PWS compared with controls may be relevant because IL-8 has been linked to angiogenesis, muscle adaptation and growth (1,38).
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532 Duran et al.
This is the first study to evaluate the recovery period IL-10 response to exercise in children with and without PWS. We originally expected to see increased IL-10 concentrations during recovery from exercise because IL-10 production helps control the magnitude and duration of the proinflammatory cytokine response to exercise (11,26). However, the duration and/or intensity of the current study’s exercise protocol may not have been enough to trigger IL-10 release from CD8+ T-cells (20,26). Further investigation is needed to determine the duration and intensity of exercise that elicits an increase in circulating IL-10 concentrations in children. Similar to the above IL-10 findings, TNF-α and IFN-γ levels were unchanged immediately post exercise and during recovery compared with baseline values. At rest, a lack of TNF-α and IFN-γ differences between PWS and controls were expected based on findings from previous researchers (17,18,37). However, a lack of TNF-α group differences in response to exercise were unexpected, as Rosa and colleagues (2011) reported that children with obesity had significantly elevated TNF-α concentrations throughout exercise compared with lean (30). With regards to IFN-γ, previous researchers reported either an increase or decrease in IFN-γ concentration in response to acute aerobic exercise in healthy young adults and adults (20,42). The incongruity between our TNF-α and IFN-γ data and those previously published could be due to pubertal maturation differences (adults vs. children), experimental differences (mode, intensity, or duration of the exercise bout), or the training status of the participants (38,43). To our knowledge, this is the first study to evaluate cytokine responses to exercise in children with PWS. Studying exercise responses in children with PWS presents with numerous challenges because of syndrome related characteristics, such as challenging behaviors, intellectual disability, poor stamina and poor cardiovascular fitness (8). Thus, obtaining maximal or peak oxygen consumption measurements in this study was not feasible. Moreover, our relatively small sample size resulted in the large cytokine concentration variability exhibited by all groups; suggesting the need for a larger sample size when evaluating cytokines. However, recruitment of child participants with PWS, a rare condition, who were also able to complete this exercise protocol was extremely challenging. Lastly, it would have been beneficial to evaluate cytokine responses at the mRNA and protein level to better understand the elevated overall integrated exercise-induced systemic IL-6 and IL-8 concentrations in children with PWS and nonsyndromic obesity. In conclusion, children with PWS had a similar pattern in the circulating cytokines’ response to acute intermittent aerobic exercise as children without the syndrome. The integrated IL-6 response to exercise was greater in children with obesity compared with lean controls; supporting the link between excess body fat, syndromic or nonsyndromic in origin, and high IL-6 in pediatric obesity. Furthermore, the larger integrated IL-8
response to exercise in PWS than controls may be due to epigenetic modifications associated with the syndrome; possibly differentiating PWS-associated obesity from simple obesity in children. Acknowledgments Thanks to participating children, Joane Less, RN, Cheryl Wilkinson, RN and Nancy Varni, RN (Children’s Hospital of Orange County). Special thanks to Dr. Robert Kersey from the Department of Kinesiology at California State University Fullerton and to Dr. Marta D. Van Loan for conducting our cytokine analyses in her laboratory, USDA ARS Western Human Nutrition Research Center (Address: 430 West Health Sciences Drive, University of California Davis, CA 95616). This project was funded by US Army Medical Research and Materiel Command Contract W81XWH-08-1-0025. Andrea M. Haqq was supported by the Canadian Institutes of Health Research and the Alberta Diabetes Institute.
References 1. Adair TH, Montani JP. Angiogenesis. San Rafael, CA: Morgan & Claypool Life Sciences; 2010. 2. Akerstrom T, Steensberg A, Keller P, Keller C, Penkowa M, Pedersen BK. Exercise induces interleukin-8 expression in human skeletal muscle. J Physiol. 2005; 563(Pt 2):507–516. PubMed doi:10.1113/jphysiol.2004.077610 3. Bar-Or O, Rowland TW. Procedures for exercise testing in children. In: O Bar-Or and TW Rowland, editors. Pediatric Exercise Medicine: From Physiologic Principles to Health Care Application. Champaign: Human Kinetics; 2004. p. 343–365. 4. Berg AH, Scherer PE. Adipose tissue, inflammation, and cardiovascular disease. Circ Res. 2005; 96(9):939–949. PubMed doi:10.1161/01.RES.0000163635.62927.34 5. Butler MG. Management of obesity in Prader-Willi syndrome. Nat Clin Pract Endocrinol Metab. 2006; 2(11):592–593. PubMed doi:10.1038/ncpendmet0320 6. Cadoudal T, Buleon M, Sengenes C, et al. Impairment of adipose tissue in Prader-Willi syndrome rescued by growth hormone treatment. Int J Obes. 2014; 38(9):1234–1240. PubMed doi:10.1038/ijo.2014.3 7. Carey AL, Steinberg GR, Macaulay SL, et al. Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase. Diabetes. 2006; 55(10):2688–2697. PubMed doi:10.2337/db05-1404 8. Cassidy SB, Schwartz S, Miller JL, Driscoll DJ. Prader-Willi syndrome. Genet Med. 2012; 14(1):10–26. PubMed doi:10.1038/gim.0b013e31822bead0 9. Christiansen T, Bruun JM, Paulsen SK, et al. Acute exercise increases circulating inflammatory markers in overweight and obese compared with lean subjects. Eur J Appl Physiol. 2013; 113(6):1635–1642. PubMed doi:10.1007/s00421013-2592-0 10. Centers for Disease Control. National Health and Nutrition Examination Survey (NHANES). Atlanta, Ga: Anthropometry Procedures Manual. NCFH Statistics; 2007.
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11. Das UN. Anti-inflammatory nature of exercise. Nutrition. 2004; 20(3):323–326. PubMed doi:10.1016/j. nut.2003.11.017 12. de Heredia FP, Gomez-Martinez S, Marcos A. Obesity, inflammation and the immune system. Proc Nutr Soc. 2012; 71(2):332–338. PubMed doi:10.1017/ S0029665112000092 13. Eliakim A, Nemet D, Zaldivar F, et al. Reduced exercise-associated response of the GH-IGF-I axis and catecholamines in obese children and adolescents. J Appl Physiol. 2006; 100(5):1630–1637. PubMed doi:10.1152/ japplphysiol.01072.2005 14. Febbraio MA, Hiscock N, Sacchetti M, Fischer CP, Pedersen BK. Interleukin-6 is a novel factor mediating glucose homeostasis during skeletal muscle contraction. Diabetes. 2004; 53(7):1643–1648. PubMed doi:10.2337/ diabetes.53.7.1643 15. Frydelund-Larsen L, Penkowa M, Akerstrom T, Zankari A, Nielsen S, Pedersen BK. Exercise induces interleukin-8 receptor (CXCR2) expression in human skeletal muscle. Exp Physiol. 2007; 92(1):233–240. PubMed doi:10.1113/ expphysiol.2006.034769 16. Galassetti P, Larson J, Iwanaga K, Salsberg SL, Eliakim A, Pontello A. Effect of a high-fat meal on the growth hormone response to exercise in children. J Pediatr Endocrinol Metab. 2006; 19(6):777–786. PubMed doi:10.1515/ JPEM.2006.19.6.777 17. Haqq AM, Muehlbauer MJ, Newgard CB, Grambow S, Freemark M. The metabolic phenotype of Prader-Willi syndrome (PWS) in childhood: heightened insulin sensitivity relative to body mass index. J Clin Endocrinol Metab. 2011; 96(1):E225–E232. PubMed doi:10.1210/jc.2010-1733 18. Hoybye C. Inflammatory markers in adults with Prader-Willi syndrome before and during 12 months growth hormone treatment. Horm Res. 2006; 66(1):27–32. PubMed doi:10.1159/000093229 19. Inzaugarat ME, Billordo LA, Vodanovich F, et al. Alterations in innate and adaptive immune leukocytes are involved in paediatric obesity. Pediatr Obes. 2013; 9(5):381–390. PubMed doi:10.1111/j.2047-6310.2013.00179.x 20. LaVoy EC, Bosch JA, Lowder TW, Simpson RJ. Acute aerobic exercise in humans increases cytokine expression in CD27(-) but not CD27(+) CD8(+) T-cells. Brain Behav Immun. 2012; 27(1):54–62. PubMed 21. Matthews JN, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in medical research. BMJ. 1990; 300(6719):230–235. PubMed doi:10.1136/ bmj.300.6719.230 22. McCarthy HD, Cole TJ, Fry T, Jebb SA, Prentice AM. Body fat reference curves for children. Int J Obes. 2006; 30(4):598–602. PubMed doi:10.1038/sj.ijo.0803232 23. McMurray RG, Zaldivar F, Galassetti P, et al. Cellular immunity and inflammatory mediator responses to intense exercise in overweight children and adolescents. J Investig Med. 2007; 55(3):120–129. PubMed doi:10.2310/6650.2007.06031 24. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity. 2014; 41(1):36–48. PubMed doi:10.1016/j.immuni.2014.05.010
25. Neels JG, Olefsky JM. Inflamed fat: what starts the fire? J Clin Invest. 2006; 116(1):33–35. PubMed doi:10.1172/ JCI27280 26. Ostrowski K, Rohde T, Asp S, Schjerling P, Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol. 1999; 515(Pt 1):287–291. PubMed doi:10.1111/j.1469-7793.1999.287ad.x 27. Pedersen BK, Febbraio MA. Muscle as an endocrine organ: focus on muscle-derived interleukin-6. Physiol Rev. 2008; 88(4):1379–1406. PubMed doi:10.1152/physrev.90100.2007 28. Petersen AC, Crockett L, Richards M, Boxer A. A self-report measure of pubertal status: Reliability, validity, and initial norms. J Youth Adolesc. 1988; 17(2):117–133. PubMed doi:10.1007/BF01537962 29. Robertson RJ, Goss FL, Bell JA, et al. Self-regulated cycling using the Children’s OMNI Scale of Perceived Exertion. Med Sci Sports Exerc. 2002; 34(7):1168–1175. PubMed doi:10.1097/00005768-200207000-00018 30. Rosa JS, Heydari S, Oliver SR, et al. Inflammatory cytokine profiles during exercise in obese, diabetic, and healthy children. J Clin Res Pediatr Endocrinol. 2011; 3(3):115–121. PubMed doi:10.4274/jcrpe.v3i3.23 31. Rosa JS, Oliver SR, Flores RL, et al. Kinetic profiles of 18 systemic pro- and anti-inflammatory mediators during and following exercise in children. J Pediatr Endocrinol Metab. 2007; 20(12):1293–1305. PubMed doi:10.1515/ JPEM.2007.20.12.1293 32. Rosa JS, Oliver SR, Flores RL, et al. Altered inflammatory, oxidative, and metabolic responses to exercise in pediatric obesity and type 1 diabetes. Pediatr Diabetes. 2011; 12(5):464–472. PubMed doi:10.1111/j.13995448.2010.00724.x 33. Rubin DA, Hackney AC. Inflammatory cytokines and metabolic risk factors during growth and maturation: influence of physical activity. Med Sport Sci. 2010; 55:43–55. PubMed doi:10.1159/000321971 34. Rubin DA, McMurray RG, Harrell JS, Hackney AC, Haqq AM. Do surrogate markers for adiposity relate to cytokines in adolescents? J Investig Med. 2008; 56(5):786–792. PubMed 35. Scheett TP, Mills PJ, Ziegler MG, Stoppani J, Cooper DM. Effect of exercise on cytokines and growth mediators in prepubertal children. Pediatr Res. 1999; 46(4):429–434. PubMed doi:10.1203/00006450-199910000-00011 36. Schwarzenberg SJ, Sinaiko AR. Obesity and inflammation in children. Paediatr Respir Rev. 2006; 7(4):239–246. PubMed doi:10.1016/j.prrv.2006.08.002 37. Sohn YB, Kwak MJ, Kim SJ, et al. Correlation of adiponectin receptor expression with cytokines and insulin sensitivity in growth hormone (GH)-treated children with Prader-Willi syndrome and in non-GH-treated obese children. J Clin Endocrinol Metab. 2010; 95(3):1371–1377. PubMed doi:10.1210/jc.2009-1489 38. Timmons BW, Tarnopolsky MA, Snider DP, Bar-Or O. Immunological changes in response to exercise: influence of age, puberty, and gender. Med Sci Sports Exerc. 2006; 38(2):293–304. PubMed doi:10.1249/01. mss.0000183479.90501.a0
PES Vol. 27, No. 4, 2015
534 Duran et al.
42. Vijayaraghava A. K R. Alteration of Interferon Gamma (IFN-gamma) in Human Plasma with Graded Physical Activity. J Clin Diagn Res. 2014; 8(6):BC05–BC07. PubMed 43. Walsh NP, Gleeson M, Shephard RJ, et al. Position statement. Part one: Immune function and exercise. Exerc Immunol Rev. 2011; 17:6–63. PubMed 44. Weiss R, Dziura J, Burgert TS, et al. Obesity and the metabolic syndrome in children and adolescents. N Engl J Med. 2004; 350(23):2362–2374. PubMed doi:10.1056/ NEJMoa031049 45. Wilson AG. Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases. J Periodontol. 2008; 79(8, Suppl.):1514–1519. PubMed doi:10.1902/jop.2008.080172
Downloaded by York Univ Libraries on 09/19/16, Volume 27, Article Number 4
39. Trinh A, Campbell M, Ukoumunne OC, Gerner B, Wake M. Physical activity and 3-year BMI change in overweight and obese children. Pediatrics. 2013; 131(2):e470–e477. PubMed doi:10.1542/peds.2012-1092 40. van Mil EG, Westerterp KR, Gerver WJ, Van Marken Lichtenbelt WD, Kester AD, Saris WH. Body composition in Prader-Willi syndrome compared with nonsyndromal obesity: Relationship to physical activity and growth hormone function. J Pediatr. 2001; 139(5):708–714. PubMed doi:10.1067/mpd.2001.118399 41. Viardot A, Sze L, Purtell L, et al. Prader-Willi syndrome is associated with activation of the innate immune system independently of central adiposity and insulin resistance. J Clin Endocrinol Metab. 2010; 95(7):3392–3399. PubMed doi:10.1210/jc.2009-2492
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