INTRODUCTION

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INSULIN-LIKE GROWTH FACTOR (IGF) I plays an important role in tissue anabolism by causing cell hypertrophy and hyperplasia in various cell types, including skeletal muscle myoblasts and tendon fibroblasts (1, 2, 11, 41). In addition, exercise training per se affects tissue anabolism (29, 30), and may provide general health benefits (6, 33), but exactly how exercise training and tissue anabolism interact and what mechanisms are responsible remain unclear. Although inconsistency exists (22), several cross-sectional studies have found significant positive correlations between fitness level and circulating IGF-I levels (9, 35). Such a correlation indicates that fitness and exercise in healthy, adolescent subjects is associated with increased activity of the IGF system, favoring an anabolic state. In contrast, prospective studies on exercise training and IGF-I have reported decreased circulating IGF-I levels (9, 10, 24, 38), mimicking responses typically found during energy-deficient and catabolic states (10, 24, 38, 39). However, these studies often had a rather short duration (3 days to 5 wk), and one paper hypothesized that the IGF-I adaptation to chronic exercise in fact consisted of a two-phase response, where an initial catabolic-type response was followed by a more chronic anabolic state after prolonged (>5-6 wk) training (10). That prolonged training does result in an increased activity of the IGF system is supported by findings from animal studies demonstrating that longer periods of training (4-9 wk) result in increased IGF-I gene expression in skeletal muscular tissue (44) and in increased circulating IGF-I levels (43). However, it is unclear to what extent the hypothesized two-phase response of IGF-I adaptations also holds true for humans during prolonged physical training.

The IGF-I action is believed in part to be mediated by circulating free IGF-I. During exercise and training, IGF binding protein (IGFBP)-3, through its proteolysis, represents a potent regulator of IGF-I bioactivity that can result in elevated concentrations of free IGF-I (37). Furthermore, increased IGFBP-3 proteolysis has been reported in one study that used an acute bout of exercise (37), whereas in a recent study on acute exercise no increase in IGFBP-3 proteolysis could be demonstrated (7). Independent of these diverse observations on responses to acute exercise, most previous studies have not addressed any potential role for IGFBP-3 proteolysis during a more chronic exposure to exercise and training. Nor has the influence of training on other IGFBPs and IGF-II been fully elucidated.

Accordingly, the aim of the present investigation was to study the regulatory changes in the circulating IGF system, including binding proteins and IGFBP-3 proteolysis, in response to prolonged physical training in young men. It was investigated whether the adaptation consisted of a two-phase response with an early adaptive effect (after 4 wk of training) and a more chronic adaptation (after 11 wk) as proposed by Eliakim et al. in 1998 (10). This was studied in both well-trained and untrained subjects to determine any potential influence of training status on responses. We hypothesized that highly standardized training would result in more extensive changes in the circulating IGF system in untrained subjects because of lower fitness level.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. Seven untrained and twelve well-trained healthy men, previously screened by the military medical board, were chosen from a larger group of conscripts who had volunteered for military duty at the Royal Danish Life Guard training camp. The Royal Danish Life Guard is considered one of the elite troop regiments within the Army, consisting of highly motivated soldiers. The training status of the subjects was determined on the basis of a fitness questionnaire that addressed the weekly amount of physical training during the last 12 mo before the study. The questionnaire also included a subjective assessment of current physical fitness level compared with that of other men at the same age, an assessment that has previously been validated by Washburn et al. (42). Each subject's individual training status was verified by a direct maximal oxygen consumption (O_2 max) determination. Subject characteristics are provided in Table 1. None of the subjects performed any training for 1 wk before the study, none was on any medication, and all were nonsmokers. Informed written consent was obtained from each subject before he was included in the study, which conformed to the Declaration of Helsinki and was approved by the Ethical Committee for medical research in Copenhagen (KF-01-118/99). Participation in the study was blinded toward the military that was responsible for the physical training.

Study design. Fasting serum samples were collected before and after 4 and 11 wk of training. The 11 wk of standardized training consisted of 2-4 h daily training, categorized into four major groups (closed-order drill and marching, general conditioning, military-specific training, and open-order combat training), and each training session was conducted by a training officer. On average, the weekly training included 7 h of marching, 4 h of general conditioning (mostly running), and 9 h of military-specific training. Overall, the physical training primarily consisted of aerobic, lower extremity weight-bearing activities, but for 5-10% of the training time, intensity was aimed to be above the anaerobic threshold. The weekly amount of vigorous physical training was quantified by review of the recruit training schedules and by training reports from the training officers.

O_2 max. O_2 max was determined during the week before training, and it was evaluated during week 11 with an incremental O_2 max test on a motor-driven treadmill. The O_2 max protocol used was designed to have the subjects reach the O_2 max point within 3-7 min. After a 15-min warm-up, the subjects ran at an individually determined constant speed (13-16 km/h) throughout the test. After the first 2 min of the test, the treadmill was elevated 2° every 90-s until the subjects were unable to keep up with the pace. In the present study, all subjects completed the 2° elevation step (3.5 min) with an exercise range of 4.5-6.5 min, ensuring that O_2 max was reached. A criterion to establish that O_2 max was reached was that the respiratory quotient exceeded 1.10. Pulmonary oxygen uptake was measured with the Innovision Amis 2001 mixing chamber, on-line equipment (Odense, Denmark).

Blood sampling. All blood sampling was conducted after an overnight fast between 0630 and 0730 (baseline, week 4, and week 11). In addition, to avoid the effects of the previous training session, the subjects were not exposed to any strenuous exercise on the day before blood sampling, and each subject was ensured a minimum of 14 h of rest. The blood samples were drawn from an antecubital vein of the nondominant arm and were immediately iced and centrifuged at 5,000 rpm for 15 min (at 4°C). The serum was stored at 80°C until analysis.

Analytic methods. Serum total IGF-I and IGF-II were determined after acid-ethanol extraction by using noncompetitive time-resolved monoclonal immunofluorometric assays as previously described (16). Serum free IGF-I and IGF-II were determined by using ultrafiltration by centrifugation (19). Serum IGFBP-1 was determined by an enzyme-linked immunoassay (Medix Biochemica, Kauniainen, Finland). Serum IGFBP-3 was measured by immunoradiometric assay (IRMA) (Diagnostic System Laboratories, Webster, TX).

Statistical analysis. All data are presented as means ± SD. One-way ANOVA for repeated measures, approached by general linear modeling, was used to test for time effect during the training period (baseline, week 4, week 11). If the ANOVA test revealed significant changes, post hoc analyses by Tukey's multiple comparison were used to compare specific pairs of means. To examine whether differences existed between untrained and well-trained subjects, the independent-samples t-test was used (effect of training status) (SPSS Standard Version 11.0). Pearson's correlation was used to address the relationship between changes in free IGF-I and changes in IGFBP-1 and IGFBP-2. An alpha level of <0.05 (2 tailed) was accepted as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The baseline characteristics of the subjects are presented in Table 1. Difference in training amounts, O_2 max, body weight, and body mass index were significant between well-trained and untrained subjects, whereas no significant differences were found between groups with regard to baseline IGF variables (Table 2, Figs. 1 and 2).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Serum total insulin-like growth factor I (IGF-I; A), free IGF-I (B), insulin-like growth factor binding protein (IGFBP)-1 (immunoradiometric assay; C), and IGFBP-2 [Western ligand blotting (WLB); D] in response to 11 wk of training (baseline, week 4, week 11) in 2 groups of young men with different initial training status. Values are means ± SD. Filled bars, well-trained individuals (n = 12); open bars, untrained individuals (n = 7).  Significantly different from baseline value, P < 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   IGFBP-3 (immunoradiometric assay; A), IGFBP-3 (WLB; B) and IGFBP-3 proteolysis (C) in response to 11 wk of training (baseline, week 4, week 11) in 2 groups of young men with different initial training status. Values are means ± SD. Filled bars, well-trained individuals (n = 12); open bars, untrained individuals (n = 7).  Significantly different from baseline value, P < 0.05.

Effect of prolonged training. In response to the training period, untrained individuals significantly improved their O_2 max by 16 ± 4%, whereas O_2 max was unchanged in the well-trained subjects, which resulted in no significant group difference being evident at the end of week 11 (data not presented). The training intervention resulted in significant decreases in both total and free IGF-I in the untrained group from baseline to week 4 [by 14 ± 6% (total IGF-I) and 27 ± 19% (free IGF-I)] and from baseline to week 11 [by 15 ± 9% (total IGF-I) and 23 ± 18% (free IGF-I)] (Fig. 1). In the well-trained group, total IGF-I decreased by 9 ± 11% (P < 0.05) and free IGF-I decreased by 20 ± 24% (P < 0.05) from baseline to week 4 but had returned to baseline levels at week 11. IGFBP-2 increased significantly in the untrained group from baseline to weeks 4 and 11, whereas no significant changes from baseline were observed in well-trained group (Fig. 1). Overall (untrained + well trained), changes in free IGF-I were found to be inversely correlated with changes in IGFBP-1 and IGFBP-2 (0.51 < r < 0.70, P < 0.05). IGFBP-3 (IRMA) and IGFBP-3 (WLB) transiently decreased in the untrained group from baseline to week 4 (P < 0.05) and returned to baseline levels at week 11, whereas no significant changes occurred in the well-trained group (Fig. 2). Compared with baseline, IGFBP-3 proteolysis increased in the untrained group (by 33 ± 26%; P < 0.05) after 4 wk and remained above baseline (by 36 ± 24%; P < 0.05) at 11 wk, whereas IGFBP-3 proteolysis in the well-trained group remained unchanged from baseline to week 11 (Fig. 2). Training caused IGFBP-4 to decrease over the 11-wk period in both the well-trained and untrained groups (Table 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, total IGF-I decreased in both untrained and well-trained subjects after 4 wk of training, which is in contrast to results from a training study on collegiate swimmers that reported an increase in total and free IGF-I levels after 2 mo of training (26). A decrease in total IGF-I is, however, in accordance with other previously published endurance-type training studies (3, 9, 10, 24) and has recently been confirmed in a study on US Army Rangers performing 8 wk of physical training combined with sleep deprivation and relative reduced food intake (15). Findings from these relatively few studies investigating the response of IGF-I to chronic training would be in accordance with the view that both intensity and duration determine the resultant IGF-I levels.

One of the central findings in the present study is that the immonureactive IGFBP-3 levels decreased in untrained subjects from baseline to week 4 and returned to baseline in week 11, whereas no change was observed for well-trained individuals. Other training studies that used different training protocols found either unchanged (3, 10, 31) or increased IGFBP-3 levels (26, 27). To our knowledge, only one study has previously demonstrated that IGFBP-3 levels decreased with 5 wk of training; however, interestingly in that study, a decrease was also observed in the control group that did not perform any training (9). The transient IGFBP-3 decrease observed during prolonged training in the untrained group, but not in the well-trained group, indicates that either initial training status or the relative physiological stress applied during training affected the response. From the present data, it may be suggested that a threshold exists with regard to the relative physiological stress needed during training to result in proteolysis of IGFBP-3. Thus it may be speculated that the standardized training (baseline to week 11) was a sufficient stimulus to cause changes in IGFBP-3 in the untrained subjects, whereas prolonged periods of training with higher intensities would be needed to exceed the threshold in well-trained subjects. Unpublished results from our group indicate that if the intensity is sufficiently high, changes in IGFBP-3 and IGFBP-3 proteolysis do occur in both well-trained and untrained subjects; however, further studies are warranted to fully elucidate this hypothesis. Interestingly, a recent study conducted on soldiers during a 4-day sustained operation was able to confirm the present findings of a decrease in IGFBP-3 in response to intense physical loading (34).

The reduction in IGFBP-3 from baseline to week 4 in the untrained subjects was indicative of IGFBP-3 proteolysis. This was confirmed by the IGFBP-3 proteolysis assay, and it supports the notion that IGFBP-3 proteolysis can be increased in response to intense exercise and training. To our knowledge, there are no other studies investigating IGFBP-3 proteolysis during chronic training, whereas two studies have investigated the proteolytic response to acute exercise. Dall et al. (7) found albumin-adjusted IGFBP-3 proteolysis in elite rowers to be unaffected by 4× 5-min submaximal rowing exercise (55-85% O_2 max) followed by an 6- to 7-min all-out test (7). In contrast, Schwarz et al. (37) found a 10-min high-intensity cycle exercise in nonathletes to increase IGFBP-3 proteolytic activity by 44%. Schwarz et al. did not adjust for shifts in plasma volume, determined by a hematocrit (increase from 44 to 50%), which theoretically would account for at least 25% of the increased concentration in plasma-soluble variables (7). However, the increase in IGFBP-3 proteolytic activity with exercise in the study by Schwarz et al. cannot solely be accounted for by changes in hemoconcentration, and a marked difference in initial training status of the individuals could partly explain the differences in results between the two studies (7, 37).

Serum free IGF-I is markedly affected by changes in levels of IGFBP-1 and -2, which both correlate inversely with free IGF-I (17) (an inverse correlation that was also found in the present study). In addition, free IGF-I may be affected by changes in IGFBP-3 proteolysis, which is believed to represent a compensatory mechanism serving to increase free IGF-I by lowering IGFBP-3 ligand affinity and thereby modifying IGF-I bioavailability by releasing IGF-I from its 150-kDa complex, consisting of IGF-I, intact IGFBP-3, and an acid-labile subunit (36). It has been proposed that exercise-induced IGFBP-3 proteolysis would contribute significantly to the anabolic effects of exercise (36, 37); however, whereas there is little doubt that IGFBP-1 and -2 are potent regulators of free IGF-I levels (18), the physiological significance of IGFBP-3 proteolysis remains to be clarified. In the present study, the IGFBP-3 proteolysis observed in untrained subjects in week 4 and week 11 was not accompanied by any increase in free IGF-I. In contrast, free IGF-I was significantly decreased at all times (Fig. 1). Conditions other than physical training have been shown to be accompanied by a marked increase in IGFBP-3 proteolysis, for example, pregnancy. However, in pregnancy, although a high degree of proteolysis has occurred, still the concomitant increase in levels of free IGF-I is only about twofold (21). IGFBP-3 proteolysis is also observed in patients with chronic renal failure. However, in this condition, serum free IGF-I is markedly reduced, a finding that has been explained by the upregulated levels of IGFBP-1 and -2 (17). Thus it can be argued that changes in IGFBP-1 and -2 have relatively greater impact on levels of free IGF-I than IGFBP-3 proteolysis. This view is supported by the present study, where the upregulated levels of IGFBP-1 and -2 may explain the reduction in free IGF-I (7, 13, 19, 36), despite an increased IGFBP-3 proteolysis.

Total and free IGF-II remained unchanged in both untrained and well-trained subjects during the 11-wk training period, which is in contrast to findings of increased total IGF-II in a 3-mo training study on young subjects with cystic fibrosis (20). This increase in total IGF-II in patients with cystic fibrosis is likely to be due to low baseline levels. In healthy elderly marathon runners, measurements of total and free IGF-II were not different from sedentary age-matched controls, indicating that there is no effect of prolonged training on IGF-II in elderly subjects (8). IGF-II may play an important role in stimulation of bone growth and development (25), but the physiological response of IGF-II to prolonged training is very sparsely reported and the biological importance has yet to be determined (37).

After 4 wk of training, IGFBP-4 had decreased in both well-trained and untrained subjects and remained lower after 11 wk also. IGFBP-4 has consistently been shown to inhibit IGF-I action but has also been shown to be able to undergo proteolytic cleavage, which has been speculated to increase the action of IGF-I (4, 5). Previous studies have shown that IGFBP-4 is degraded only in the presence of exogenous IGFs, but Fowlkes et al. (14) have shown that, in vitro, IGFBP-4 can be degraded in the absence of IGFs, an effect that was almost entirely inhibited by the addition of IGFBP-3 to the medium. Fowlkes et al. therefore speculated that IGFBP-3 has the potential to regulate IGFBP-4 proteolysis in vivo. The interaction between IGFs and IGFBP-3 and -4 is undoubtedly very complex, and even though the probable role is to regulate the IGF interaction at the cell surface, the in vivo physiological role has not yet been elucidated. In a recent paper by van Doorn et al. (40), the clinical relevance of IGFBP-4 in various pathological conditions was examined and the conclusion was that IGFBP-4 might be of minor clinical importance in patients with hyperthyroidism, hypothyroidism, growth hormone deficiency, acromegaly, and chronic renal failure. However, during intense physical training that seems to reflect a systemic catabolic state, proteolysis of IGFBP-4 could have an important compensatory local effect. Therefore, the reduction in IGFBP-4, found in the present study, could have the physiological role of counteracting the concomitant decrease in free and total IGF-I that was seen after 4 wk of training and thereby reduce the catabolic-like early effect of training and maybe even increase a local anabolic effect.

It could be argued that the training regimen used in this study would lead to a general state of tissue catabolism. However, interestingly, it was shown in the same subjects that both degradation and synthesis of collagen type I were increased around the Achilles tendon after 4 wk of training followed by a net synthesis after 11 wk of training (29). This evidently does not allow for any quantitative statements regarding whole body anabolism or catabolism, but at least it indicates that enzymatic degradation of connective tissue primarily is dominant in the early training phase, indicative of a more catabolic state than later during the training period. Such a phenomenon may have been more pronounced in untrained vs. trained subjects, and it could support the view of a two-phased response in the IGF system with regard to prolonged training (7). Finally, it cannot be excluded that local tissue IGF-I levels were increased despite reduced circulating levels, that paracrine or autocrine growth promotion exists, or that increased numbers of cell-surface IGF receptors (or altered sensitivity) could be responsible for more differentiated local anabolic responses (13, 32, 36).

In conclusion, the results from the present study indicate that 11 wk of physical training affect serum levels of total and free IGF-I, IGFBP-2, and IGFBP-3 and affect IGFBP-3 proteolysis differently in untrained and well-trained subjects, with more extensive changes seen in the untrained subjects. Furthermore, the training-induced reduction in IGFBP-3 and concomitant increase in IGFBP-3 proteolysis in previously untrained individuals suggest the existence of a threshold for relative physiological stress to be exceeded in order for IGFBP-3 proteolysis to occur.