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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2234
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 5 1988-1994
Copyright © 2008 by The Endocrine Society

Evidence for Acyl-Ghrelin Modulation of Growth Hormone Release in the Fed State

Ralf Nass1, Leon S. Farhy1, Jianhua Liu, Catherine E. Prudom, Michael L. Johnson, Paula Veldhuis, Suzan S. Pezzoli, Mary Clancy Oliveri, Bruce D. Gaylinn, H. Mario Geysen and Michael O. Thorner

Department of Medicine (R.N., L.S.F., J.L., S.S.P., M.C.O., B.D.G., M.O.T.), Division of Endocrinology and Metabolism, and Departments of Pharmacology (M.L.J., P.V.) and Chemistry (C.E.P., H.M.G.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. Michael O. Thorner, Department of Medicine, University of Virginia, Box 801411, Charlottesville, Virginia 22908. E-mail: mot{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The timing and frequency of GH secretory episodes is regulated by GHRH and somatostatin. This study provides evidence for amplification of these GH pulses by endogenous acyl-ghrelin.

Design: Blood was sampled every 10 min for 26.5 h during a fed admission with standardized meals and also during the final 24 h of a 61.5-h fast. GH secretion profiles were derived from deconvolution of 10-min sampling data, and full-length acyl-ghrelin levels were measured using a newly developed two-site sandwich assay.

Setting: The study was conducted at a university hospital general clinical research center.

Participants: Participants included eight men with mean (± SD) age 24.5 ± 3.7 yr (body mass index 24 ± 2.1 kg/m2).

Results: Correlations were computed between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-min interval preceding each GH burst. In the fed state, the peak correlations were positive for all subjects and significantly higher than in the fasting state when acyl-ghrelin levels declined [mean (± SEM): 0.7 (0.04) vs. 0.29 (0.08), P = 0.017]. In addition, long-term fasting was associated with an increase in the GH secretory pulse mass and amplitude but not frequency [fed vs. fasting pulse mass: 0.22 (0.05) vs. 0.44 (0.06) µg/liter, P = 0.002; amplitude: 5.2 (1.3) vs. 11.8 (1.9) µg/liter/min, P = 0.034; pulses per 24 h: 19.4 (0.5) vs. 22.0 (1.4), P = 0.1].

Conclusion: Our data support the hypothesis that under normal conditions in subjects given regular meals endogenous acyl-ghrelin acts to increase the amplitude of GH pulses.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Ghrelin is a 28-amino acid acylated peptide that, acting through the GH secretagogue receptor (GHS-R), has a strong dose-dependent GH-releasing effect (1, 2). A role for endogenous ghrelin in the regulation of GH release has been postulated but has not been established. GH secretion is pulsatile and characterized by periodic secretory bursts, with nocturnal augmentation of GH secretion (3). The most reproducible GH pulse occurs shortly after sleep onset (4). GH secretion is controlled via feedback by IGF-I and two neurohormones: stimulatory GHRH and inhibitory somatostatin (5). In humans with an inactivating mutation of the GHRH receptor, rhythmic GH secretion persists, which supports a role for an additional GH-releasing factor besides GHRH in the regulation of GH (6). This is further supported by the inability of the somatostatin analog octreotide to inhibit the nocturnal increase in GH release (7, 8, 9). Thus, a factor that antagonizes the effects of somatostatin might be involved in the regulation of GH.

Studies to determine the effects of endogenous ghrelin on the control of GH have yielded conflicting results: a relationship between GH and ghrelin (10); no relationship (11); or a relationship under certain conditions or times of the day such as fasting or during the night (12). These inconsistent findings may be due, at least in part, to differences in study design and methodology. With the exception of one study by Avram et al. (11), none of the reported research has distinguished between acyl- and des-acyl ghrelin. The only data using deconvolution analysis to study the relationship between ghrelin (total) and GH were published by Misra et al. (13) in healthy adolescents and adolescent girls with anorexia nervosa. Other studies analyzed circulating GH concentrations instead of secretion events (10, 12).

To test the hypothesis that acyl-ghrelin amplifies GH secretion, a novel mathematical approach was used (14). In eight healthy young men fed standardized meals, the individual GH secretion pulses were determined from a 24-h time series by deconvolution (15). The amplitude of each GH secretory pulse was then compared with the average ghrelin concentration over a fixed time interval during and before each GH pulse. We chose a 60-min interval because serum GH levels peak 30–60 min after a ghrelin injection, which has been well documented (16, 17). Rather than comparing the entire GH and ghrelin concentration profiles, the method relates the amplitude of an individual GH pulse to the concentrations of acyl-ghrelin accompanying the development of this pulse. This approach has the advantage that it disregards those parts of the ghrelin profile during which there is no pulsatile GH secretory activity. Consequently, the method is suitable for analyzing endocrine relationships in which one of the hormones (ghrelin) is presumed to be an amplifier/modulator and not the sole regulator of the secretory activity of the second hormone (GH).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects and experimental design

The study was approved by the institutional review boards of the University of Virginia and the General Clinical Research Center (GCRC), and all volunteers gave written informed consent before participating. Eight healthy men were recruited by advertisement. Mean (± SD) age was 24.5 ± 3.7 yr (range 18–28) and mean body mass index was 24 ± 2.1 kg/m2 (range 20.6–26.2). Strenuous daily exercise was restricted to less than 1 h per day. Screening included a medical history questionnaire, physical examination, and a fasting blood profile. Exclusion criteria included smoking, acute illness, or medications known to affect GH release.

Each subject underwent a fed and a fasting admission a minimum of 4 wk apart in random order. For the fed admission, volunteers were admitted for dinner and allowed to sleep after 2100 h. In the morning two forearm indwelling venous cannulae were placed at 0600 h, and blood sampling was performed every 10 min for 26.5 h, beginning at 0800 h. Standardized meals were served at 0800, 1300, and 1800 h and were consumed within 30 min with no snacks allowed. The calories supplied at each meal were calculated using the Harris-Benedict equation as 20% protein, 30% fat, and 50% carbohydrate. Sampling ended at 1030 h, 2.5 h after breakfast on the second day. On the fasted admission, after a standardized dinner, subjects fasted for a total of 61.5 h (except for water ad libitum). Sampling occurred during the last 24 h of the fast and continued for 2.5 h after the fast was ended with a standardized breakfast.

GH assay

Serum GH concentrations were measured in duplicate by fluoroimmunometric assay on an Immulite 2000 analyzer (Diagnostic Products Corp., Flanders, NJ). The assay sensitivity was 0.01 µg/liter, with an intraassay coefficient of variation (CV) of 3.4% at 2.4, 2.6% at 4.8, and 2.3% at 12 µg/liter; the interassay CV was 3.8% at 2.5, 3.5% at 5.0, and 3.3% at 12.6 µg/liter. Data collection and quality control validation were performed by the GCRC Core Laboratory.

Acyl-ghrelin sample collection

Blood (1.3 ml) was added to chilled 3-ml EDTA Vacutainer tubes preloaded with 4-[2-aminoethyl benzene] sulfonylfluoride (Alexis Biochemicals, San Diego, CA) (4 mM final concentration) and stored on ice. The blood was centrifuged for 10 min at 2000 x g at 4 C within 1 h of collection, the plasma was separated, and 0.5 ml plasma was acidified with 100 µl of 1 N HCl; samples were stored at –20 C until assay.

Sandwich assay

Plasma acyl-ghrelin was measured with an in-house two-site sandwich ELISA specific for full-length acyl-ghrelin (18). The assay sensitivity was 6.7 pg/ml, with an intraassay CV of 9.2% at 30, 12.7% at 100, and 16.8% at 300 pg/ml: the interassay CV was 17.8% at 50 pg/ml. All samples from a specific subject admission (160 time points) were run together in duplicate in a single 384-well plate.

Data analysis

GH secretion parameters were determined according to deconvolution analysis (15). The specific algorithm used was the recently introduced, automated multiparameter deconvolution analysis (AutoDecon: algorithm developed by a coauthor of this paper, Michael L. Johnson; available online at http://mljohnson.pharm.virginia.edu/home.html), which allows simultaneous determination of quantitative properties of underlying secretory bursts (including burst amplitudes and timings), endogenous hormone half-life, and a subject-specific basal hormone secretion (19). AutoDecon is maximally assumption free, using rigorous statistical tests to define secretion events. It includes a parameter-fitting module, which performs weighted nonlinear least-squares parameter estimation, an insertion module, which establishes the location of presumed secretion events, and a triage module, which removes nonsignificant events.

The relationship between acyl-ghrelin and GH secretion was estimated in accordance with the method recently presented by Farhy et al. (14).

The method relates the amplitude of an individual GH pulse to the concentrations of acyl-ghrelin preceding or accompanying the development of this pulse. Thereby it disregards parts of the ghrelin profile not related to pulsatile GH activity. It also does not take into account the relative changes in ghrelin (or lack of changes) preceding the GH pulses by considering only average ghrelin concentration levels. Specifically, for each subject the following was performed. First, the amplitudes a1, a2, ... and the corresponding timing t1, t2, ... of the GH pulses were determined by deconvolution (Fig. 1Go, top panel). For a given (ti) and lag ({Delta}) of 0, 10, ... min, the average ghrelin concentration (

Formula

) over a 60-min interval [(ti – {Delta}) – 30, (ti – {Delta}) + 30] was calculated. This is illustrated in Fig. 1Go (middle panel) for the first pulse at time = t1. The signs

Formula

span the lagged 60-min intervals, point at their centers (ti – {Delta}), {Delta} = 0, 10, ..., and have heights equal to

Formula

({Delta} = 0, 10, ...). Finally (Fig. 1Go, bottom panel), for each lag ({Delta}), we computed the correlation between the log of the amplitudes of the GH pulses (log10a1, log10a2, ...) and the corresponding average acyl-ghrelin concentrations (

Formula

), which we identified as the level of amplification (LA) for a given lag ({Delta}). Because LA is based on correlation, it has values between –1 and 1, and values close to 1 imply a stronger (positive) relationship. For each subject the LA was calculated at all lags (0, 10, ..., 120 min), which produced the sequence LAi (0), LAi (10), ..., LAi (120), where LAi({Delta}) is the LA for subject no. i at a fixed lag = {Delta}. Next, for each subject we determined the maximum LA for lags {Delta} that are less than 1 h and the corresponding lag time when this maximum was achieved. Formally, if the subject’s number was (i = 1, ..., 8), the maximal LA was computed according to the formula:

Formula
This was further aggregated across all eight subjects by computing the following: 1) the average of the highest LA: average (LA1, ..., LA8); and 2) the average LA at all lags {Delta} from 0 to 120 min expressed by the function LAmean({Delta}) = average [LA1({Delta}), ..., LA8({Delta})], {Delta} = 0, 10, ..., 120.


Figure 1
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  		FIG. 1. Schematic illustration of the estimation of the LA for a given subject and lag. Top, Amplitudes (log) a1, a2, ... and corresponding timing t1, t2, ... of individual GH pulses determined by deconvolution analysis Middle, Illustration of pulse at t1. Computation of 1-h average acyl-ghrelin (g) for a given time (t1) and lag ({Delta}) of 0, 10, ... min. The height ( Figure 1) of the rightmost sign Figure 1 that points at t1 represents the average acyl-ghrelin from –30 min to +30 min from t1 and corresponds to a lag of 0 (the sign spans this 0-lag interval). Analogously, the other symbols Figure 1 indicate the 1-h average acyl-ghrelin concentrations (Figure 1) computed over 60-min intervals obtained by a left shift of the 0-lag interval at 10, 20, 30, 40, 50, or 60 min. The signs Figure 1 indicate the center of the corresponding intervals and have height equal to the corresponding average acyl-ghrelin concentration in this interval. Bottom, Correspondence between log GH pulse amplitudes a1, a2,... and average acyl-ghrelin levels used in the computation of LA in a given subject for a fixed lag {Delta} (see text).

 
Finally, we explored the dependence of LAmean on the lag {Delta} and tested whether LAmean was significantly positive in the 1-h period before the GH pulses ({Delta} {epsilon} [0.60]). The expectations were that LAmean would start from low values at lag = 120 and would increase the closer the chosen ghrelin interval gets to the GH secretion pulse and may reach a plateau somewhere in the middle. High individual LA and aggregated LAmean would imply the existence of a strong nonlinear relationship between GH pulses and previous acyl-ghrelin concentration consistent with a model in which acyl-ghrelin acts to amplify GH pulses. The lag {Delta} would estimate the apparent individual delay between acyl-ghrelin action and the observed effect on GH secretion.

Statistical analysis

Unless otherwise stated, data were expressed as mean ± SEM, and statistical comparisons were performed using a Wilcoxon signed rank test. P < 0.05 was considered statistically significant. The Bonferroni type I error rate adjustment was used when multiple tests were conducted in the entire 1-h interval preceding the GH pulses.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Pulsatility analysis of GH

Deconvolution analysis showed that the increase in mean GH levels observed with fasting is explained by an increase in mean GH burst mass and amplitude (fed vs. fasting burst mass 0.22 ± 0.05 vs. 0.44 ± 0.06 µg/liter; P = 0.002 and amplitude: 5.2 ± 1.3 vs. 11.8 ± 1.9 µg/liter /min; P = 0.034) but with no change in pulse frequency (fed vs. fasting: 19.4 ± 0.5 vs. 22.0 ± 1.4 pulses per 24 h; P = 0.1).

Relationship between acyl-ghrelin and GH

The average circulating acyl-ghrelin concentrations (on a linear scale) and GH concentrations (on a log scale) during the fed and fasting states are shown in Fig. 2Go. When averaging across eight subjects, it is not possible to locate individual ghrelin and GH pulses and the relationships between them. However, the plots indicate a good coincidence between the two profiles in the fed state (Fig. 2AGo), suggesting a nonlinear dose-response relationship between ghrelin concentration and GH secretion. In contrast, in the fasting state, acyl-ghrelin concentration was reduced by 58% (Fig. 2Go B), GH levels were elevated, and there was no apparent relationship between GH and ghrelin.


Figure 2
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  		FIG. 2. Mean (± SEM) plasma acyl-ghrelin (picograms per milliliter) and serum GH levels (micrograms per liter, shown on log scale); n = 8 young men during the fed (A) and fasting (B) admissions.

 
The profiles in a representative subject shown in Fig. 3Go illustrate the subject-specific relationship between average (smoothed) acyl-ghrelin concentration (Fig. 3Go, upper dashed line) and GH secretion (Fig. 3Go, lower solid line) in the fed state. The plots indicate that GH secretory events share a similar pattern with ghrelin concentrations. To test this objectively, the amplitudes of GH pulses were correlated with previous average ghrelin concentrations, and the LA was computed for each subject, examining different lags between GH pulses and ghrelin concentrations. For each of the eight subjects in the fed state, the computed LA was positive for all lags from 0 to 60 min, and the mean of the individual peak correlations was 0.70 ± 0.04. The average lag at which the peak correlations were achieved was 31.4 ± 8.8 min. However, in the fasting state, not all LAs were positive for lags from 0 to 60 min, and the mean of the individual peak correlations was 0.29 ± 0.08; this was significantly lower than in the fed state (P = 0.017). The average lag of the peak correlations in the fasting state was 21.4 ± 8.8 min. The individual maximal LAs in the fed and fasting states, and the corresponding lags during which they occurred, are shown in Table 1Go.


Figure 3
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  		FIG. 3. Relationship between averaged (smoothed) acyl-ghrelin concentration (upper dashed line) and GH secretion rate (shown on log scale, lower solid line) from deconvolution in one representative subject.

 

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  		TABLE 1. Maximal LA and the lag at which it occurred in the fed and fasting states for each individual

 
Figure 4Go shows the mean LA at each fixed lag in both states. Because LA is a measure of correlation and lag is a measure of time proximity, the hypothesis that acyl-ghrelin amplified GH peak height would be consistent with an increased LA (correlation) when looking at ghrelin levels measured closer to the GH peak (lower lag). As expected, the LA starts from low values [LA = 0.14 (fed) and LA = –0.1 (fast)] at lag = 120 and increases the closer a given ghrelin interval gets to the GH secretion pulse. The apparent plateau is reached at lag = 40 min at LA = 0.57 in the fed state and at lag = 10 min at LA = 0.23 in the fasting state.


Figure 4
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  		FIG. 4. Mean (±SEM) LA at different lags ({Delta} = 0, ..., 120 min). An asterisk indicates the three values that were significantly positive. The rectangles indicate the mean LA values in the 1-hr lag interval before the GH pulses. The asterisk over the line connecting the two rectangles indicates a significant difference in LA between the two 1-h intervals.

 
In the fed state, if only lags within 1 h of the corresponding GH pulse are considered, the LA varies between 0.5 (at lag = 60 min) and 0.57 (the maximum at lag = 40 min). The LAs at lags 0, 30, and 60, which correspond to lags that span the 1-h period before the GH pulses, were significantly positive (P = 0.012). Applying Bonferroni correction, we concluded that the LA is positive for lags in this 1-h interval (P < 0.05). In the fasting state, the LA was significantly positive only at lags = 0 (P = 0.05), 10 (P = 0.04), or 20 min (P = 0.05). At lags 0, 30, and 60 min, the LA was higher in the fed vs. the fasting state (lag = 0: P = 0.017, lag = 30: P = 0.012, and lag = 60: P = 0.017). Again, applying Bonferroni correction, we concluded that the LA was higher in the fed vs. the fasting state for lags in this 1-h interval (P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In contrast to earlier studies, appropriate conditions for sample collection, well-validated assays, and adequate sample frequency have revealed a clear relationship between acyl-ghrelin and GH in subjects given standardized meals (Fig. 2AGo). This relationship has been evaluated quantitatively in each subject using sophisticated mathematical methods that examine the ghrelin tone preceding each GH burst. Acyl-ghrelin and GH exhibit similar changes over 24 h, with the highest acyl-ghrelin and GH levels occurring between 2300 and 0100 h in the absence of a meal or meal anticipation. During the day, there were coordinated meal-related changes of acyl-ghrelin and GH at breakfast, lunch, and dinner, strongly suggesting that under fed conditions acyl-ghrelin acts as a positive modifier of GH release. This relationship was not apparent under long-term fasting conditions.

Ghrelin is a natural ligand for GHS-R and potently stimulates GH release when administered exogenously. Human studies of continuous 24-h infusion with GH-releasing peptide (20) showed that GH secretagogues increase GH pulse amplitude but not GH pulse frequency. Similar results have been shown with an orally active, long-acting GH secretagogue (MK-677) in humans (21), which also acts through the GHS-R. Animal studies by Zizzari et al. (22) using a GHS-R antagonist support our findings that endogenous acyl-ghrelin modifies circulating GH release during the fed state. Both sc and intracerebroventricular infusion of the rat GHS-R antagonist BIM-28163 did not alter the pulsatile pattern of GH secretion but lowered the GH pulse amplitude. In humans a GHS-R missense mutation, which impairs the constitutive activity of the GHS-R, is associated with short stature (23). Consistent with these results, GHS-R-null mice have lower IGF-I levels when compared with wild-type animals (24), and ghrelin-null mice also showed a tendency to lower IGF-I concentrations when compared with the control animals (25); however, this did not reach statistical significance. This body of data implies that endogenous ghrelin plays a role in GH regulation.

Several studies have investigated the relationship between ghrelin and GH. Muller et al. (12) showed that fasting induced a diurnal rhythm for total ghrelin similar to that observed in GH levels, but the diurnal rhythm was not present when subjects were tested under fed conditions. However, in their study blood samples were measured only every 8 h and their assay measured total ghrelin.

Koutkia et al. (10) used cross-approximate entropy analysis and found that there is a significant regularity in cosecretion between ghrelin and GH in the fasted state. However, this was found only during nighttime and blood was sampled only every 20 min.

Espelund et al. (26) as well as Norrelund (27) and Natalucci et al. (28) did not find a correlation between ghrelin and GH under fasting conditions. Misra et al. (13), using deconvolution analysis for GH and total ghrelin in healthy adolescents and adolescents with anorexia, found that fasting ghrelin is an independent predictor of basal GH secretion and GH secretory burst frequency. Blood samples were measured overnight for 12 h (2000–0800 h) every 30 min.

All the above-mentioned studies have measured total ghrelin and did not distinguish between acyl-ghrelin, des-acyl ghrelin, and inactive fragments. Avram et al. (11) used an acyl-ghrelin RIA, but did not observe any relationship with GH under fed or fasting conditions. Their different results may relate to protocol differences, a less specific single-site ghrelin assay, and less stringent precautions to preserve ghrelin acylation. Furthermore, their study population included men and women and they used the Cluster program rather than deconvolution analysis.

Previous studies from our group had found an increased number of GH pulses in the pattern of enhanced GH secretion seen on fasting when compared with the fed state (29, 30). It was speculated that this could be due to a limitation of the GH assays available at that time because many samples in the fed admission had undetectable levels. Some of these old GH RIAs had a sensitivity of only 0.5 µg/liter. The GH assay used in this study has a sensitivity of 0.01 µg/liter. To illustrate the importance of GH assay sensitivity, we point out that almost 50% of all pulses detected in our study during the fed state (75 of 145) reflect circulating levels less than 0.5 µg/liter, indicating that a great deal of biology is occurring below this concentration. The increased sensitivity of the current two-site fluoroimmunometric GH assay and the improved mathematical methods now available have resulted in the finding of more, previously undetected, small pulses in the fed state. Thus (similar to the effects of GH secretagogues cited above), we now confirm previous speculations (30) that there is only an increase in GH pulse amplitude but no increase in the frequency of GH pulses during fasting.

In the fed state, our results reveal a clear nonlinear relationship between acyl-ghrelin and GH (Fig. 2AGo); nonlinear relationships are common in endocrine regulatory systems (31, 32). Therefore, GH amplitudes were modified with a log transformation (to mimic the log scale presentation in Fig. 2Go) and then were correlated with previous acyl-ghrelin levels. In all subjects the amplitudes of GH pulses were significantly positively correlated (P < 0.05) with the acyl-ghrelin levels during or within 1 h preceding these pulses, with a mean maximal LA of 0.7 (± 0.04). It is expected that for each subject the highest LA will be achieved with a lag that is specific for this individual, reflecting intersubject variability, and in fact, we observed a relatively high SEM of the average value of this parameter in both states (31.4 ± 8.8 min in the fed and 21.4 ± 8.8 min in the fasting state). Various factors may contribute to this variability, including mismatch between acyl-ghrelin increase and the timing of the GHRH pulses. In the fasting state, the LA was significantly positive only when the distance between previous mean ghrelin concentration, and the following GH pulse was less than 20 min, and the LA was significantly higher in the fed vs. the fasting state in the 1-h interval mentioned above (P < 0.05). Consequently, in the fed state, the significant relationship between the GH secretion peak and the mean circulating acyl-ghrelin levels support the hypothesis that in addition to somatostatin and GHRH, acyl-ghrelin modulates the regulation of GH secretion, leading to higher GH peaks. However, during fasting, which was accompanied by significant reduction in mean acyl-ghrelin concentration, we were not able to document a strong relationship between acyl-ghrelin and GH, suggesting that other factors dominate the control of GH release in this state.

This conclusion is also supported by studies showing that the increase in fasting GH levels is caused by a decrease in central somatostatin release (33, 34), which underlines our conclusion that GHRH and somatostatin are the main regulatory factors of the timing of GH release.

Our conclusions are also consistent with the hypothesis that the mechanism by which ghrelin amplifies GH is, at least partially, based on antagonizing somatostatin action both at the pituitary and in the central nervous system (35). Given this presumption, low ghrelin levels during fasting would not necessarily result in reduction of the GH concentration levels if the dominant suppressor of GH is withdrawn at this time.

Whereas a decrease in central somatostatin release might be responsible for the increase in fasting GH levels, a fasting-induced increase in free fatty acids could be responsible for the decrease in circulating acyl-ghrelin levels. Ho et al. (29) demonstrated that after 1 d of fasting, circulating β-hydroxybutyrate increases, and Gormsen et al. (36) showed that an acute increase in free fatty acid levels decreases circulating acyl-ghrelin levels.

The possibility that changes in des-acyl ghrelin could affect GH release is unlikely based on the findings of in vitro studies showing that des-acyl ghrelin is neither an agonist nor an antagonist for the GHS-R1a (37). In addition in vivo studies in humans (38) show that administration of des-acyl ghrelin has no impact on GH release, irrespective whether it was given alone or in combination with acyl-ghrelin.

Because our study was not interventional and the analysis is based on correlation, we cannot exclude the existence of one common or several separate factors that control both GH release and circulating ghrelin levels simultaneously. For example, Luque et al. (39) showed in a series of in vitro studies using primate pituitaries that glucocorticoids enhance GH release at the pituitary level. Glucocorticoids rise during fasting and have been shown to decrease circulating ghrelin levels (40). Therefore, a possible role for glucocorticoids as a common regulator of circulating ghrelin and GH during fasting cannot be excluded, even though the effects of glucocorticoids are stimulatory for GH acutely, but chronic elevation leads to suppression. On the other hand, the work by Zizzari et al. (22) using a GHS-R antagonist favors a direct modulatory role of circulating ghrelin on GH release as do studies with ghrelin mimetics (21).

Finally, based on our data, we cannot exclude the possibility that there is a negative feedback loop between GH and circulating ghrelin levels. However, the current literature on this subject is controversial (41), and our findings from previous animal studies, together with findings of others, do not support the existence of such a negative feedback loop (36, 42, 43).

The relationship between acyl-ghrelin, which increases food intake, and GH found during the fed state would be beneficial to man because the anabolic changes induced by GH require the presence of adequate nutrition. During long-term fasting, the role of GH is thought to preserve protein by partitioning metabolic fuels (29). The protein-sparing effect of GH is likely to be augmented by the decrease in fasting acyl-ghrelin levels, which would otherwise counteract the lipolytic effects of GH.

Conclusions

Whereas previous studies have attempted to correlate circulating GH levels with circulating total or acyl-ghrelin levels measured by single-site ELISA or RIA, this study is unique in that a specific two-site sandwich assay was used to measure full-length acyl-ghrelin in carefully preserved samples. In addition, we correlated acyl-ghrelin levels with the amplitudes of the proximal GH secretory peaks derived from deconvolution analysis, not simply with the total circulating GH levels. With this advanced methodology (including the use of a highly sensitive GH assay), a significant relationship between the GH secretion peak amplitudes and the mean circulating acyl-ghrelin levels during the fed condition in healthy young men was observed. These results support the hypothesis that in addition to somatostatin and GHRH, ghrelin plays a role in the regulation of GH secretion, leading, in particular, to higher GH peaks before times of food intake.


   Acknowledgments
 
We thank the University of Virginia GCRC nursing staff for their excellent support when conducting this study as well as the GCRC Core Laboratory staff and our volunteers who made this work possible. We also thank Dr. Boris Kovatchev for stimulating discussions and critical advice on data analysis.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants MO1 RR 00847 (to the General Clinical Research Center at the University of Virginia), RO1 DK32632 (to M.O.T.), K23 RR018770 (to R.N.), R01 RR019991 (to M.L.J., P.V., and L.S.F.), R25 DK064122 (to M.L.J.), R21 DK072095, P30 DK063609, and RO1 DK51562 (to L.S.F.); an unrestricted grant from Bristol-Myers Squibb; and a gift to the laboratory from Mr. Salvatore Ranieri.

Disclosure Statement: The authors have declared that no conflicts of interest exist, although M.O.T. reports that he is a paid consultant for Novo Nordisk and Tercica.

First Published Online March 11, 2008

1 R.N. and L.S.F. participated equally in this work. Back

Abbreviations: {Delta}, Lag; CV, coefficient of variation; GCRC, General Clinical Research Center; GHS-R, GH secretagogue receptor; LA, level of amplification; t, time.

Received October 4, 2007.

Accepted March 3, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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