INTRODUCTION

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THE INSULIN-LIKE GROWTH factor (IGF)-I system is composed of IGF-I itself (a 7.6-kDa polypeptide), a family of six binding proteins (BPs) (i.e., BPs 1-6, ranging in size from 22.8 to 31.4 kDa), an acid-labile subunit (ALS; 80-86 kDa), and an IGF protease (19, 37). IGF-I can circulate either unbound (i.e., free) or bound to one of the BPs. The majority (~75%) of IGF-I circulates primarily in a ternary complex (~150 kDa) consisting of IGF-I, insulin-like growth factor binding protein (IGFBP)-3, and ALS. In addition to its well-known mitogenic, anabolic, and cell cycle progression properties, the IGF-I system has also been implicated as playing a role in mediating the growth-promoting actions of physical exercise. For example, two studies have reported a positive relationship between serum IGF-I and aerobic fitness (20, 36). Furthermore, Eliakim et al. (9) reported that 5 days of aerobic training in rats increased muscle IGF-I without corresponding increases in systemic IGF-I or muscle IGF-I mRNA expression. Most recently, Adams et al. (1) confirmed this observation by demonstrating that, during compensatory hypertrophy after overload via unilateral ablation of synergists, muscle IGF-I was elevated without concomitant increases in IGF-I mRNA. The increase in muscle IGF-I without mRNA remains unexplained but could be attributed to a number of potential factors, including IGF-I production from infiltrating immune cells, increased translational efficiency of IGF-I message, or tissue sequestration of IGF-I from the systemic circulation (1, 7, 9, 27, 45).

Resistance exercise is known to dramatically increase protein synthesis, accretion, and muscle hypertrophy (10, 12). Whereas Adams et al. (1) reported that muscle IGF-I is elevated early in the time course of myogenesis in overloaded skeletal muscle, the precise role that IGF-I plays in mediating this process (systemic vs. local) is still poorly defined (12). Even less defined are the responses of the individual components of the circulating IGF-I system (i.e., IGF-I, BPs, and ALS) after resistance exercise stress. A greater understanding of the impact of resistance exercise on the systemic changes in the circulating IGF-I system would further elucidate the current knowledge of endocrinological adaptations to exercise that lead to muscle development (i.e., resistance training). No studies to date have collectively assessed the individual components of the ternary complex (IGF-I, IGFBP-3, ALS), or smaller molecular weight BPs after resistance exercise. Schwarz et al. (40) reported an increase in proteolytic activity after aerobic exercise, demonstrating a dissociation of the ternary complex within the systemic circulation. Additionally, Chadan et al. (6) recently reported that aerobic exercise in the elderly increased immediate postexercise concentrations of IGFBP-2 and -3. These data suggest that acute exercise can modulate IGF-I access to the IGF-I receptor, thus impacting bioavailability.

Only two previous reports have temporally examined circulating IGF-I at multiple time points after acute exercise. Cappon et al. (5) tracked serum total IGF-I every 4 h for 24 h after a series of three high-intensity aerobic bouts. Kraemer et al. (24) also examined serum total IGF-I every 4 h for 12 h on the recovery day after a multiset, total-body resistance workout. Neither study reported a sustained effect of exercise on total IGF-I concentrations. It would also seem worthwhile to employ a prolonged serial sampling scheme to characterize whether other components (i.e., BPs) of the circulating IGF-I system are influenced by the stress imposed by acute exercise. To glean more information on the effect of resistance exercise on the IGF-I system, we examined total IGF-I, free IGF-I, IGFBP-3, IGFBP-2, and ALS every hour for 13 h after a heavy, acute-resistance exercise bout. Based on recent findings from our laboratory that demonstrated that acute, heavy-resistance exercise can alter the temporal pattern of overnight growth hormone (GH) release (B. C. Nindl, W. C. Hymer, D. R. Deaver, and W. J. Kraemer, unpublished observations), we hypothesized that local-resistance exercise would also lead to alterations in the IGF-I system that would be detected in the systemic circulation.

	METHODS

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Subjects. Ten young, healthy, fit men (age: 21.0 ± 2.1 yr, height: 177.1 ± 7.4 cm, weight: 78.6 ± 6.4 kg, body fat: 11.3 ± 4.1%, maximal oxygen uptake: 51 ± 1 ml · kg¹ · min¹) participated in this investigation, which was approved by The Pennsylvania State University's Human Use Institutional Review Board for Use of Human Subjects in Research and the University Park Research Center [General Clinical Research Center (GCRC)] Scientific Review Committees. All subjects were instructed as to the risks of the investigation and subsequently read and signed the institutionally approved informed consent document. Each subject was medically screened by a physician before inclusion in the study. Percent body fat and maximal volume of oxygen consumption were measured by methods previously described (24). Inclusion criteria were age, <25 yr; percent body fat, <20%; maximal oxygen uptake, >45 ml · kg¹ · min¹; and one-repetition maximum (RM) squat strength >1.5 × body mass.

Strength assessment. One RMs were tested for the following exercises: squat, bench press, leg press, and lat pull-down using Universal (Universal Equipment, Omaha, NE) and York Barbell equipment (York Barbell, York, PA). Warm-up consisted of performing 5-10 repetitions at 40-60% perceived maximum, a 3- to 5-min rest and stretching period, and the completion of 3-5 repetitions at 60-80% maximum. Subsequent single lifts were then made to determine the 1 RM with 5-min rests between lifts. An attempt was considered successful when it was completed through a full range of motion without deviating from proper technique and form. The mean ± SD was 135 ± 12 kg for the squat, 110 ± 8 kg for the bench press, 196 ± 11 kg for the leg press, and 84 ± 3 kg for the lat pull-down.

Dietary control. All subjects completed 3-day dietary intake diaries (before each overnight trial). Subjects were asked to replicate, as much as possible, their first 3-day dietary intake for the second 3-day period on their overnight visits. Dietary analyses (Nutritionist IV, First DataBank, San Bruno, CA) of these records verified that the caloric content and composition were similar for the 3 days before each overnight stay. On the day of the subjects' overnight trials, the entire day's meals were provided for the subjects. These meals were prepared by registered dietitians at GCRC and conformed to a "GH-constant diet," which had the following criteria: no caffeine, aspartame, or snacks; macronutrient distribution was 50% carbohydrate, 20% protein, and 30% fat; and sodium was controlled at 3 g. Calories were based on the Harris Benedict standard formula, plus an appropriate activity factor for the subject's age, gender, and physical activity. Meal times were breakfast at 0630, lunch at 1130, and dinner at 1900. The macronutrient breakdown of the dietary intakes and GCRC diets are given in Table 1.

Acute, heavy-resistance exercise protocol. The acute, heavy-resistance exercise protocol (AHREP) was designed to be a high-volume workout that recruited and activated a large amount of muscle tissue. This was accomplished by the performance of multijoint exercises that required the use of major muscle groups in both the lower and upper body (i.e., the squat, leg press, bench press, and lat pull-down). The relative loads for each exercise alternated between 10- and 5-RM loads. The 10- and 5-RM loads were calculated as 70 and 85%, respectively, of the exercise 1 RM. Subjects terminated the exercise set on the completion of the number of repetitions or muscle failure. In the event that the desired number of repetitions (at either 10- or 5-RM loads) was not achieved for a given set, the load was subsequently reduced before the next set of the exercise. A 90-s rest period was given after each exercise, as shown in Table 2. All subjects completed the entire workout. The mean ± SE time for completion of the AHREP was 125.3 ± 3.4 min.

Overnight trials. Subjects underwent two randomized, counterbalanced overnight trials to facilitate familiarization with the facility, and all subjects slept in the GCRC the night before each overnight serial blood draw. Conditions were mimicked exactly to include the taping (but not puncture of the skin) of a catheter near the antecubital vein for the night. One of these overnights served as the control trial, when the subject reported to the GCRC and rested quietly until 1700, whereupon a catheter was inserted into the antecubital vein and serial blood was drawn at a rate of one draw every 10 min from 1700 to 1800 and one draw every hour thereafter until 0600 the following morning. Venous blood sampling followed the same procedures during the exercise condition. During the exercise condition, the subject performed the AHREP from 1500 to 1700. Blood was obtained as soon as possible after the completion of the last set (mean ± SE time after exercise for the first blood draw was 4.3 ± 0.7 min). During each overnight trial, subjects were allowed to move freely, watch television, receive telephone calls, study quietly, and so forth. Bedroom lights were turned off at 2200, and the television was turned off at 2300. Serial blood draws were performed throughout the night every hour. Self-reported measures of sleep quality and minutes until onset of sleep did not differ between the exercise and control trials (data not shown). Blood was collected in glass vacutainers, allowed to clot at room temperature, and centrifuged for 30 min at 800 g at 4°C. After centrifugation, serum was aliquoted into microfuge tubes, flash frozen in liquid nitrogen, and stored at 80°C until later analysis.

IGF-I system assays. All IGF-I system assays were performed with immunoassays (Diagnostic Systems Laboratories, Webster, TX). Radioimmunoassays were performed on a Cobra gamma counter (Packard Instruments, Downers Grove, IL). The enzyme-linked immunoabsorbent assay for ALS was performed with the aid of an automated plate washer (Dynex Technologies, Chantilly, VA), and absorbance was read on a plate reader (Dynex Technologies). All samples for a given subject were run in the same assay batch to eliminate interassay variance. Total IGF-I, free IGF-I, and IGFBP-3 concentrations were determined by using two-site immunoradiometric assays. The sensitivity of the assays was 2.06, 0.03, and 0.5 ng/ml for total IGF-I, free IGF-I, and IGFBP-3, respectively. The intra-assay variances were <5% for total IGF-I, free IGF-I, and IGFBP-3. IGFBP-2 concentrations were determined by a radioimmunoassay. The sensitivity and intra-assay variance for IGFBP-2 were 0.5 ng/ml and <5%, respectively. The sensitivity and intra-assay variance for ALS were 0.7 ng/ml and <5%, respectively.

Statistical analyses. An analysis of variance with repeated measures was used to assess condition (control vs. exercise) and time (i.e., throughout the night) effects. Interaction means were graphed for all variables, whereas marginal main-effect means were tabled for condition comparison. Also, molar volume ratios between selected IGF-I system components were calculated and compared between the exercise and control conditions. For the calculation of molar volume ratios, the following formula was used: 1) moles of protein 1 = [sample volume (µl) · measured concentration · 1 × 10¹²]/ mass of hormone (kDa); 2) moles of protein 2 = [sample volume (µl) · measured concentration · 1 × 10¹²]/ mass of hormone (kDa); 3) molar ratio = moles protein 1/moles of protein 2.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Table 3 shows the marginal means for the control vs. exercise conditions for the first hour post-AHREP. No exercise effects were observed for total and free IGF-I or the ALS. Significant increases after exercise were observed for marginal means for IGFBP-3, whereas IGFBP-2 approached significance (P = 0.07).

Figure 1 shows the 13-h overnight serial measures for total IGF-I (A), free IGF-I (B), IGFBP-3 (C), IGFBP-2 (D), and the ALS (E). Table 4 shows the marginal means for the control vs. exercise conditions for the overnight measures. No exercise effects were observed for total or free IGF-I. Significant exercise increases were evident for IGFBP-2, whereas significant exercise decreases were evident for the ALS.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Overnight circulating concentrations of total insulin-like growth factor I (IGF-I; A), free IGF-I (B), insulin-like growth factor binding protein (IGFBP)-3 (C), IGFBP-2 (D), and acid-labile subunit (ALS; E) from 1800 (hour 1) to 0600 (hour 13) in control and exercise conditions. Values are means ± SD. * For the marginal main-effect means due to condition, see Table 4.

Figure 2 depicts the molar volume ratios of selected components of the IGF-I system for the 13-h overnight sampling period. The ratios of total IGF-I to IGFBP-2 (IGF-I/IGFBP-2), ALS/IGFBP-2, and ALS/IGFBP-3 decreased after exercise. Table 5 lists the marginal main-effect means for the ratios.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Ratio comparison between selected components of the IGF-I system for the control and exercise conditions: total IGF-I/IGFBP-3 (A), total IGF-I/IGFBP-2 (B), total IGF-I/ALS (C), ALS/IGFBP-3 (D), ALS/IGFBP-2 (E), and IGFBP-3/IGFBP-2 (F). Values are means ± SD. * For marginal main-effect means due to condition, see Table 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is the first study to collectively evaluate the individual components of the circulating IGF-I system (i.e., total and free IGF-I, IGFBP-2 and -3, and the ALS) overnight for 13 h after acute, heavy-resistance exercise. The primary findings of this study were as follows: 1) no changes were observed for either total or free IGF-I over the 13-h sampling period; 2) IGFBP-3 increased immediately postexercise, returning to baseline and remaining stable for the 12 h afterward; 3) exercise increased IGFBP-2 concentrations for 13 h postexercise; and 4) exercise decreased the ALS concentrations for 13 h postexercise. These data suggest that the effect that acute, high-volume, heavy-resistance exercise exerts on the circulating IGF-I system is not in the alterations of the amount of IGF-I, but rather in the alteration of the manner in which IGF-I is partitioned among its family of BPs. Thus this study has demonstrated that local-resistance exercise can lead to alterations in the IGF-I axis that are detected in the systemic circulation.

There were no differences between the control and exercise conditions for the first sampling hour immediately postexercise for total or free circulating IGF-I. There is only one previous report in the literature for free IGF-I responses after acute-resistance exercise. In contrast to the findings of this study, Bermon et al. (4) reported an elevation in free IGF-I for older subjects immediately and 6 h postexercise. Previous studies from our laboratory have reported either no increase or transient, inconsistent increases in total IGF-I after different acute-resistance exercise protocols (24-28, 34). The acute protocol in this study was composed of a higher volume (50 sets) and longer duration (~2 h) than previous acute-resistance protocols (30 sets and ~60 min). In contrast, other researchers, using short-duration (~10 min), aerobic-type protocols, have consistently reported postexercise elevations in total IGF-I (5, 38). Schwarz et al. (40) demonstrated that the total IGF-I increase after 10 min of cycling was intensity dependent but unrelated to endogenous GH. The data in the present study support a previous finding from our laboratory in that the large elevation in GH after acute-resistance exercise is not accompanied by IGF-I increases (24). Knowing the phenotypic differences between aerobically and resistance-trained athletes, one may find this paradox counterintuitive, and, on initial consideration, it may appear that the mode of exercise explains the divergent responses observed immediately postexercise. However, an alternative explanation may be that IGF-I elevations temporally appear early in exercise, and, because resistance exercise protocols are typically longer (1 h) in duration, these transient increases have been missed by not sampling during exercise. Preliminary data from another study in our laboratory support this contention, as we have recently observed significant increases in IGF-I after a short-duration (~12 min), acute-resistance exercise protocol (34). Recent findings from Nguyen et al. (31) further illustrate the variable IGF-I response after exercise, as they reported a 12% increase, a 15% decrease, and no change for incremental ergometer cycling exercise, long-distance Nordic ski race, and a treadmill-simulated soccer game, respectively.

For the thirteen 1-h overnight serial samples, no differences in total or free IGF-I were detected between control and exercise conditions. This finding supports earlier reports by Cappon et al. (5), who reported no effect of exercise on circulating IGF-I concentrations over a 24-h period of observation (sampled every 4 h), and by Kraemer et al. (24), who also reported no exercise effect over a 24-h recovery period (sampled every 6 h). It is important to note that all of these protocols elicited significant rises in endogenous GH, and the lack of an IGF-I response clearly illustrates a departure from the classic GH-mediated liver release of IGF-I. Despite some reports of IGF-I increases immediately after acute exercise (5, 40), the available data fail to demonstrate that this is a sustained effect. Interestingly, the literature is also conflicting regarding the effects of chronic training on circulating IGF-I concentrations, with some studies showing a positive effect (10, 23, 36, 38), whereas others show no effect (4, 27, 32, 34, 43). Future efforts are needed to reconcile these disparate results to fully elucidate the influence of mode and duration of exercise on circulating IGF-I.

IGFBP-3 showed a transient, postexercise increase but then returned to baseline and remained stable throughout the night. Other studies have reported a postexercise increase for IGFBP-3 (6, 40, 41), but this study demonstrates that this increase is not sustained for any significant length of time. The ternary complex is known to undergo proteolysis after exercise via an IGFBP-3 protease present in the circulation (11, 14, 19, 29, 37). This results in a decreased affinity of IGFBP-3 for IGF-I in the ternary complex. That there were no overnight differences after exercise in IGFBP-3 can be attributed to the fact that proteolysis of intact IGFBP-3 still results in a lower molecular-weight immunoreactive fragment (11, 19, 37).

Two new findings emerging from this study were that IGFBP-2 remained elevated and ALS decreased throughout the night after resistance exercise. Although Eliakim et al. (8) recently reported an increase in IGFBP-2 after a 5-wk physical exercise intervention in adolescent men, few other data are available examining the effects of exercise on IGFBP-2. In addition to IGFBP-3 proteolysis, our finding of decreased ALS concentrations provides another mechanism whereby the ternary complex could become destabilized. Unfortunately, no other reports are available in the literature for ALS responses after exercise. Taken together, by increased IGFBP-3 proteolysis and lowered ALS concentrations, exercise should make more free IGF-I available. However, from the data in this study, this was clearly not the case. It is conceivable that IGF-I released from the ternary complex either could quickly escape the intravascular space or could bind to one of the smaller molecular-weight BPs, whereby free IGF-I would be kept relatively constant (11, 14, 19, 30, 31, 37). It is tempting to suggest that the increase in IGFBP-2 could act to bind the free IGF-I disassociated from the ternary complex and increase the proportion of IGF-I aggregated in binary complexes. The fact that IGFBPs circulate in molar excess makes this a possible occurrence (11). Alternatively, the free IGF-I findings should be viewed cautiously, as they were not measured with column extraction.

Theoretically, the increase in IGFBP-2 could either augment or inhibit IGF-I action. Allowing more IGF-I to penetrate capillary fenestrations and traffic in the extravascular space toward the IGF-I receptor could potentiate IGF-I action. This model would dictate that tissue-specific regulation of binary-complex proteolysis would act to increase site-specific bioavailability of serum IGFs, depending on local tissue demands. IGFBP-2 has an Arg-Gly-Asp integrin recognition sequence, implying that it can interact with cell surfaces and deliver IGF-I to adjacent IGF-I receptors (19). It is also possible that IGFBPs exert their own direct biological impact (11, 14, 19, 29, 31, 37). On the other hand, the hypothesis that the elevation in IGFBP-2 would work to potentiate IGF-I action must be tempered by the fact that, if the binary complex were not locally degraded, then the tissue effect of IGF-I might actually be inhibited by preventing access of IGF-I to the IGF-I receptor.

Total IGF-I/IGFBP-3 and total IGF-I/ALS have been used as biomarkers for IGF-I bioavailability (2, 44). These ratios have been shown to increase with GH administration (42). These ratios were not altered with acute, heavy-resistance exercise, nor was the molar ratio of total IGF-I/IGFBP-3 altered with 5 mo of swim training (23). Ratios involving ALS (relative to IGFBP-2 and -3) and total IGF-I/IGFBP-2 decreased after exercise. Total IGF-I/IGFBP-2 and total IGF-I/IGFBP-3 have been used as indexes to detect GH doping. Kidman et al. (22) showed that both of these ratios increased several hundredfold after only 3 days of GH administration. The data from the present study suggest that indexes of ALS/BP-2, ALS/BP-3, and total IGF-I/BP-2 may be useful biomarkers of metabolic stress imposed by exercise (2, 14, 33). These ratio indexes could be related to the homeostatic regulation of glucose metabolism. Further studies are needed to determine whether an exercise dose-response relationship exists for changes in these ratios and whether these ratios correlate with protein synthesis and myogenesis.

It cannot be determined from the present data whether the changes observed for circulating ALS and IGFBP-2 are reflective of exercise-mediated anabolic or metabolic effects at the tissue level. IGF-I is locally produced and operates in an autocrine and/or paracrine manner, and it may be that tissue-specific IGF-I is more "reactive" to exercise stress than is circulating IGF-I (43). To this end, Eliakim et al. (9) showed increases in muscle IGF-I but no changes in circulating IGF-I in the rat after 5 days of endurance training, whereas Yan et al. (46) demonstrated increases in rat muscle IGF-I immunoreactivity 4 days after eccentric contractions. Henriksen et al. (16) established that exercise potentiates the action of IGF-I in skeletal muscle for the activation of the glucose transport and system A neutral amino acid transport. Thus, whereas the exercise impact on circulating IGF-I remains equivocal, the data linking exercise and locally produced IGF-I action remain compelling.

In conclusion, this study has provided the first look at how individual components of the IGF-I system respond for 13 h after the perturbation of an acute, heavy-resistance exercise bout. Local resistance exercise can result in changes in the IGF-I axis that are detected in the systemic circulation. Whether these systemic alterations potentiate IGF-I action at the tissue and/or cellular level has yet to be elucidated. These adaptations may be one of the many biological processes occurring in the hormonal milieu that augment muscular development.