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Original Studies |
Medical Department M (R.D., J.O.L.J., J.S.C., A.F.), Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark; Sports Medicine Research Unit (K.H.W.L., M.K.), Bispebjerg Hospital, DK-2400 Copenhagen, Denmark; and Medical Research Laboratories (H.Ø., A.F.), Aarhus University, DK-8000 Aarhus C, Denmark
Address all correspondence and requests for reprints to: Rolf Dall, M.D., Medical Department M, Aarhus Kommunehospital, Norrebrogade 42–44, DK-8000 Aarhus C, Denmark. E-mail: rd{at}dadlnet.dk
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Abstract |
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Our findings do not support the hypothesis that short-term strenuous exercise induces major acute changes in the GH/IGF axis. To what degree the protein anabolic effects of regular exercise are associated with acute alterations in the GH/IGF axis remains unclear.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eight female and eight male rowers from the Danish National Team
were included in the study. Subject characteristics are presented in
Table 1
. Informed consent was obtained
according to the Helsinki 2 declaration, and the study protocol was
approved by the Ethical Committee for Medical Research in Copenhagen
(KF 01–109/97).
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After weighing (Sega 708, Vogel & Halke GmbH, Hamburg, Germany) and determination of body fat by the measurement of skinfolds (Harpenden skinfold caliper; Baty International, Burges Hill, UK) (23), subjects were tested on a rowing ergometer (Concept II, Inc., Morrisville, VT). The test was part of routine tests performed by rowers of the national team, and the protocol consisted of 4 times 5-min submaximal stages with a 1-min break between stages. After the final submaximal stage, subjects were allowed a 10-min rest, after which an all-out test (6 min for males and 7 min for females) was performed. The submaximal stages corresponded to approximately 55, 65, 75, and 85% of VO2 max.
An AMIS 2001 system (Innovision, Odense, Denmark) was used to measure ventilation and expiratory O2 and CO2 concentrations. Before each protocol, the gas analyzers were calibrated using gases of known composition, and the external volume sensor was calibrated using an external syringe of known volume. Heart rate was measured continuously by a heart rate monitor (Polar Sport Tester, Kempele, Finland). VO2 max was chosen as the highest VO2 attained during the test. Subjects were allowed to drink and eat freely.
Blood sampling
Serum. Thirty minutes before the rowing test, a catheter was inserted into a superficial forearm vein. The catheter was kept patent by flushing with 3 mL isotonic NaCl. Sampling was performed 5 min before commencement of the test (baseline); immediately after termination of the test (0); and at 15, 30, 60, 90, and 120 min post exercise. Blood was sampled and allowed to clot for 30 min at room temperature and centrifuged at 5000 rpm for 15 min (at 4 C). Serum was stored at -80 C until analysis.
Blood. Blood lactate was sampled by a micropipette at baseline and 1 min after termination of the test.
Analytical methods
Blood lactate was measured on a YSI-Sport (YSI, Inc., Yellow Springs, OH). Serum albumin was analyzed with a modified bromcresol green binding assay (Hoffman-La Roche Diagnostics GmbH, Mannheim, Germany).
Serum total IGF-I and total IGF-II were measured by an in-house noncompetitive, time-resolved immunofluorometric assay after acid-ethanol extraction of serum, as previously described (24). Serum free IGF-I and IGF-II were measured by ultrafiltration, as previously described (20), and were analyzed at baseline, 0, 60, and 120 min. Serum IGFBP-1 was measured by enzyme-linked immunosorbent assay (Medix Biochemica, Kainainen, Finland). IGFBP-3 was measured by immunoradiometric assay (IRMA), and IGFBP-2 and IGFBP-6 were measured by RIAs (Diagnostic Systems Laboratories, Inc., Webster, TX).
Western ligand blotting (WLB), SDS-PAGE, and ligand blot analysis were performed in serum, according to the method of Hossenlopp et al. (25), as previously described (26). Two microliters of serum was subjected to SDS-PAGE (10% polyacrylamide) under nonreducing conditions. Specificity of the IGFBP-3 and IGFBP-4 bands was supported by competitive coincubation with unlabeled recombinant human IGF-I purchased from Bachem, Budendorf, Switzerland.
The 125I-IGFBP-3 degradation assay was performed as previously described (27). 125I-IGFBP-3 (30,000 cpm) (Diagnostic Systems Laboratories, Inc.) was incubated for 18 h at 37 C. Two microliters of serum from the athletes and controls was subjected to SDS-PAGE as described above. On each gel, serum samples from a healthy nonpregnant subject and term-pregnant woman were used as internal controls. Gels were fixed in a solution of 7% acetic acid, dried, and autoradiographed. The degree of proteolysis was calculated as a ratio of the absorbency of fragmented 125I-IGFBP-3 over the sum of all 125I-IGFBP-3-related optical densities in that lane and was expressed as a percentage.
Autoradiograms from WLB and the IGFBP-3 protease assay were quantified by densitometry using a Shimadzu CS-9001 PC dual-wavelength flying spot scanner (Shimadzu Europa GmbH, Duisburg, Germany). The relative density of the bands was measured as arbitrary absorbency units (AU).
Variables, measured in serum, were calculated with and without correction for changes in plasma volume during and after exercise, except for free IGF-I and IGF-II, which diffuse freely between the fluid compartments. The measured variables were divided by the albumin ratio, calculated as serum albumin at a given time point divided by serum albumin at baseline. All data given in the text are albumin-adjusted unless otherwise specified.
Statistical analyses
ANOVA for repeated measures, approached by general linear modeling, was used to test for a possible time effect during exercise; except for GH levels, for which Friedman’s test was used because GH recordings were not normally distributed. The ANOVA model also included gender as a between-subjects factor to test whether exercise-induced responses were gender-dependent. If the ANOVA test revealed significant changes, post hoc analyses by multiple paired t tests, with Bonferroni correction, were performed, comparing baseline levels with postexercise levels. Post hoc analyses of GH recordings were tested with Wilcoxon signed-ranks tests with Bonferroni correction. A P-value less than 5% was considered significant. Data are presented as mean ± SEM.
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Results |
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At baseline, gender differences were recorded for GH, total IGF-I,
IGFBP-2, IGFBP-3, and IGFBP-4 as shown in Table 2. However, gender did not significantly
influence the exercise response in any of the variables. Therefore, the
results are shown without gender stratification.
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) increased, during
exercise, from a baseline level of 2.6 ± 0.8 µg/L to a peak
level of 12.7 ± 2.7, recorded 15 min after the end of exercise
(Friedman; P < 0.0005 both with and without albumin
correction). Total IGF-I increased during exercise [(ANOVA;
P < 0.0005) 249 ± 17 µg/L (baseline)
vs. 289 ± 22 (t = 0); P <
0.00002], but the increase was not significant when total IGF-I levels
were albumin-adjusted (ANOVA; P < 0.3). Free IGF-I
decreased 25% post exercise with nadir at t = 60 min [(ANOVA;
P < 0.05) 0.77 ± 0.09 µg/L (baseline)
vs. 0.58 ± 0.06 (t = 60); P <
0.012)], which was coincident with the observed IGFBP-1 peak (Table 3). Total IGF-II levels increased
during exercise (ANOVA; P < 0.0005) but not when
albumin-adjusted (ANOVA; P < 0.1) (Fig. 1). Free
IGF-II decreased post exercise (ANOVA P < 0.05).
Post hoc analysis with Bonferroni correction did not reach
statistical significance at any specific time point post exercise
(Table 3). IGFBP-1 levels, with or without albumin correction,
increased during exercise (ANOVA; P < 0.0005), and the
peak was observed 60 min after the end of exercise [4.0 ± 0.8
µg/L (baseline level) vs. 10.2 ± 1.1(t = 60);
P < 0.0006] (Fig. 2).
The increase was significant from 30 min after end of exercise and
lasted through the remaining sampling period. IGFBP-2 levels did not
change significantly during exercise (P < 0.6) (Fig. 2). IGFBP-4, determined by WLB, increased significantly (ANOVA;
P < 0.02), but post hoc analysis did not
achieve significance at any specific time point [20.7 ± 2.5
µg/L (baseline) vs. 28.2 ± 4.1 (end of exercise);
P < 0.2]. After albumin adjustment, no change was
recorded by ANOVA (P < 0.2) (Fig. 2). IGFBP-6 levels
increased after exercise (ANOVA; P < 0.0005), also
when albumin adjusted (ANOVA; P < 0.0005) (Fig. 2).
The peak IGFBP-6 level was observed immediately after exercise
[199 ± 11 µg/L (baseline) vs. 234 ± 17 (end
of exercise); P < 0.006] (Fig. 2). IGFBP-3 increased
during exercise (ANOVA; P < 0.0005), and the peak was
seen at the end of exercise [3900 ± 128 µg/L (baseline)
vs. 4570 ± 156 (end of exercise); P <
0.0005]; but IGFBP-3 levels, adjusted for albumin, did not change
significantly during exercise (ANOVA; P < 0.4) (Fig. 3). Further, IGFBP-3 determined by WLB
(arbitrary units) did not change significantly (ANOVA;
P < 0.07 and P < 0.5 with and without
albumin adjustment, respectively) (Fig. 3). IGFBP-3 protease activity
did not change post exercise (ANOVA; P < 0.5) (Fig. 3). Because this analysis is a ratio between intact IGFBP-3 and
proteolysed IGFBP-3, no albumin adjustment is applied.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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It is well known that exercise induces a rapid (within minutes)
decrease in plasma volume and that the magnitude of this decrease is
related to exercise intensity (29). With high exercise
intensity, plasma volume may decrease by more than 20%, and a
considerable amount of this fluid is taken up by muscle cells
(29). It is difficult to obtain correct estimates for
rapid changes in plasma volume because measurements of hemoglobin,
hematocrit, serum albumin, and total protein are associated with errors
(30). In the present study, changes in plasma volume was
estimated by measuring serum albumin. Clearly, this method is based on
the assumption that albumin does not escape the intravascular
compartment. However, some studies indicate that transcapillary plasma
fluid loss may lead to a simultaneous and significant, mainly
convective, loss of intravascular albumin (31).
Accordingly, the decrease in plasma volume may have been underestimated
in the present study, although a 19% reduction in plasma volume, as
determined by the increase in serum albumin, was found immediately
after termination of exercise. In general, the interpretation of small
increments (
20%) in plasma concentrations in conjunction with
exercise should be performed with caution, because changes of this
magnitude most likely are explained by fluid shifts. It is difficult to
estimate precisely to which degree the components of the GH/IGF axis
are trapped intravascularly. IGFBP-3 and bound IGF-I and IGF-II
(99%) are molecules of the size or greater than albumin and can
readily be albumin corrected. Smaller molecules, as GH (free and bound
to GH-binding protein), IGFBP-1, IGFBP-2, IGFBP-4, and IGFBP-6, may
(to some degree) follow the fluid shift; whereas free IGF-I and free
IGF-II are freely diffusible.
In a study by Schwarz et al. (22), a 44% increase in IGFBP-3 proteolytic activity was reported after 10 min of lasting strenuous exercise in nonathletes, whereas less strenuous exercise induced no IGFBP-3 proteolysis. In the present study, we were unable to detect any change in IGFBP-3 proteolysis in a group of elite athletes during strenuous exercise. Neither could we show any increase in IGFBP-3 (measured by immunoassay) nor a fall in intact IGFBP-3 (by WLB) when comparisons were adjusted for hemodynamic changes. The differences in fitness level between the subjects of the two studies could be of importance for the IGFBP-3 proteolytic activity. Furthermore, in the present study, the subjects were allowed their normal diet during the study day; whereas, in the study by Schwarz et al., the subjects were examined in the fasting state. Dietary effects on the IGF axis, in relation to exercise, have not been investigated in detail, but Cappon et al. (32) showed that intake of a single meal with a high-fat content before exercise caused a 54% reduction of the GH peak value obtained post exercise. Because IGFBP-3 proteolysis is related to catabolic conditions, the shortage of energy supply in the study by Schwarz et al. could theoretically be of importance for the outcome of their results and may explain the workload dependency demonstrated in that study (15, 21, 33). This hypothesis is supported by Davies et al. (16), who found that the degree of IGFBP-3 proteolysis induced by heart surgery was dependent on whether the patients were fasting or nonfasting. If the degree of IGFBP-3 proteolysis present was correlated to the workload per se, a pronounced IGFBP-3 proteolysis would have been expected in the present study as the elite rowers worked to exhaustion.
There is substantial evidence, in the literature, that GH and IGFBP-1 levels increase post exercise (2, 3, 4, 5, 6, 7, 8, 9, 10, 22, 32, 34, 35, 36, 37, 38, 39). In the present study, the GH peak, post exercise, was seen 15 min after the cessation of the exercise period; whereas the IGFBP-1 peak was seen after 60 min. IGFBP-1 is known to be inversely correlated to insulin levels, and a study from Hopkins et al. (12) showed that a high intake of carbohydrate during exercise diminished the IGFBP-1 increase, from a factor 12 to 6. The teleological benefit of these changes could be that elevated GH levels increase lipolysis and elevated IGFBP-1 levels protect against IGF-I induced hypoglycemia post exercise. Actually, we found decreased free IGF-I levels post exercise coincidently with the IGFBP-1 peak. In contrast, during increased IGFBP-3 proteolysis, an increase in free IGF-I levels would be expected, because less IGF-I would be bound to IGFBP-3. The literature is ambiguous about changes in total IGF-I levels during exercise, because some studies have shown increased levels (22, 34, 37), unaltered levels (12, 38, 40), and decreased levels (3, 28, 41). The variability of exercise-induced IGF-I changes is readily explained by the very different exercise protocols used. In the present study, albumin adjusted levels of total IGF-I and total IGF-II were similar at all timepoints, whereas unadjusted total IGF-I and IGF-II levels increased significantly. In the study by Schwarz et al. (22), hematocrit actually changed from 44% to 50%, which is a 14% increase. This hemoconcentration alone will increase the plasma concentration of soluble variables by 25%.
Earlier studies have reported increased IGFBP-2 levels after exercise (10, 28), a finding not supported by the present data. Likewise, we did not detect significant changes in IGFBP-4 levels. Eliakim et al. (42) found unchanged resting IGFBP-4 levels, after 5 weeks of endurance training, in adolescent females; whereas there are no previous reports about IGFBP-4 changes after acute exercise. Finally, we found increased IGFBP-6 levels during exercise, also after albumin correction. IGFBP-6 has a much higher affinity for IGF-II than for IGF-I (43), thereby being a potential regulator of IGF-II bioactivity. Further, IGFBP-6 has recently been investigated in healthy persons and patients, and the most striking finding here was a 2.9-fold increase in patients with nonislet cell tumor-induced hypoglycemia (44). Nonislet cell tumor-induced hypoglycemia is characterized by increased secretion of IGF-II (45); and accordingly, IGFBP-6 could be related to IGF-II regulation. Theoretically, the increase in IGFBP-6 levels may protect against IGF-II-induced hypoglycemia as a parallel to the hypothesis that increased IGFBP-1 levels protect against IGF-I induced hypoglycemia post exercise (3, 34). In the present study, free IGF-II levels decreased by 18% immediately post exercise coincidently with the IGFBP-6 peak.
In conclusion, we were unable to demonstrate any increased IGFBP-3 proteolysis, either measured by a specific IGFBP-3 protease assay or measured by immunoassays and WLB, in response to short-term strenuous exercise. As for total IGF-I, total IGF-II, IGFBP-2, and IGFBP-4 levels, we found no robust change during exercise, whereas a rise in IGFBP-6 was observed. Finally, free IGF-I and IGF-II decreased significantly.
Put together, our findings do not support the hypothesis that post exercise is acutely associated with major changes in the GH/IGF axis. To what degree the protein anabolic effects of regular exercise are regulated by alterations in the GH/IGF system, therefore, remains unclear.
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Acknowledgments |
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Footnotes |
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Received May 26, 2000.
Revised August 10, 2000.
Accepted October 18, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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—, data with
albumin adjustment; GH, P < 0.0005 (Friedman test)
without and with albumin adjustment; IGF-I, P <
0.0005 (ANOVA) without and P < 0.3 with albumin
adjustment; IGF-II, P < 0.0005 (ANOVA) without and
P < 0.1 with albumin adjustment. If the Friedman
test or ANOVA analyses were significant, post hoc
analyses with Bonferroni correction were performed. 
,
Significant without albumin adjustment; *, significant with albumin
adjustment.
