Circulating bioactive and immunoreactive IGF-I remain stable in women, despite physical fitness improvements after 8 weeks of resistance,

aerobic, and combined exercise training

Bradley C. Nindl, Joseph A. Alemany, Alexander P. Tuckow, Kevin R. Rarick, Jeffery S. Staab, William J. Kraemer, Carl M. Maresh, Barry A. Spiering, Disa L. Hatfield, Allan Flyvbjerg and Jan Frystyk
J Appl Physiol 109:112-120, 2010. First published 15 April 2010; doi:10.1152/japplphysiol.00025.2010 You might find this additional info useful... This article cites 42 articles, 24 of which can be accessed free at: http://jap.physiology.org/content/109/1/112.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles A novel, noninvasive transdermal fluid sampling methodology: IGF-I measurement following exercise D. E. Scofield, H. L. McClung, J. P. McClung, W. J. Kraemer, K. R. Rarick, J. R. Pierce, G. J. Cloutier, R. A. Fielding, R. W. Matheny, Jr., A. J. Young and B. C. Nindl Am J Physiol Regul Integr Comp Physiol, June , 2011; 300 (6): R1326-R1332. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://jap.physiology.org/content/109/1/112.full.html Additional material and information about Journal of Applied Physiology can be found at: http://www.the-aps.org/publications/jappl

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J Appl Physiol 109: 112120, 2010. First published April 15, 2010; doi:10.1152/japplphysiol.00025.2010.

Circulating bioactive and immunoreactive IGF-I remain stable in women, despite physical tness improvements after 8 weeks of resistance, aerobic, and combined exercise training
Bradley C. Nindl,1 Joseph A. Alemany,1 Alexander P. Tuckow,1 Kevin R. Rarick,1 Jeffery S. Staab,1 William J. Kraemer,2 Carl M. Maresh,2 Barry A. Spiering,2 Disa L. Hateld,2 Allan Flyvbjerg,3 and Jan Frystyk3
Military Performance Division, US Army Research Institute of Environmental Medicine, Natick, Massachusetts; 2Human Performance Laboratory, University of Connecticut, Storrs, Connecticut; and 3Medical Research Laboratories, Clinical Institute of Medicine, Aarhus University Hospital, Aarhus, Denmark
Submitted 13 January 2010; accepted in nal form 14 April 2010
1

Nindl BC, Alemany JA, Tuckow AP, Rarick KR, Staab JS, Kraemer WJ, Maresh CM, Spiering BA, Hateld DL, Flyvbjerg A, Frystyk J. Circulating bioactive and immunoreactive IGF-I remain stable in women, despite physical tness improvements after 8 weeks of resistance, aerobic, and combined exercise training. J Appl Physiol 109: 112120, 2010. First published April 15, 2010; doi:10.1152/japplphysiol.00025.2010.Insulin-like growth factor-I (IGF-I) is regulated by a number of IGF-binding proteins (IGFBPs) and proteases that inuence IGF-I bioactivity. A specic IGF-I kinase receptor activation assay (KIRA) has been developed that determines the ability of IGF-I to activate the IGF-I receptor by quantication of intracellular receptor autophosphorylation on IGF-I binding. KIRA-assessed IGF-I bioactivity has not been utilized within the context of chronic exercise training paradigms. This study measured total and free immunoreactive IGF-I, bioactive IGF-I, and IGFBP-1, -2, and -3 before (Pre), during (Mid), and after (Post) 8 wk of exercise training in young, healthy women, who were randomized into one of four groups: control (n 10), resistance (n 18), aerobic (n 13), and combined (n 15) exercise training. The training programs were effective in improving physical tness specic to the exercise mode engaged in: increases were observed for lean mass ( 2%), aerobic tness (6 7%), and upper (20 24%) and lower (15 48%) body strength (all P values 0.05). By contrast, no time, group, or interaction effects were observed for the circulating IGF-I system, as immunoreactive total (Pre 264 16 g/l; Mid 268 17 g/l; Post 271 17 g/l), free (Pre 0.70 0.1 g/l; Mid 0.63 0.1 g/l; Post 0.63 0.2 g/l) and bioactive (Pre 2.35 0.3 g/l; Mid 2.25 0.3 g/l; Post 2.33 0.3 g/l) IGF-I were unchanged throughout the study. All IGFBP measures were also unchanged. We conclude that increased lean mass, aerobic tness, and upper and lower body strength resulting from an 8-wk exercise training programs can occur without concomitant increases in either circulating bioactive or immunoreactive IGF-I, as well as associated IGFBPs. In terms of reecting positive anabolic neuromuscular outcomes, these data do not support a role for endocrine-derived IGF-I. kinase receptor activation assay; insulin-like growth factor binding proteins; physical training; hormones
INSULIN-LIKE GROWTH factor-I (IGF-I) is an important anabolic and metabolic hormone that is thought to mediate many of the benecial outcomes of physical activity (1, 9, 31). IGF-I is a ubiquitous hormone that can act in both systemic and local

Address for reprint requests and other correspondence: B. C. Nindl, Military Performance Division, The U.S. Army Research Institute of Environmental Medicine, Natick, MA 01760 (e-mail: Bradley.nindl@us.army.mil). 112

modes of action (1, 36). Local (i.e., muscle) IGF-I has been consistently demonstrated to be increased after both acute and chronic exercise, whereas results for circulating IGF-I have been more equivocal (increases, decreases, and no changes have been reported) (1, 8, 10, 18, 27, 29, 30, 35, 37, 39, 40). These disparate ndings for circulating vs. local IGF-I have yet to be fully understood and have led some to question the physiological signicance of circulating IGF-I with regard to training-induced adaptations. It is important to point out, however, that a wealth of scientic data from basic, applied, clinical, and epidemiological approaches have been accumulating, suggesting that the utility of circulating IGF-I may reside in the potential for measured concentrations to provide insight with regard to tness, health, training, metabolic, and disease status (31). For example, elevated IGF-I concentrations have been positively associated with aerobic tness (8), muscle volume (10), bone and tendon health (17, 32, 34), cognitive function (31), and decreased mortality (4). Conversely, not all studies have shown positive associations, and, in fact, elevated IGF-I has also been reported to be a cancer risk factor (31). It is clear that the precise and relative role of systemic vs. locally produced IGF-I in mediating the outcomes of different physical activity modalities are far from fully understood. Adding further ambiguity to the role that IGF-I plays in mediating chronic exercise-induced training adaptations and counterintuitive to IGF-Is anabolic role, a number of studies have actually reported that circulating IGF-I declines during longitudinal exercise training, despite positive neuromuscular and body composition changes (8, 10, 11, 29, 39). Decreased IGF-I concentrations have been observed after training for adolescent boys and girls who experience increased thigh muscle volume (8, 10), end-stage renal disease patients who experience improved strength (29), and soldiers who experience improved aerobic tness (39). Furthermore, in a recent study from our laboratory, where diet, sleep, and physical activity levels were strictly controlled, circulating IGF-I declined 30% during an increase in physical activity over a 7-day period, regardless of whether subjects were in an energy decit (1,000 kcal/day) or in energy balance. This nding leads us to conclude that unexplained, exercise-associated mechanisms, such as increased energy ux, are associated with declines in circulating IGF-I concentrations (38). In an attempt to reconcile this seemingly paradoxical phenomena, Eliakim et al. have postulated that circulating IGF-I exhibits a biphasic response pattern, whereby IGF-I declines
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during the initial stages of increased physical activity, dependent on the nutritional and metabolic demands placed on individuals, and, after a chronic adjustment, circulating IGF-I increases (9). This is an intriguing hypothesis that is partially supported by short-term studies reporting decreases and longer term studies reporting increases (8, 10, 29, 39, 40). However, few studies have actually been able to capture this potential biphasic IGF-I response pattern during physical training programs. Additionally, most studies to date have only examined singular physical training programs, with no studies available in the literature comparing diverse training programs (i.e., resistance, aerobic, and combined physical training) involving contrasting metabolic demands, muscle ber recruitment and contractile patterns, and phenotypic outcomes. Another important aspect of IGF-I biology that has largely been unexplored with regard to longitudinal exercise training is the measurement of IGF-I bioavailability and bioactivity. A family of at least six different binding proteins, as well as various proteases in the systemic circulation, can modify the extent to which IGF-I can interact with cellular IGF-I receptors and exert a biological outcome (7, 11, 12). Traditional immunoassays only provide immunoreactive IGF-I concentrations, which are difcult to interpret with respect to bioactivity, due to the complex interactions of IGF-I with the binding proteins (which can inhibit or stimulate IGF-I-mediated effects). Chen et al. (7) have developed a highly sensitive and IGF-I-specic kinase receptor activation assay (KIRA), which determines the ability of circulating IGF-I to activate the IGF-I receptor by quantication of intracellular receptor autophosphorylation on IGF-I binding. This IGF-I bioassay represents an important tool that can broaden our understanding of the IGF-I system in health and disease. For example, circulating bioactive IGF-I has been shown to be more sensitive than total IGF-I relative to fasting declines (42) and with regard to decreased mortality and cardiovascular disease risk (4). Using an acute exercise model, Kanaley et al. (18) have reported the only data on IGF-I bioactivity in which no changes were observed after 45 min of cycle ergometry exercise. The potential for longitudinal exercise training to alter bioactive IGF-I has not been addressed. We reasoned that an assessment of bioactive IGF-I during chronic exercise training might reveal a response pattern of greater physiological relevance than traditional measurement of immunoreactive IGF-I and could provide new insight with regard to IGF-I biology and exercise. Therefore, the purpose of the present study was twofold: 1) to determine the extent to which an IGF-I bisphasic response pattern could be observed in three 8-wk exercise training programs consisting of different metabolic and neuromuscular demands [resistance, aerobic, and resistance aerobic (combined exercise)], and 2) comprehensively assess aspects of the circulating IGF-I system: total immunoreactive IGF-I, free immunoreactive IGF-I, bioactive IGF-I, IGF-binding proteins (IGFBP)-1, -2, and -3, and the proportion of IGFBP-1 complexed to IGF-I. We hypothesized that a physical training program involving a greater exercise volume and metabolic demand (i.e., combined exercise training) would result in greater perturbations in the IGF-I system compared with singular modes of exercise training (resistance or aerobic), and that IGF-I bioactivity would prove to be superior than immunoreactive IGF-I in reecting training-associated tness improvements.
J Appl Physiol VOL

METHODS

Experimental Design This study was a prospective, between-group design utilizing a control (C) group and matched subject cohorts to reduce experimental variance. Before experimental treatments, 58 women were matched for age, body mass, height, strength, and peak O2 consumption (VO2peak) and randomly assigned to a C group, a resistance exerciseonly (R) group, an aerobic endurance exercise-only (E) group, or a combined resistance and aerobic exercise (CB) group. Each of the three treatment groups (E, R, and CB) trained 3 days/wk for 8 wk, with testing conducted at pre- (Pre), mid- (Mid), and posttraining (Post). These groups allowed for direct comparison of the inuence of each training modality on measured components of the circulating IGF-I system. Each subject was directly supervised by a certied strength and conditioning specialist to ensure program compliance. The data presented herein were part of a larger study examining the effects of training on bone biomarkers (23). Subjects Fifty-eight women from the ages of 18 to 26 yr were recruited from a state university community and completed the study. There were no signicant differences between the experimental groups for descriptive characteristics at baseline (20.2 2.1 yr, 165.3 6.3 cm, 64.2 9.1 kg). Each subject was briefed concerning all potential risks of the experimental protocol, and voluntarily signed an informed consent document approved by the Institutional Review Boards of the US Army Research Institute of Environmental Medicine and the University of Connecticut before participation in the investigation. The study was approved by both the US Army Research Institute of Environmental Medicine and the University of Connecticut Human Use and Review boards. All subjects were required to meet a strict set of inclusion criteria: regularly menstruating, not pregnant, not currently lactating, not participating in any structured physical training program more than two times per week for the preceding 6 mo, and willing to participate in a experimental physical training study requiring a 12-wk commitment. Additionally, all subjects were required to undergo a comprehensive physical exam by a physician to identify and eliminate confounding endocrine, orthopedic, or other adverse pathologies that would endanger the volunteers or impact their response to training. Due to potential inuences of estrogen levels on IGF-I concentrations, all women were tested in approximately the same phase of their menstrual cycles, with blood draws scheduled according to the womens self-reports of their menstrual status. Training Groups E training group (n 13). An outline of the endurance training program is listed in Table 1. Subjects in the E and CB groups performed running-based endurance exercise sessions on 3 alternating days/wk. Endurance sessions were periodized between continuous running and sprint-type interval training on different days. All sessions began with a 5- to 10-min warmup that included light jogging and dynamic range of motion exercises before training and ended with a 5- to 10-min cool down. The weekly training was composed of 20 30 min of continuous running at a prescribed target heart rate of 70 85% maximum heart rate, or interval running consisting of 400-, 800-, 1,200-, and 1,600-m runs conducted close to maximal intensity with a 1:1 recovery time. This program was designed specically to improve 3.2-km run time. R training group (n 18). Subjects in the R and CB groups trained on 3 alternating days, and each session lasted 60 min. A nonlinear periodized model was used in which loads and repetitions were varied on a daily basis over each week. Table 2 contains an outline of the
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Table 1. Program variables for the endurance exercise training program

Week No. Monday Easy SS, Longer Wednesday Interval and TT Friday SS, Threshold

1 2 3 4 5 6 7 8 9 10 11 12 13

Pretesting and familiarization Pretesting and familiarization (2 mi TT) 2030 min SS or jog/walk (100 m jog/100 m walk) 2030 min SS 2030 min SS 30 min total SS, 75% HRmax 30 min total (1 mi alternating straights fast; jog/walk corners) 30 min SS, 8085% HRmax 30 min total SS, 75% HRmax (1 800 m, 1 400 m, 2 200 m) 30 min SS, 8085% HRmax 30 min total SS, 75% HRmax (3 800 m) 30 min SS, 8085% HRmax Midtesting Midtesting 2 mi TT 30 min total SS, 75% HRmax (8 400 m) 30 min SS, 8085% HRmax 30 min total SS, 75% HRmax (1 1 mi, 1 800 m, 2 400 m) 30 min SS, 8085% HRmax 30 min total SS, 75% HRmax 2 1 mi (5 min rest in between) 30 min SS, 8085% HRmax 30 min total SS, 75% HRmax 3 mi SS 2 mi TT Posttesting Posttesting

SS, steady state; TT, time trial; mi, mile; HRmax, intensity of exercise relative to maximal heart rate. All interval workouts used 2-mile TT intensity and had 1:1 work-to-rest ratios between multiple bouts.

resistance training program. Following a 2-wk pretesting and familiarization period, the initial training (weeks 35) consisted of light days using 12-repetition maximum (RM) loads, moderate days using 8- to 10-RM loads, and heavy days using 6- to 8-RM loads. During weeks 6 12, light days utilized 12-RM loads, moderate days utilized 6- to 8-RM loads, and heavy days utilized 3- to 5-RM loads. CB training group (n 15). Subjects in the CB group performed both the aerobic and the resistance exercise programs on the same day, during the same session, and exercised 3 alternating days/wk utilizing the same periodization plan used by the R and E groups. Lightresistance training days always corresponded to the interval sprint days to not limit the intensity of the resistance training session. Resistance training sessions were performed rst and were immediately followed by the aerobic training session. The CB group performed resistance training rst so that lower body resistance exercises were performed in a nonfatigued state to ensure high-quality loading conditions. Individuals in the CB group strictly performed the same prescribed workouts as subjects in the E and R groups (i.e., they still performed the prescribed warmup exercises for the endurance training session, despite having just completed the resistance training session).

In addition, subjects in this group trained at the same time as the respective R and E groups to ensure that they received the same encouragement and coaching. C group (n 10). This group did not undergo any formalized physical training. They completed all testing that the experimental groups performed, while maintaining their current activity levels. Strength Assessments 1 RMs were tested on the squat and bench-press exercises on a MaxRack (Max Rack, Columbus, OH) using previously described methods (23). Warm up consisted of performing 510 repetitions at 40 60% perceived maximum, a 3- to 5-min rest and dynamic stretching period, and the completion of three to ve repetitions at 60 80% maximum. Three to ve subsequent lifts were then made to determine the 1 RM, with a 5-min rest interval between lifts. VO2peak Subjects completed a 4-min warmup period at 8 km/h. If the subjects heart rate was 140 beats/min following 3 min of running, treadmill speed was increased to 8.9 or 9.7 km/h to elicit a heart rate

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Table 2. Program variables within periodized resistance training program

Weeks Acute Program Variables

12 35 Sets, n Reps, n Rest, s Total time, min 69 Sets, n Reps, n Rest, s Total time, min 1011 Sets, n Reps, n Rest, s Total time, min 1213 Exercises

Light 3 12 90 40 Light 3 12 90 40 Light 3 12 90 40 Monday: squat, stiff-leg deadlift, bench press, lat pull down, upright row*, calf exercises, abdominal work

Pretesting and familiarization Moderate 3 810 120 48 Moderate 3 68 150 57 Moderate 3 68 150 57 Posttesting Wednesday: leg press*, stiff-leg deadlift, incline bench press, seated row, shoulder press*, calf exercises, abdominal work

Heavy 3 67 120 47 Heavy 3 35 180 63 Heavy 3 35 150 63 Friday: squat, stiff-leg deadlift, bench, lat pull down, upright row*, calf exercises, abdominal work

n, Number; reps, repetitions; lat, lateral. *During week 6, the following exercises were changed: leg press to deadlift; upright row to high pull; shoulder press to push press. J Appl Physiol VOL
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150 beats/min. Following the warm-up period, treadmill grade was increased to 5% and was increased an additional 2.5% every 2 min. Expired air was measured continuously for oxygen and carbon dioxide concentrations using a calibrated metabolic measurement system (ParvoMedics, Salt Lake City, UT). Measurements were averaged over 20-s periods. The criteria for VO2peak was an increase of 2.0 1 1 mlkg min in oxygen uptake with an increase in workload and respiratory exchange ratio 1.10, or when the subject could no longer maintain the pace. Dual-energy X-ray Absorptiometry Body composition was assessed by whole body scans using a fan-beam densitometer (Prodigy, GE Medical Systems, USA). Total body estimates of percent fat and nonbone fat-free mass were determined using manufacturer-described procedures and supplied algorithms (Total Body Analysis, version 3.6, Lunar, Madison, WI). The coefcient of variation (CV) has been reported to be 1% for fat-free mass and 2% for fat mass with this method (23). Dietary Intake Food frequency, nutrition, and weight history questionnaires were administered during the pre- and posttesting phases to measure dietary change. Five-day food records were also collected on each subject to monitor dietary intake at baseline (week 0), Mid (week 4), and Post (week 9). During baseline screening, all subjects were instructed by registered dieticians on the proper method of recording food intake (19). These educational sessions included examples and illustrations of how to quantify food intake. Food diaries were analyzed for energy and macro/micronutrient content (NUTRITIONIST PRO, version 1.1, First Databank, The Hearst, San Bruno, CA). Body weight was monitored weekly. IGF-I System Assays Blood was drawn via venipuncture at baseline, Mid, and Post. Participants were returned their dietary intake records Mid and Post so that they could eat the same food on the day before each resting blood draw. All blood draws were obtained after a minimum 12-h fast and in the approximate same phase of the menstrual cycle and with at least 48 h after their last exercise session to mitigate any effects from the last bout of exercise. Blood was allowed to sit for 30 min at room temperature and immediately centrifuged at 4C at 2,000 g for 15 min. Serum samples were then

separated and stored at 80C until analysis. Total IGF-I concentrations were measured by an in-house noncompetitive, timeresolved immunouorometric assay (TR-IFMA) after acid-ethanol extraction of serum with mean intra- and interassay CVs of 5 and 10%, respectively (13). Free IGF-I concentrations were determined by an ultraltration method, as described by Frystyk et al. (15, 16). Briey, centrifugation of the serum sample is utilized in coordination with a semipermeable membrane, which allows only free IGF-I to pass through, leaving any IGF-I bound to a binding protein behind. After this ultraltration process, the ultraltrate was analyzed for IGF-I immunoreactivity with a sensitivity of 0.020 g/l and a mean intra- and interassay CVs of 15 and 20%, respectively. Bioactive IGF-I was measured by an IGF-I KIRA based on human embryonic renal cells (EBNA 293) transfected with the human IGF-I receptor (IGF-IR) gene. In this assay, cultured cells were stimulated with either IGF-I standards or unknown serum samples. After 15 min, samples were removed, and the cells were lysed. Then the crude cell lysates were transferred to an assay that detected the concentration of phosphorylated (i.e., activated) IGF-IRs. This assay used a monoclonal antibody against the extracellular IGF-IR for coating and a Europium-labeled monoclonal anti-phosphotyrosine antibody (PY20) as tracer. The sensitivity of the KIRA assay was 0.08 g/l. IGF-II cross-reacts by 12%, and proinsulin, insulin, and insulin analogs by 1% compared with IGF-I. The intra- and interassay CVs were 7 and 15%, respectively. IGFBP-1 was assessed by an in-house RIA, and IGFBP-2 by an in-house TR-IFMA (20). Mean intra- and interassay variances were 6 and 12%, respectively. IGFBP-3 was analyzed by a commercial immunoradiometric assay from Diagnostic Systems Laboratories (Webster, TX). IGFBP-1 associated with IGF-I (binary complex of IGF-I and IGFBP-1) was determined by an in-house TR-IFMA using an IGFBP-1 antibody for coating, and a Europium-labeled IGF-I antibody as tracer (14). Mean intra- and interassay variance was 5 and 15%, respectively. Statistics All data are presented as means SE. Appropriate statistical assumptions for each analysis were tested before evaluation of the data. Descriptive and parametric statistical analyses were performed with STATISITCA 7 for Windows (StatSoft Tulsa, OK). A four (C, R, E, CB) by three (Pre, Mid, Post) analysis of variance (ANOVA) with repeated measures was employed for statistical analyses. When a signicant F-ratio was identied, post hoc comparisons were conducted using Tukeys honestly signicant difference test. Signicance in this investigation was set at P 0.05 for all measurements.

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Table 3. Physical performance data

Pre Mid Post Statistical Effect

1 RM bench press, kg Control Resistance Aerobic Combined 33.8 31.1 32.1 33.8 1.8 1.4 1.6 1.5 33.4 33.1 32.1 36.5 1.8 1.4 1.5 1.4 33.9 38.0 32.8 40.6 2.0 21.5* 2.3* 2.2* Time: P 0.2; Group: P 0.001; Interaction: P 0.001

1 RM back squat, kg Control Resistance Aerobic Combined 61.6 53.8 53.8 57.2 3.5 2.7 3.0 3.0 69.4 68.0 59.8 68.7 3.3 2.5 2.8 2.8 68.2 3.5 80.7 2.7* 61.4 1.0* 77.1 1.1* Peak VO2, ml kg 1 min 38.3 38.9 42.4 41.5 1.4 1.1 1.4* 1.2* Time: P 0.1; Group: P 0.001; Interaction: P 0.001

Control Resistance Aerobic Combined

38.0 38.2 40.0 38.2

1.5 1.1 1.3 1.5

NA NA NA NA

Time: P

0.03; Group: P

0.2; Interaction: P

0.03

Pre, pretraining; Mid, midtraining; Post, posttraining; 1 RM, one-repetition maximum; VO2, O2 consumption; NA, not applicable. *Statistical signicance between Pre and Post values. Statistical signicance between Mid and Post values; Statistical signicance between Pre and Mid values. J Appl Physiol VOL
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RESULTS

BIOACTIVE IGF-I AND EXERCISE TRAINING

IGF-I System Data for total, free, and bioactive IGF-I are presented in Table 4. All groups had similar mean concentrations of total (264.4 16.7 g/l), free (0.7 0.1 g/l), and bioactive (2.4 0.6 g/l) IGF-I concentrations at the pretraining time point. There were no signicant changes in total IGF-I concentrations over time (P 0.4), nor did total IGF-I concentrations vary among the groups over time (P 0.9). Similar statistical occurrences were evident for free and bioactive IGF-I, as concentrations did not vary over the training period, and no signicant interactions occurred for free (P 0.7) or bioactive IGF-I (P 0.9). Total IGF-I signicantly correlated with bioactive IGF-I at Pre (r 0.61, P 0.05), Mid (r 0.49, P 0.05), and Post (r 0.49; P 0.05) time points. Also, free IGF-I signicantly correlated with bioactive IGF-I at Pre (r 0.62, P 0.05), Mid (r 0.52, P 0.05), and Post (r 0.46, P 0.05). Data for the IGFBPs, binary IGFBP-1, and IGFBP-1 saturation are presented in Table 5. There were no statistical differences at baseline among the four groups. There were no signicant group time interactions for IGFBP-1 (P 0.5), IGFBP-2 (P 0.4), IGFBP-3 (P 0.9), binary IGFBP-1 (P 0.7), or IGFBP-1 saturation (P 0.4), in that concentrations of the aforementioned variables were consistent over the training period, among the groups. Similarly, IGFBP concentrations were stable over the training period, as no time effects were observed for mean IGFBP-1 (P 0.2), IGFBP-2 (P 0.8), and IGFBP-3 (P 0.8), binary IGFBP-1 (P 0.2), or IGFBP-1 saturation (P 0.5).
DISCUSSION

Physical Performance All physical performance measurement outcomes are presented in Table 3. A group time interaction occurred for the 1-RM bench press (P 0.001). Both the R and CB groups experienced signicant increases in 1-RM bench-press strength by the end of the study (as compared with their initial values) of 6.3 and 7.0 kg, respectively. There was a signicant interaction of group time for the 1-RM squat (P 0.001). The results suggested that there was no increase in squat 1 RM in the C group, and that there was a progressive increase in squat 1 RM in E ( 7.6 kg), R ( 26.8 kg), and CB ( 19.9 kg) exercise groups. There was no signicant change in VO2peak for the C or R exercise groups. However, a group time inter action (P 0.001) denoted that mean VO2peak was signicantly higher (compared with baseline) by 6% in the E exercise group and 8% in the CB exercise group. Body Composition/Dietary Intake Although there was no signicant interaction of group time for body mass (P 0.5), there was a signicant time effect (P 0.004), indicating a signicant increase ( 1 kg) Pre to Post (Pre: 61.6 1.1; Mid: 61.9 1.8; Post 62.6 1.3 kg). Fat-free mass had similar outcomes to body mass as no interaction occurred (P 0.8), but there was a signicant time effect (P 0.0001). Aggregated across all groups, fat-free mass was signicantly increased ( 0.8 kg) from baseline values (Pre: 41.3 0.6; Mid: 41.8 0.5; Post 42.1 0.7 kg). There were no signicant statistical effects for fat mass. Energy intake was statistically similar across the training program for the C (Pre: 1,874 476; Mid: 1,916 677; Post: 1,911 377 kcal/day), R (Pre: 2,005 412; Mid: 1,928 500; Post: 1,924 428 kcal/day), E (Pre: 1,876 488; Mid: 1,744 516; Post: 1,870 552 kcal/day), and CB (Pre: 1,842 358; Mid: 1,766 404; Post: 1,828 397 kcal/day) groups.

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Whereas all previous circulating IGF-I/exercise training studies in the literature have focused on conventional immunoassays to measure total and free IGF-I, this is the rst study that has assessed IGF-I bioactivity within a longitudinal exercise training paradigm by using a cell-based, in vitro IGF-I bioassay (KIRA) that quanties phosphorylation of tyrosine residues of the activated IGF-I receptor (the requisite prelim-

Table 4. Serum concentrations of IGF-I system components during chronic exercise training
Pre Mid Post Statistical Effect

Total IGF-I Control Resistance Aerobic Combined 268.6 272.3 247.7 269.0 20.4 14.9 17.0 14.8 273.1 283.2 242.6 274.2 21.7 15.8 18.0 15.8 269.0 291.3 250.2 276.1 20.4 15.0 17.0 15.0 Time: P 0.4; Group: P 0.4; Interaction: P 0.9

Bioactive IGF-I Control Resistance Aerobic Combined 2.2 2.5 2.1 2.6 0.3 0.2 0.3 0.2 1.9 2.4 2.1 2.5 0.4 0.3 0.3 0.3 2.0 2.5 2.3 2.5 Free IGF-I Control Resistance Aerobic Combined Values are means assay). 0.7 0.6 0.8 0.7 0.2 0.1 0.1 0.1 0.7 0.5 0.8 0.7 0.1 0.1 0.2 0.1 0.6 0.5 0.6 0.8 0.2 0.2 0.2 0.1 Time: P 0.6; Group: P 0.9; Interaction: P 0.7 0.3 0.3 0.3 0.3 Time: P 0.5; Group: P 0.5; Interaction: P 0.9

SE in g/l. IGF-I, total insulin-like growth factor I; bioactive IGF-I, bioassayable IGF-I (as determined by the kinase receptor activation

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Table 5. Serum concentrations of IGF-binding proteins during chronic exercise training

Pre Mid Post Statistical Effect

IGFBP-1, g/l Control Resistance Aerobic Combined 91.8 78.2 74.5 73.3 15.7 11.4 13.1 11.4 82.3 79.3 59.5 63.3 14.4 10.5 12.0 10.5 84.7 60.0 64.9 70.2 13.9 10.1 11.6 10.1 Time: P 0.2; Group: P 0.6; Interaction: P 0.5

Binary IGFBP-1, g/l Control Resistance Aerobic Combined 14.7 35.2 28.3 20.2 8.8 6.3 3.8 6.2 12.0 30.8 26.8 15.3 8.0 5.6 6.0 5.4 15.8 23.3 26.9 15.4 7.0 4.9 5.2 4.7 Time: P 0.2; Group: P 0.1; Interaction: P 0.7

IGFBP-1 saturation*, % Control Resistance Aerobic Combined 18.1 39.7 29.2 30.4 7.4 5.1 5.6 5.1 14.3 35.2 32.9 25.8 6.9 4.8 5.3 4.8 21.3 36.4 28.6 24.2 IGFBP-2, Control Resistance Aerobic Combined 75.6 123.8 88.5 99.1 28.6 20.9 23.9 20.9 71.6 106.3 97.2 99.4 18.3 13.3 15.2 13.3 69.0 101.8 105.0 94.2 5.5 3.8 4.2 3.8 g/l 18.8 13.7 15.6 13.7 Time: P 0.8; Group: P 0.5; Interaction: P 0.4 Time: P 0.5; Group: P 0.07; Interaction: P 0.4

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IGFBP-3, mg/l Control Resistance Aerobic Combined 4.8 4.8 4.6 4.7 0.3 0.1 0.2 0.2 4.8 4.8 4.6 4.7 0.3 0.2 0.2 0.2 4.7 4.9 4.6 4.7 0.2 0.2 0.2 0.1 Time: P 0.8; Group: P 0.9; Interaction: P 0.9

Values are means SE. IGFBP-1, -2, and -3, insulin-like growth factor binding proteins 1, 2, and 3, respectively. Binary IGFBP-1 represents the total amount of IGF-I that is complexed with IGFBP-1. IGFBP-1 saturation (%) represents the molar ratio of IGF-I to IGFBP-1. *Trend for a main between-group effect (P 0.07) for IGFBP-1 saturation.

inary step in IGF-I-mediated intracellular signal transduction). The KIRA in vitro bioassay has the advantage over traditional immunoassays in that it considers important modifying elements (IGFBPs and proteases) known to inuence the biological activity of IGF-I by quantifying a biological outcome (IGF-I receptor activation) rather than simply an immunological outcome (antibody specicity toward an epitope) (7, 11). This study also measured total and free IGF-I, IGFBP-1, -2, and -3, and the proportion of IGFBP-1 complexed to IGF-I after three separate, 8-wk exercise training programs (resistance, aerobic, and combined exercise) performed on 3 alternating days/wk in young, healthy, previously untrained women. Overall, the training programs were effective in improving physical tness specic to the exercise mode engaged in. Contrary to our study hypotheses that bioactive IGF-I would prove superior to immunoreactive IGF-I in reecting training-associated tness improvements and that the combined training would result in greater perturbations in the IGF-I system, our results indicate that the IGF-I system remained stable over the course of all three training programs. We conclude that increased lean mass ( 2%), aerobic tness (6 7%), and upper (20 24%) and lower (15 48%) body strength resulting from an 8-wk exercise training programs can occur without concomitant increases in resting concentrations of either circulating bioactive or immunoreactive IGF-I, as well as associated IGFBPs. In terms of reecting positive anabolic neuromuscular outcomes, these data support a growing consensus that locally produced IGF-I may be of greater relative importance than endocrine-derived IGF-I.
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Our results differ from other short-term ( 111 wk) exercise training studies that have reported decreases in total IGF-I for such diverse populations as adolescent boys and girls, soldiers, and end-stage renal disease patients (8, 10, 29, 39). Also, our study was not able to conrm Eliakims theory of a biphasic (decrease and then increase) IGF-I response pattern during exercise training (9), at least not when comparing samples collected with 4-wk intervals. Based on the literature and two published studies from our laboratory (29, 38), we had anticipated observing a decrease in IGF-I at some point during the 8 wk of training. It is well known that IGF-I declines during a negative energy decit, often coinciding with weight loss (25, 26, 28 33). Furthermore, using regression analyses, Nemet et al. (25) have suggested that, even under conditions of energy balance, IGF-I declines following short-term physical training. In synthesizing the literature from available studies, Rarick et al. (38) recently postulated that IGF-I declines under highenergy ux conditions (high levels of both energy expenditure and energy intake under conditions of energy balance) by yet unexplained exercise-associated mechanisms. In providing a potential explanation for the lack of change in IGF-I for the present study, we propose that our young, healthy subjects were training under low-to-moderate energy ux conditions. The 3 alternating days/wk of the training programs were well tolerated by the subjects, as the training was progressive in nature, allowed for adequate recovery between sessions, ranged in total duration from 20 90 min/session, and ranged in estimated caloric expenditure of 300 600 kcal/session. As evidenced by body composition changes (signicant 1.6 and
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1.9% increases for body mass and lean mass, respectively) and diet records (both caloric and macronutrient intake from selfreported diet records were similar throughout training), our subjects were in a slight positive energy balance. The weekly training volume in the present study was signicantly less than those above-mentioned studies by Eliakim et al. (8, 10) and Rosendahl et al. (39), in which training was conducted 5 days/wk, ranging from 90 to 240 min/workout, depending upon the study. Hence, other training studies of similar length to our study, which have reported a decline in IGF-I, have had total training durations/volumes 230 times higher than those of the present study. It is, therefore, suggested that exercise training volume, especially as it related to individuals tness level, training background, and nutritional status, is an important determinant of the IGF-I response to chronic training. The underlying physiological mechanisms whereby exercise volume would exceed an individuals metabolic homeostatic capacity and perturb the IGF-I system remain unknown. Interestingly, Lavoie (21) has speculated that, during exercise, the energetic ux within the hepatocyte (changes in the ATP-to-Pi ratio) may allow the liver to act as an afferent organ on the central nervous system, contributing to endocrine regulation. In support of this hypothesis, Lavoie et al. (22) have demonstrated that exercise-induced IGFBP-1 increases are linked to liver glycogen depletion. Future studies might be well served by considering that changes in the circulating IGF-I system during exercise offer greater insight into global metabolic demands, rather than local anabolic processes. Studies reporting changes in IGF-I concentrations also typically demonstrate changes in IGFBPs. Variations in IGFBPs will inuence the extent to which IGF-I is complexed among its free, binary, and ternary molecular isoforms, ux from the circulation to cellular receptors, and ultimately its bioactivity (11, 12, 30, 31). The lack of changes in IGFBPs in this study is consistent with the lack of changes for bioactive IGF-I. One of the major advantages for the KIRA is that it considers the modifying actions of IGFBPs in the quantication of bioactive IGF-I. In validation of the assay, Chen et al. (7) reported that the addition of IGFBPs dose-dependently reduced the KIRA signal. During fasting, Chen et al. (6) have also reported that the signicant elevations in IGFBP-1 and -2 correspond with bioactive IGF-I declines and precede immunoreactive IGF-I declines. Although IGFBP-1 was unchanged in this longitudinal training study, we have recently reported that IGFBP-1 was a sensitive biomarker for assessing the physiological strain of acute physical exercise, with increases perceptible for up to 24 h (27). In the only other study to report KIRA assessed IGF-I bioactivity, but using acute exercise (45 min of cycle ergometry at the lactate threshold), Kanaley et al. (18) reported that signicant increases in IGFBP-1 and -2 were associated with a trend (P 0.065) toward decreased IGF-I bioactivity. Other studies in the literature reporting acute exercise responses for immunoreactive IGF-I (2, 11, 18, 27, 30) have been equivocal with increases, decreases, and no changes being observed. While our present data do not support an argument for the use of bioactive IGF-I over immunoreactive IGF-I in providing additional insight into exercise-associated adaptations, other observations have suggested a utility for measures of bioavailable (i.e., free) and bioactive IGF-I. Measures of immunoreactive IGF-I in
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the present study only explained 25% of the variance for measurement of bioactive IGF-I. These relationships are essentially the same as reported by Brugts et al. (5) in a study comparing IGF-I measures in a population of 426 healthy individuals (310 men and 116 women) aged 18 79 yr. That study suggested that the IGF-I KIRA does, in fact, provide information on a different dimension about the IGF-I system. In another intriguing epidemiological report, Brugts et al. (4) observed that elevated IGF-I bioactivity in elderly men was associated with decreased mortality and reduced cardiovascular disease. In other studies involving situations (i.e., aging and military operational stress) where growth hormone secretion is known to be altered (decreases in aging and increases in acute military stress) (31, 33), measures of immunoreactive free IGF-I have been associated with better cognitive function in older men and related to fat-free mass losses during military operational stress. Thus it appears the potential for measures of bioactive/free IGF-I to offer different information than provided for immunoreactive IGF-I occur during situations where there is either an alteration in GH secretion or dynamic changes in IGFBPs. The precise physiological importance of this information remains to be claried. While both circulating and local IGF-I clearly have anabolic actions, it is difcult to ignore the mounting experimental evidence marginalizing the importance of circulating IGF-I in mediating local muscle hypertrophy/remodeling. For example, the transgenic liver-IGF-I-decient mouse, in which there is an 80% reduction in circulating IGF-I, have been shown to have normal body weight and growth phenotypes (43). Recently, Matheny et al. (24) conducted a 16-wk training study in the liver-IGF-I-decient mouse and reported a doubling in strength, muscle mass, IGF-I mRNA, and IGF-I receptor phosphorylation. Furthermore, Spangenburg et al. (41) have experimentally demonstrated that the IGF-I receptor is not even necessary for load-induced skeletal muscle hypertrophy. With regard to local mechanisms and the fact that exercise has been reported to increase IGF-I receptor phosphorylation (24), it might be prudent to measure bioactive IGF-I in muscle interstitial uid using microdialysis techniques (2, 3, 17) to determine the extent to which local anabolism might be reected. In summary, in young, healthy women who participated in either resistance, aerobic, or combined training on 3 alternating days/wk for 8 wk under low-to-moderate exercise volume/ energy ux conditions, we were able to demonstrate favorable body composition, strength, and aerobic tness adaptations without changes in the circulating IGF system. Based on the present data and previous studies, we conclude that chronic exercise-induced changes in the circulating IGF system typically occur under conditions of increased exercise volume/ energy ux and are indicative of global metabolic demands, rather than local anabolic processes.
ACKNOWLEDGMENTS The authors thank the volunteers from the University of Connecticut for their time and dedication. The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as ofcial or as reecting the views of the Army or the Department of Defense. Citations of commercial organizations and trade names in this report report do not constitute an ofcial Department www.jap.org

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BIOACTIVE IGF-I AND EXERCISE TRAINING of the Army endorsement or approval of the products or services of these organizations. 19. GRANTS This research was supported by a grant from the US Army Medical Research and Material Command Bone Health and Military Medical Readiness Research Program to BCN. DISCLOSURES No conicts of interest, nancial or otherwise, are declared by the author(s). REFERENCES 1. Adams GR. Invited Review: Autocrine/paracrine IGF-I and skeletal muscle adaptation. J Appl Physiol 93: 1159 1167, 2002. 2. Berg U, Bang P. Exercise and circulating insulin-like growth factor-I. Horm Res 62: 50 58, 2004. 3. Berg U, Gustafsson T, Sundberg CJ, Kaijser L, Carlsson-Skwirut C, Bang P. Interstitial uid in exercising skeletal muscle in women. Eur J Endocrinol 157: 427435, 2007. 4. Brugts MP, van Beld W, Hoand LJ, van der Wansem K, van Koetsveld PM, Frystyk J, Lambert SWJ, Janssen JAMJL. Low circulating insulin-like growth factor-I bioactivity in elderly men is associated with increases mortality. J Clin Endocrinol Metab 93: 25152522, 2008. 5. Brugts MP, Ranke MB, Hoand LJ, van der Wansem K, Weber K, Frystyk J, Lamberts SWJ, Janssen JAMJL. Normal values of circulating insulin-like growth factor-I bioactivity in the healthy population: comparson with ve widely used IGF-I immunoassays. J Clin Endocrinol Metab 93: 2539 2545, 2008. 6. Chen J, Hojlund K, Beck-Nielsen H, Christiansen JS, Orskov H, Frystyk J. Free rather than total circulating insulin-like growth factor-I determines the feedback on growth hormone release in normal subjects. J Clin Endocrinol Metab 90: 366 371, 2005. 7. Chen J, Ledet T, Orskov H, Jessen N, Lund S, Whittaker J, De Meyts P, Larsen MB, Christiansen JS, Frystyk J. A highly sensitive and specic assay for determiniation of IGF-I bioactivity in human serum. Am J Physiol Endocrinol Metab 284: E1149 E1155, 2003. 8. Eliakim A, Brasel JA, Mohan S, Wong WLT, Cooper DM. Increased physical activity and the growth hormone-IGF-I axis in adolescent males. Am J Physiol 44: R308 R314, 1998. 9. Eliakim A, Nemet D, Cooper DM. Exercise, training, and the GH-IGF-I axis. In: The Endocrine System in Sports and Exercise, edited by WJ Kraemer and AD Rogol. Malden, MA: Blackwell, 2005, p. 165179. 10. Eliakim A, Scheett TP, Newcomb R, Mohan S, Cooper DM. Fitness, training, and the growth hormone/insulin-like growth factor-I axis in prepubertal girls. J Clin Endocrinol Metab 86: 27972802, 2001. 11. Frystyk J. Exercise and the growth hormone-insulin-like growth factor-I axis. Med Sci Sports Exerc 42: 58 66, 2010. 12. Frystyk J. Utility of free IGF-I measurements. Pituitary 10: 181187, 2007. 13. Frystyk J, Dinesen B, Orskov H. Non-competitive time-resolved immunouorometric assays for determination of human insulin-like growth factor I and II. Growth Regul 5: 169 176, 1995. 14. Frystyk J, Hojlund K, Rasmussen KN, Jorgensen SP, Wildner-Christensen M, Orskov H. Development and clinical evaluation of a novel immune-assay for the binary complex of insulin-like growth factor-I and IGF-binding protein-1 in human serum. J Clin Endocrinol Metab 87: 260 266, 2002. 15. Frystyk J, Ivarsen P, Stoving RK, Dall R, Bek T, Hagen C, Orskov H. Determination of free insulin-like growth factor-I in human serum: comparison of ultraltration and direct immunoradiometric assay. Growth Horm IGF Res 11: 117127, 2001. 16. Frystyk Skjaerbaek JC, Dinesen B, Orskov H. Free insulin-like growth factors (IGF-I and IGF-II) in human serum. FEBS Lett 348: 185191, 1994. 17. Hansen M, Koskinen SO, Petersen SG, Doessing S, Frystyk J, Flyvbjerg A, Westh E, Magnusson SP, Kjaer M, Langberg H. Ethinyl oestradiol administration in women suppress synthesis of collagen in response to exercise. J Physiol 586: 300530016, 2008. 18. Kanaley JA, Frystyk J, Moller N, Dall R, Chen JW, Nielsen SC, Christiansen JS, Jorgensen JOL, Flyvberg A. The effect of submaximal exercise J Appl Physiol VOL 20.

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