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Medical Department M (Endocrinology and Diabetes) (E.T.V., R.D., J.S.C., J.O.L.J.), Aarhus University Hospital, Dk-8000 Aarhus C, Denmark; Department of Anesthesiology (K.H.W.L.), Hillerod Hospital, Dk-3400 Hillerod, Denmark; and Institute of Sports Medicine (M.K.), Bispebjerg Hospital, Dk-2400 Copenhagen NV, Denmark
Address all correspondence and requests for reprints to: Esben Thyssen Vestergaard, Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital, Dk-8000 Aarhus C, Denmark. E-mail: etv{at}dadlnet.dk.
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Abstract |
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Objective: The objective of this study was to characterize systemic ghrelin levels after exercise with and without concomitant GH administration.
Design, Participants, and Intervention: Group A: Twenty-nine elite athletes (age, 18–37 yr) were studied after a maximal exercise test. Group B: In a double blind, placebo-controlled, parallel study, 32 healthy subjects (age, 18–33 yr) were randomized to placebo, GH 0.1 IU/kg per day, or GH 0.2 IU/kg per day for 4 wk. These subjects performed a multistage fitness test to assess maximum oxygen uptake at baseline and after 4 wk. We measured total circulating ghrelin levels before and immediately after exercise and at 15, 30, 60, 90, and 120 min after exercise.
Results: Group A: Serum ghrelin levels after exercise decreased significantly (P < 0.01). Group B: Exercise at baseline was associated with a significant lowering of ghrelin levels after exercise (P < 0.0001). In addition, 4 wk of high-dose GH were followed by a further approximately 20% reduction in basal and after exercise serum ghrelin (micrograms per liter): 0.78 (range 0.52–1.17) vs. 0.63 (range 0.50–0.91), P < 0.05.
Conclusions: 1) Ghrelin levels decrease significantly after exercise in elite athletes and healthy subjects. 2) High-dose GH suppresses ghrelin levels. 3) These data support the hypothesis that GH feedback inhibits ghrelin secretion.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Whereas ghrelin administration in humans elicits a pronounced increase in pituitary GH secretion, a stimulatory effect of gut-derived ghrelin on GH release remains to be convincingly demonstrated. There is more evidence in support of a moderate suppressive effect of GH on ghrelin levels based on data in acromegaly (8, 9) and from patients with GH deficiency (10, 11, 12). Ghrelin in serum is also suppressed by systemic exposure to somatostatin (13).
Physical exercise of even moderate intensity stimulates GH release after a lag phase of 10–20 min, and GH levels usually remain elevated for at least 1 h after exercise (14). In a study comprising eight patients with GH deficiency undergoing submaximal aerobic exercise for 45 min with and without concomitant exposure to an iv GH bolus, we did not detect significant alterations in ghrelin levels nor did we detect ghrelin changes in healthy control subjects who exercised without GH administration (12). However, we did record significantly lower mean ghrelin levels when the patients were exposed to GH, and there was a trend towards lower ghrelin levels after exercise in all groups. Disclosure of significant relations between serum ghrelin (s-ghrelin) levels and GH in relation to exercise is not only of physiological interest, but could have implications for detection of GH abuse in athletes.
We hypothesized that circulating ghrelin is suppressed by the exercise-induced GH release. Thus, in the present study, ghrelin levels were studied in relation to exercise with and without GH administration in healthy and fit subjects.
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Subjects and Methods |
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This study was part of the international GH-2000 project and concerns analysis of samples from the Danish study population.
Group A.
A total of 29 elite athletes (21 males and 8 females) age 24.5 ± 0.8 (range 18–37) yr practicing one of four different kinds of sport at a national or international level (long-distance cycling, sprint cycling, rowing, and weight lifting) participated in the study. Clinical characteristics are shown in Table 1
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. Subjects were fit, but not athletes at a competitive level, and exercising at least twice a week for a minimum of 1 yr duration. Women were studied in different phases of their menstrual cycle, but data are paired, and the interval between the two exercise tests (28 d) was approximately the length of a normal menstrual cycle.
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The study was approved by the local ethics committee and was performed according to Good Clinical Practice guidelines.
Study protocol
Group A. The participants were examined as previously described (15). Sampling was performed after a maximal exercise test performed under laboratory conditions after a standardized maximum oxygen uptake (VO2-max) test. The athletes were tested in different ways according to type of sport.
Rowing test. Rowers were tested on a Concept II rowing ergometer (Concept II, Inc., Morrisville, VT). The protocol consisted of four times 5-min submaximal stages with a 1-min break between the stages. After the final submaximal stage, the 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 roughly to 55, 65, 75, and 85% of VO2-max, respectively.
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 commercial gases of known volume. The highest VO2 attained during the test was registered as the VO2-max value. Heart rate (HR) was measured continuously by a HR monitor (Polar Sport tester; Polar Electro Oy, Kempele, Finland).
Cycling test (long distance). Long-distance cyclists were tested on a biking ergometer (Ciclotraining Olympionic; Politecnica 80, Padova, Italy). The protocol consisted of four 5-min submaximal stages as described in the rowing test. After a 10-min rest, a 5-min all-out test was performed. HR and VO2 were measured as previously described.
Cycling test (sprint). Sprinters were tested on a biking ergometer (Monark, Vansbro, Sweden). The protocol consisted of three 15-second all-out sprints separated by 15-min rest.
Weight-lifting test. The weight-lifting tests were performed on a contact carpet (Newtest, Oulu, Finland). The protocol consisted of five sets of three maximal jumps on the carpet: 1) squat jump, 2) counter movement jump, 3) counter movement jump with 50% body weight overload, 4) counter movement jump with 100% body weight overload, and 5) counter movement jump. Counter movement is a concentric dynamic muscle contraction that is preceded by an eccentric stretching of the muscle. The subject starts from an upright position.
A forearm vein was cannulated 30 min before the test, and a baseline sample was drawn immediately before the start of the test. Samples were taken at the end of the test and thereafter at 15, 30, 60, 90, and 120 min after exercise. During the 2-h sampling period neither exercise nor food intake was allowed, and according to the protocol, a maximum intake of 0.2 liter water was allowed.
Group B. Each subject was examined on two occasions: d 0 and 28. On d 0 subjects underwent blood sampling for routine biochemistry and assessment of body composition by bioelectrical impedance (16). At 0900 h, each subject exercised to exhaustion in a multiple fitness test (MFT), and VO2-max was calculated from validated performance tables for the test (17). Blood was sampled before and after the MFT. Results of blood samples drawn before the MFT are referred to as basal values, whereas blood samples drawn after the MFT are referred to as postexercise values. Blood was drawn immediately after the MFT (t = 0) and at t = 15, 60, 90, and 120 min for measurement of ghrelin levels.
After the initial MFT, the subjects were randomized to a 28-d treatment period with either placebo ("placebo group," PL), 0.1 IU/kg/d GH (Norditropin Novo Nordisk, Copenhagen, Denmark; "low dose," LD), or 0.2 IU/kg/d GH ("high dose," HD) by means of sc self injections at approximately 2200 h. To minimize side effects, only 50% of the target dose was given during the first week. On d 28, another MFT was performed in all subjects.
Assays
S-ghrelin was measured with an in-house assay, which uses a polyclonal antibody and measures both octanoylated and des-octanoylated ghrelin (18). We have ascertained that ghrelin concentrations in serum samples remain stable during up to 11 freeze-thaw cycles (19). Samples were collected within a limited time span, stored at –80 C, and analyzed together. Hence, any potential degradation is expected to affect all samples equally. Serum IGF-I was determined after acid-ethanol extraction using a commercial assay (Nichols Institute Diagnostics, San Clemente, CA). Serum GH was measured by an immunoradiometric assay (Pharmacia Biotech, Uppsala, Sweden).
Statistical analysis
Results are expressed as mean ± SE or geometric mean and range. Ghrelin was log transformed (base e, indicated by ln) to obtain normality. The trapezoidal rule was used to estimate area under the concentration curve (AUC) levels for the 120-min postexercise total ghrelin levels. To account for interindividual differences in baseline values, the impact of GH treatment on postexercise ghrelin levels was analyzed in terms of relative changes. Comparisons between groups were carried out with one-way ANOVA (study group B). Effect of the time course on serum concentrations were made by univariate ANOVA for repeated measurements with Bonferroni correction. In study group A, the between-subject effect of sex and sport category was estimated by univariate ANOVA for repeated measurements with time as within subject variable. Correlations were calculated by using Pearson’s linear regression coefficient. A P value < 0.05 was chosen as level of significance. The number of subjects is indicated by n. All analysis was performed using SPSS version 13.0 for Windows.
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Results |
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Baseline characteristics of the two study groups are provided in Tables 1
and 2. In group B, no significant differences were observed regarding the distribution of age, height, weight, body mass index (BMI), or body fat (BF) between the three subgroups.
Baseline results
Group B.
Unexpectedly, the baseline ghrelin levels differed significantly between the three subgroups, which was not attributed to a gender difference [ghrelin (micrograms per liter): 0.64 (0.44–1.17) (males) vs. 0.63 (0.40–1.14) (females) (not significant, NS) (Fig. 1)], P = 0.02.
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Basal GH as well as IGF-I levels were comparable between groups on d 0 (GH (milliunits per liter): PL 4.7 ± 2.1 vs. LD 6.7 ± 2.9 vs. HD 2.0 ± 1.6, NS; IGF-I (micrograms per liter): PL 291.1 ± 16.5 vs. LD 374.9 ± 40.8 vs. HD 299.0 ± 21.2, NS). No correlation was evident between ghrelin levels and either BMI or total BF (data not shown).
Ghrelin response to exercise
Group A.
Both raw and albumin-corrected s-ghrelin levels decreased significantly after exercise (Fig. 2A; P < 0.01). We observed no significant interaction of either sport category or of sex.
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). This postexercise decrease in ghrelin became highly significant when pooling the values from the three groups on d 0 (Fig. 2B, P < 0.0001). The increase in GH levels preceded the decrease in ghrelin levels; GH levels peaked with a lag phase of 15 min, whereas ghrelin levels reached nadir levels with a lag phase of 30 min.
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Basal ghrelin levels on d 0 and 28 were similar in the PL and LD groups, whereas a significant decrease on d 28 was observed after high-dose GH treatment (Fig. 1, P = 0.04).
The postexercise s-ghrelin AUC levels were comparable on d 0 and 28 in the PL group (in minutes times micrograms per liter) (164.1 ± 23.3 vs. 149.8 ± 18.4, NS) (Fig. 3A) and the LD group (195.2 ± 12.8 vs. 177.6 ± 10.8, NS) (Fig. 3B). In contrast, ghrelin AUC levels were significantly reduced after 28 d of high-dose GH administration (206.1 ± 41.1 vs. 155.2 ± 32.9, P = 0.02) (Fig. 3C).
The effect of GH administration was assessed by comparison of the relative changes in postexercise ghrelin levels and revealed a significant decrease in the HD group, P = 0.04 (Fig. 3D).
BMI and BF were both comparable on d 0 and 28 (Tables 2 and 3).
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We tested correlations between the changes in IGF-I and changes in both basal as well as postexercise s-ghrelin levels to determine whether any association exists between the increases in IGF-I levels and decrease in ghrelin levels after GH administration (group B). 
IGF-I levels inversely correlated with basal -ghrelin (Fig. 4) and postexercise ghrelin AUC levels (r = 0.54; P = 0.09 (PL) and r = –0.59; P = 0.03 (GH-treated groups). Moreover, correlation analysis of the excursions of GH and ghrelin levels (AUC from t = 0 to t = 30 min) on d 0, group B, after exercise revealed an inverse relationship r = –0.35, P = 0.05. We found no correlation between changes in cortisol and ghrelin levels (data not shown). No correlation was revealed between the changes in BMI and ghrelin between d 0 and 28.
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Discussion |
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We have previously observed in a smaller sample that the exercise-induced GH increase was not preceded by an elevation in circulating ghrelin levels (12). On the contrary, the present data support the hypothesis that the exercise-induced increase in GH levels feedback inhibits ghrelin secretion. In fact, ghrelin levels decreased 21 and 34% after exercise in the elite athletes and the healthy subjects, respectively. This decrease is comparable to that observed postprandially (20, 21). Exercise per se results in a decrease in plasma volume. We estimated plasma volume changes by measuring serum albumin concentrations and found a decrease in plasma volume by approximately 16% (group A, data not shown). Therefore, the conclusions drawn by observations of small changes in plasma concentrations (
20%) during exercise should be interpreted cautiously (22). The observed s-ghrelin changes could, in part, be attributable to hemoconcentration, but the decrease prevailed after correction for fluid shifts, and in group B, we observed an even more robust decrease in circulating ghrelin after exercise.
The notion that ghrelin is suppressed by the exercise-induced GH release, is supported by our observation of a further suppression of basal as well as postexercise ghrelin levels by high-dose GH administration, and the negative correlation between changes in IGF-I and ghrelin levels. Our results are also in line with a recent report (23) in which ghrelin levels were lower in participants who reported more frequent exercise. In the absence of exogenous GH administration, exercise result in an approximately 10- to 15-fold increase in circulating GH with a minor increase in circulating IGF-I (24), which at first glance, contrasts the more moderate changes in circulating GH concentrations (3-fold increase) after exogenous high-dose administration of GH. Nevertheless, the concomitant marked rise in circulating IGF-I (by 3-fold) indicates that GH levels are persistently elevated well above physiological concentrations. Even though the two conditions thus differ in terms of GH patterns, the data support that GH may contribute to the exercise-induced decrease in ghrelin.
Besides being a potent GH secretagogue (1), ghrelin is considered the only peripheral orexigenic hormone. Preprandial ghrelin surges and postprandial troughs suggest that ghrelin is a meal initiator (20). In rodent models, ghrelin partitions metabolism towards the use of glucose rather than fat (25). Furthermore, ghrelin decreases locomotor activity (26), thus appearing to be involved in the conservation of energy expenditure. In human studies, ghrelin enhances both appetite and energy intake (5, 7). Ghrelin has been linked to reduced insulin sensitivity (27) and increased fasting blood glucose levels (28). Recent reports on ghrelin knockout adult mice demonstrate that lack of ghrelin protects from rapid weight gain induced by early exposure to a high-fat diet, decreases adiposity, and increases energy expenditure and locomotor activity (29), and adult mice lacking the ghrelin receptor, when fed a high-fat diet, eat less food, and accumulate less weight. The physiological significance of an exercise-induced decrease in ghrelin levels is unknown, but it merits to be investigated whether it provides an added benefit by suppressing appetite and directing substrate use toward lipid oxidation. Reports indicate that obese people, indeed, increase energy expenditure more than intake during treadmill exercise in an experimental setting (30) and that normal-weight young adults are in negative caloric balance during an exercise period (31).
Our results are in line with the results of Kraemer et al. (32), where 80% of maximal concentric exercise decreased ghrelin levels 15 min after exercise. However, no effects on postexercise ghrelin levels were apparent in clinical trials comprising graded exercise of 50–90% of VO2-max (33), 30–59% of VO2-max (34), and 60–100% of VO2-max (35). However, in the latter trial, recovery blood samples were drawn only 15 and 30 min after exercise, after which a possible further ghrelin decrease could not be detected.
We revealed a negative correlation between IGF-I and ghrelin levels, but other metabolic changes during exercise, such as free fatty acids, may influence ghrelin levels. In support of this, we have recently reported that free fatty acids suppress ghrelin levels independent of ambient GH and insulin levels (36).
The effects of pharmacological GH exposure are of potential relevance in relation to GH doping by athletes. In the development of a test to detect GH abuse, one strategy could be to integrate measurements of specific and sensitive GH biomarkers into one equation. In that regard, ghrelin is a potential candidate. However, the suppression of ghrelin after GH exposure is moderate compared with, e.g. the robust increase in circulating bone markers and IGF-I (37, 38). Moreover, s-ghrelin levels are also determined by factors such as nutritional status, food intake, and insulin, all of which need to be accounted for.
IGF-I levels were distinctly increased in both the low- and high-dose GH-treated groups. The moderate suppressive effects of GH on ghrelin levels became evident in the high-dose GH group only. Hence, this part of the study does not apply directly to normal human physiology regarding the role of GH levels on ghrelin secretion.
Potential nongeneralizability of data from the elite athletes with extreme conditioning and decreased fat should be noted as well. However, the present observations are in line with the observations in healthy, young adults studied in group B.
In conclusion, this report adds to the hypothesis that GH feedback inhibits ghrelin secretion. This clinical study does not allow conclusions to be drawn about the mechanisms underlying the putative GH feedback on ghrelin secretion. Furthermore, we have shown that ghrelin levels decrease significantly up to 120 min after exercise in healthy subjects. Whether this exercise-associated decrease in ghrelin is of physiological importance remains to be studied.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: E.T.V., R.D., K.H.W.L., and M.K. have nothing to declare. J.O.L.J. consults for and received lecture fees from Novo Nordisk and Pfizer. J.S.C. consults for and received lecture fees from Novo Nordisk, Pfizer, Ipsen, and Roche.
First Published Online October 10, 2006
Abbreviations: AUC, Area under the concentration curve; BF, body fat; BMI, body mass index; HD, high-dose group; HR, heart rate; LD, low-dose group; MFT, multiple fitness test; NS, not significant; PL, placebo group; s-ghrelin, serum ghrelin; VO2-max, maximum oxygen uptake.
Received July 5, 2006.
Accepted October 2, 2006.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, P < 0.05.
) and after 28 d of treatment (
). Overall P value ANOVA for repeated measurements is less than 0.001 d 0 and less than 0.05 d 28 for each of the three groups. Asterisks refer to post hoc paired t test (Bonferroni corrected) comparing t = 0 to the other time points. *, P < 0.05. The printed P values refer to post hoc comparison of the s-ghrelin AUC d 0 and 28 (paired t test). D, Displayed is the ratio between d 28 and d 0 s-ghrelin AUC after exercise. The printed P value refers to comparison by one-way ANOVA.