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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0620
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 9 3597-3603
Copyright © 2008 by The Endocrine Society

Twenty-Four Hour Continuous Ghrelin Infusion Augments Physiologically Pulsatile, Nycthemeral, and Entropic (Feedback-Regulated) Modes of Growth Hormone Secretion

Johannes D. Veldhuis, George Ann Reynolds, Ali Iranmanesh and Cyril Y. Bowers

Endocrine Research Unit (J.D.V.), Department of Internal Medicine, Clinical Translational Science Unit, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905; Division of Endocrinology (G.A.R., C.YB.), Department of Internal Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112; and Endocrine Section (A.I.), Department of Medicine, Salem Veterans Affairs Medical Center, Salem, Virginia 24153

Address all correspondence and requests for reprints to: Cyril Y. Bowers, Endocrine Research Unit, Department of Internal Medicine, Clinical Translational Science Unit, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Ghrelin is a 28-amino acid acylated peptide that potentiates GHRH stimulation and opposes somatostatin inhibition acutely. Whether prolonged ghrelin administration can sustain physiological patterns of GH secretion remains unknown.

Hypothesis: Continuous delivery of ghrelin will amplify physiological patterns of GH secretion over 24 h.

Subjects: Men and women ages 29–69 yr, body mass indices 23–52 kg/m2, were included in the study.

Location: The study was performed at an academic medical center.

Methods: Twenty-four hour continuous sc infusion of saline vs. ghrelin (1 µg/kg·h) with frequent sampling was examined. Deconvolution and entropy analyses were performed.

Outcomes: IGF-I concentrations were determined. Basal, pulsatile, nycthemeral, and entropic measures of GH secretion were calculated.

Results: Ghrelin infusion compared with saline infusion for 24 h elevated (median) acylated ghrelin, GH, and IGF-I concentrations by 8.1-fold (P < 0.001),11-fold (P < 0.001), and 1.4-fold (P = 0.002). GH secretory-burst mass and frequency increased by 6.6-fold (P = 0.004) and 1.7-fold (P < 0.001), respectively, resulting in a 12-fold increase in pulsatile GH secretion (P < 0.001). Interpulse variability decreased significantly (P = 0.046), whereas GH secretory-burst shape and half-life did not change. The amplitude of the nycthemeral GH rhythm increased by 3.4-fold (P < 0.001), and GH patterns became more irregular (higher approximate entropy P < 0.001). Combining GHRH with ghrelin was not an additive in driving GH secretion.

Conclusions: Continuous ghrelin infusion for 24 h elevates acylated ghrelin, GH and IGF-I concentrations, and stimulates pulsatile, nycthemeral, and entropic modes of GH secretion. The consistency of outcomes in a heterogeneous cohort of adults suggests potentially broad utility of this physiological secretagogue in hyposomatotropic states.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Ghrelin is the most potent naturally occurring GH secretagogue known, which uniquely requires a fatty acid moiety esterified to the third N-terminal amino acid (serine) for full biological activity (1). Several lines of evidence support the physiological importance of this novel acylated peptide. Combined knockout of the ghrelin gene and its receptor leads to a lean body phenotype, increased energy expenditure, and heightened motor activity in mice (2). Silencing either the peptide or cognate receptor gene is associated with impaired glucose homeostasis in animals subjected to fasting or given a high-fat diet (3, 4). Transgenic repression of neuronal ghrelin-receptor expression reduces pulsatile GH secretion and IGF-I concentrations in the female rodent (5), and pharmacological antagonism of the ghrelin receptor suppresses the amplitude of GH pulses in the male rat (6). In humans, mutations of the ghrelin receptor are associated with short stature (7). Conversely, acute infusion of ghrelin stimulates GH secretion, enhances appetite, reduces diastolic blood pressure, and inhibits insulin secretion (8, 9, 10, 11, 12, 13). What remains unknown is whether hypothalamo-pituitary responses become refractory to sustained ghrelin stimulation. In addition, whereas the ghrelin analogs GH-releasing peptide (GHRP)-2 and MK0677 are able to promote pulsatile GH secretion for up to 30 d (14, 15), the greater instability of acylated ghrelin might restrict the utility of the native peptide (16, 17). To address these issues, we investigated the capability of continuous sc infusion of ghrelin to drive physiological patterns of GH secretion over 24 h. To extend generality of inference, men and women were studied over the age span of 29–69 yr with body mass indices (BMIs) of 23–52 kg/m2.


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

There were 15 adults that participated in the 24-h dose-finding study, nine of whom received an infusion of 1 µg/kg·h ghrelin. Inclusion criteria were: a normal medical history, physical examination, and screening biochemical measures of hepatic, renal, metabolic, endocrine, and hematological function; active, ambulatory, healthy, community based living status; and provision of witnessed written informed consent approved by the institutional review board. Exclusion criteria were the use of psychoactive or neuroactive drugs; substance abuse; acute or chronic illness; systemic inflammatory, cardiopulmonary, hepatic, renal, endocrine, or metabolic disease; anemia; and the lack of provision of institutionally approved voluntary witnessed, written informed consent. Protocol-specific peptide use was first reviewed by the U.S. Food and Drug Administration.

Infusion protocols

Volunteers were admitted to the General Clinical Research Center at Tulane-Charity-Louisiana State University in the evening. Blood sampling and peptide infusions were performed the next day after an overnight fast. Thrice-daily meals were provided during the 24-h infusions.

Subjects each underwent blood sampling every 20 min for 24 h during constant sc infusions of saline (n = 9), ghrelin (n = 9), GHRH (n = 8), or both (n = 7) (each 1 µg/kg·h) beginning at 0900 h. Ghrelin was purchased from Clinalfa (Laufelfingen, Switzerland) and GHRH-1,29 from Bachem Laboratories (Torrance, CA). Six additional subjects received saline, and subsets of two, three, and four volunteers received, respectively, 0.1, 0.3, and 3.0 µg/kg·h ghrelin. Peptide was dissolved in sterile water and 3.5% mannitol, and delivered continuously via a portable insulin infusion pump. Volunteers were allowed to lie in bed, sit in a chair, and ambulate in the room.

Hormone assays

GH concentrations were measured in duplicate by automated immunochemiluminescence assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) (18, 19). Assay sensitivity is 0.005 µg/liter based on 22-kDa recombinant human GH standard. Median intraassay and interassay coefficients of variation are 5.4 and 6.7%, respectively. No serum GH concentrations decreased at or less than 0.020 µg/liter in this study. IGF-I (Nichols Institute Diagnostics) was quantitated by RIA in duplicate. Intraassay and interassay coefficients of variation were 6.8 and 8.5%, respectively. Total and acylated ghrelin were assayed in 20-min plasma samples, which were collected in chilled tubes containing EDTA and then rapidly acidified with HCl (LINCO Research, Inc., St. Charles, MO).

Deconvolution analysis

GH concentration time series were analyzed using a recently developed deconvolution method (20). The automated MATLAB (The MathWorks, Inc., Natick, MA) algorithm first detrends the data and normalizes concentrations to the unit interval (0, 1) (21). Second, successive potential pulse-time sets, each containing one fewer burst, are created by a smoothing process (a nonlinear adaptation of the heat-diffusion equation). Third, a maximum-likelihood expectation method estimates all secretion and elimination rates simultaneously for each candidate pulse-time set. The deconvolution model specifies basal secretion (β0), two half-lives ({alpha}1, {alpha}2), secretory-burst mass ({eta}0, {eta}1), random effects on burst mass ({sigma}A), procedural/measurement error ({sigma}{epsilon}), and a three-parameter flexible {gamma}-secretory-burst waveform (β1, β2, β3). The fast half-life was represented as 3.5 min and constituted 37% of the decay amplitude (22). Finally, model selection is performed to distinguish among the candidate pulse-time sets using the Akaike information criterion (23). Observed interpulse intervals are described by a two-parameter Weibull process (more general form of a Poisson process). The parameters (and units) are frequency (number of bursts per 24 h, {lambda} of Weibull distribution), regularity of interpulse intervals (unitless {gamma} of Weibull), fast and slow half-lives (min), basal and pulsatile secretion rates (concentration units per 24 h), mass secreted per burst (concentration units), and waveform mode [time delay to maximal secretion after burst onset (min)] (20, 21).

Twenty-four hour rhythmicity of GH concentrations was assessed by cosine regression (24). Outcomes are the acrophase (time of daily maximum), amplitude (50% of the zenith-nadir difference), and mesor (regression mean) of the daily rhythm.

Approximate entropy (ApEn) (1, 20%) was used as a scale- and model-independent regularity statistic to quantify the orderliness of GH release (25, 26, 27, 28). Higher ApEn denotes greater disorderliness of the secretion process. Mathematical models and clinical experiments establish that greater irregularity signifies decreased feedback control with high sensitivity and specificity (both >90%) (29, 30, 31). GH ApEn is elevated in puberty compared with prepuberty, in older compared with young individuals, and at any age in women compared with men.

Statistics

Derived (deconvolution, cosine, and ApEn) measures were transformed logarithmically before analysis to limit dispersion of variance. Data were analyzed by one-way ANOVA with repeated measures followed post hoc by Tukey’s honestly significantly different (HSD) test to contrast means (32). Weighted linear regression was performed with segmentation analysis. Data are cited as the mean ± SEM. P < 0.05 was construed as significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Subject characteristics for all study subgroups are summarized in Table 1Go. There were 48 sampling sessions (26 done by men and 22 by women) of 24-h each, yielding 3456 samples in total. Dose-finding 24-h infusion studies were performed in a total of 15 individuals (age 50 ± 2.6 yr, range 29–69; BMI 31 ± 2.1 kg/m2, range 23–52). The protocol used ghrelin doses of 0, 0.1, 0.3, 1.0, and 3.0 µg/kg·h. Total and acylated ghrelin concentrations increased by 5.4- and 8.1-fold, respectively (both P < 0.001) (Table 2Go). By ANOVA, administration of ghrelin stimulated pulsatile GH secretion significantly at the two highest infusion rates (P < 0.001). Responses to 1.0 and 3.0 µg/kg·h did not differ. Figure 1Go depicts linear regression analysis, which corroborated dose dependence up to 1 µg/kg·h (R2 = 0.99; P = 0.0029). The pulsatile GH response to 3 µg/kg·h significantly departed from this relationship (P < 0.01), suggesting possible down-regulation or an asymptotic maximum. Basal GH secretion also increased linearly (R2 = 0.88; P = 0.017). The slope of the response of pulsatile GH secretion on ghrelin dose was 7.9-fold greater than that for basal GH secretion (P < 0.001). Similar data were obtained by regression on total or acylated ghrelin concentrations (data not shown). Three-parameter logistic regression analysis revealed that incremental IGF-I concentrations (end minus beginning of infusion) increased curvilinearly from 17 ± 4 (saline) to 78 ± 8.9 (1.0 µg/kg·h) and 103 ± 35 (3.0 µg/kg·h) [P < 0.001].


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  		TABLE 1. Volunteer characteristics

 

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  		TABLE 2. Mean 24-h ghrelin concentrations1

 

Figure 1
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  		FIG. 1. Dose-related stimulation of 24-h pulsatile and basal GH secretion (top) and incremental IGF-I concentrations (bottom) across a 30-fold range of ghrelin infusion rates in a total of 15 volunteers. The results of linear (top) and curvilinear logistical (bottom) regression analyses are given. The GH response to 3.0 µg/kg·h ghrelin departs markedly (P < 0.001) from the strongly linear initial response, suggesting feedback inhibition, response down-regulation, or an asymptotic maximum (rate limiting step) in the GH response.

 
Five women and four men received both placebo and ghrelin (1 µg/kg·h) infusions. Age ranged from 29–69 yr and BMI from 24–52 kg/m2. A subset of eight subjects (lacking one woman) and a subset of seven subjects (lacking one man and one woman) also received GHRH and combined ghrelin/GHRH infusions, respectively. Mean GH concentration profiles are shown in Fig. 2Go. In the placebo session, fasting (0800 h) total IGF-I concentrations averaged 138 ± 13 µg/liter (Fig. 3Go), and mean and peak 24-h GH concentrations were 0.56 ± 0.14 and 4.8 ± 0.93 µg/liter, respectively (Fig. 4Go). GHRH infusion did not elevate IGF-I or GH concentrations significantly (Figs. 3Go and 4Go). Ghrelin administration increased IGF-I concentrations to 254 ± 36 µg/liter (P < 0.001 vs. placebo), and mean and peak GH concentrations to 2.9 ± 0.60 and 12 ± 1.8 µg/liter, respectively (both P < 0.001 vs. placebo). Combined ghrelin/GHRH compared with ghrelin infusion was associated with nonsignificant further GH increments yielding 24-h mean and peak GH concentrations of 4.9 ± 1.2 and 22 ± 5.1 (P = 0.064 and P = 0.29, respectively, vs. ghrelin alone).


Figure 2
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  		FIG. 2. Mean (± SEM) plasma GH concentration profiles over 24 h for all four interventions (Subjects and Methods). N, Cohort size; MN, midnight.

 

Figure 3
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  		FIG. 3. Stimulatory effects of ghrelin and combined ghrelin/GHRH on IGF-I concentrations. IGF-I was measured in serum collected at 0900 h just before and again at 0900 h at the end of each 24-h infusion. Comparisons are by paired Student’s t tests. Data are the mean ± SEM. N, Number of subjects.

 

Figure 4
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  		FIG. 4. Mean (top) and maximal (bottom) GH concentrations in adults administered ghrelin (1 µg/kg·h) or GHRH (1 µg/kg·h) or both compared with saline (placebo) for 24 h. Data are the mean ± SEM. P values were determined by ANOVA. Columns with different (unshared) alphabetic superscripts differ significantly by Tukey’s post hoc test. N, Number of subjects.

 
Deconvolution analysis was used to quantify the impact of ghrelin on physiological properties of GH secretion. Figure 5Go summarizes deconvolution estimates of GH secretory responses to the four interventions. Mean basal GH secretion was not significantly affected by GHRH, ghrelin, or both peptides at this ghrelin dose. GHRH did not augment mean pulsatile GH secretion (placebo 25 ± 7.2 vs. GHRH 38 ± 7.9 µg/liter·24 h), but ghrelin did so by 5.6-fold (P < 0.001) (Fig. 5AGo). Combined ghrelin/GHRH infusion evoked 7.6-fold stimulation over placebo (P < 0.001), which did not differ from ghrelin alone (P = 0.44, paired Student’s t test). Augmentation of pulsatile GH secretion was attributable primarily to a 3.5-fold (ghrelin) and 4.8-fold (ghrelin/GHRH) increase in the mass of GH secreted per burst (each P < 0.001 vs. placebo and vs. GHRH). Analyses restricted to the overnight fasting (8 h) sampling window revealed that infusion of each of GHRH (1 µg/kg·h), ghrelin (1 µg/kg·h), and the combined peptides increased GH concentrations over saline by, respectively, 2.5-, 9.2-, and 12-fold [viz., to respectively 0.63 ± 0.16 (P = 0.022), 2.30 ± 0.34 (P = 0.001), and 2.98 ± 0.66 (P < 0.001) vs. 0.25 ± 0.044 (saline)].


Figure 5
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  		FIG. 5. A, Augmentation of 24-h pulsatile GH secretion by administration of ghrelin (1 µg/kg·h) and combined ghrelin/GHRH (both 1 µg/kg·h) over placebo and GHRH (1 µg/kg·h). B, Predominant (77%) increase in GH secretory-burst mass (top) with lesser (23%) increase in detectable burst frequency (bottom) induced by ghrelin and ghrelin/GHRH infusions. See Fig. 4 format. NS, P > 0.05.

 
There was a small (1.7 fold) increase in detectable GH burst frequency during ghrelin and ghrelin/GHRH administration compared with placebo (P < 0.001) (Fig. 5BGo). The slow half-life of GH (17 ± 1.9 min) and estimated mode (31 ± 1.8 min) of GH secretory bursts (time delay to maximal secretion from burst onset) were not affected by peptidyl stimulation. Ghrelin increased interpulse-interval regularity compared with placebo (P < 0.046), as quantified by higher {gamma} of the Weibull distribution (2.2 ± 0.20 for ghrelin vs. 1.9 ± 0.15 for placebo). The effects of GHRH and ghrelin/GHRH were intermediate (not different from either ghrelin or placebo).

Cosinor analysis of the 24-h rhythmicity of GH concentrations is summarized in Fig. 6Go. Infusion of ghrelin with or without GHRH elevated the mesor (cosine mean) by 5-fold (P < 0.001) and nycthemeral cosine amplitude by 5-fold (P < 0.001). None of the peptide interventions influenced the timing (acrophase) of the nighttime GH maximum.


Figure 6
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  		FIG. 6. Twenty-four hour GH rhythmicity defined by the amplitude (top), phase (time of zenith, middle), and cosine mean (bottom) in the four interventions. Data are presented as described in Fig. 4.

 
All three peptide infusions elevated GH ApEn (P = 0.001), a measure of process randomness or pattern irregularity, indicating reduced negative feedback. ApEn changes were comparable among the secretagogues (Fig. 7Go).


Figure 7
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  		FIG. 7. Effects of ghrelin, GHRH, and both peptides on the relative randomness (irregularity, ApEn) of the GH secretory process over 24 h. Higher ApEn denotes less feedback inhibition. The format is given in Fig. 4.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Continuous sc infusion of ghrelin for 24 h increased fasting morning IGF-I and 24-h mean GH concentrations dose dependently. Significant and equivalent effects were achieved at infusion rates of 1 and 3 µg/kg·h. The natural GHRP stimulated physiologically pulsatile, nycthemeral, and entropic (feedback sensitive) modes of GH secretion in women and men. The secretagogue did not alter basal GH secretion, half-life, or secretory-burst shape. The prespecified heterogeneity of age and BMI in the cohort investigated and the consistency of stimulatory responses in all nine individuals suggest that these outcomes should be applicable to other groups of adults without acute illness.

Infusion of ghrelin (1 µg/kg·h) augmented mean 24-h pulsatile GH secretion by 5.6-fold compared with placebo, whereas this dose of GHRH peptide had no significant effect (1.3 fold). Amplification of pulsatility was due primarily (77%) to greater GH secretion per burst and to a lesser degree (23%) more detectable GH pulses. In contrast, ghrelin at this infusion rate did not modify basal (nonpulsatile) GH secretion significantly. The acylated secretagogue elevated 24-h rhythmic GH production (both the amplitude and mean), and increased the regularity of the GH pulse-renewal process. Despite more regular pulse timing, ghrelin evoked more disorderly GH secretory patterns consistent with significant muting of negative feedback (27, 29, 31). Accordingly, natural ghrelin can drive sustained GH pulsatility over 24 h, which closely resembles physiological patterns in puberty, the late-follicular phase of the menstrual cycle, and aerobically conditioned individuals (33, 34, 35).

Ghrelin and combined ghrelin/GHRH infusions were more effective than placebo or GHRH in all endpoints examined except in increasing GH ApEn. Albeit not designed to test for synergy, in the subgroup of seven subjects who completed all four infusions, combined ghrelin/GHRH stimulation tended to be more effective than ghrelin in elevating 24-h mean GH concentrations (P = 0.064). If corroborated in larger series, combined peptide stimulation might provide a method to amplify physiological patterns of GH secretion for prolonged intervals.

Ghrelin markedly augmented 24-h GH rhythmicity without displacing the timing of the daily maximum. In particular, the nycthemeral amplitude and the cosine mean of GH concentrations increased by 5-fold over placebo. Addition of GHRH to ghrelin did not enhance these effects.

All three peptide combinations induced a marked increase in irregularity of GH secretory patterns, reflected by an increase in ApEn (Fig. 7Go). In interlinked biological and mathematical systems, greater irregularity denotes reduced negative feedback (25, 31). More disorderly secretion (elevated ApEn) also characterizes GH responses to GHRP2, GHRH, estrogen, and testosterone administration (29, 36, 37, 38, 39). Because the ApEn measure is scale independent, these outcomes are not explained by higher mean GH concentrations or larger GH pulses per se (27, 28). Indeed, an inactivating GHRH-receptor mutation, visceral adiposity, and hypopituitarism are also associated with greater disorderliness of the GH secretion process in the setting of low GH and/or IGF-I feedback (40, 41, 42). Thus, collective data are consistent with the hypothesis that ghrelin drives GH secretion while attenuating negative feedback possibly by reducing hypothalamic somatostatin outflow.

Caveats include the relatively small cohort studied despite 48 sampling sessions to establish proof of principle, the need for more extended investigation beyond 24 h, and the limitations of 20 min compared with more frequent sampling because the latter permits more precise pulsatility estimates. However, total GH secretion is well estimated at both sampling rates (43). Overall 24-h GH responses to GHRH were not significant, but 8-h nighttime fasting values exceeded those of saline infusion. This difference could reflect suppression of GHRH action by food during the day and/or relief of somatostatin restraint at night.

In conclusion, continuous sc infusion of native ghrelin elevates IGF-I concentrations and sustains physiologically pulsatile, 24-h rhythmic and entropic (feedback-sensitive) patterns of GH secretion in a cohort of adults with deliberately dissimilar ages and BMIs. The outcomes demonstrate the potential utility of prolonged ghrelin administration to amplify GH production in conditions of reversible hyposomatotropism, such as aging or obesity.


   Acknowledgments
 
We thank Kay Nevinger for support of manuscript preparation, Ashley Bryant for data analysis and graphics, the Mayo Immunochemical Laboratory for assay assistance, and the Mayo research nursing staff for implementing the protocol.


   Footnotes
 
This work was supported in part via the Center for Translational Science Activities Grant No. 1 UL 1 RR024150 to the Mayo Clinic and Foundation and General Clinical Research Grant No. RR05096 to Tulane University School of Medicine from the National Center for Research Resources (Rockville, MD), R01 NIA AG19596 and AG29362 from the National Institutes of Health (Bethesda, MD).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 1, 2008

Abbreviations: ApEn, Approximate entropy; BMI, body mass index; GHRP, GH-releasing peptide.

Received March 17, 2008.

Accepted June 23, 2008.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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