AJP - Endo Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E1829-E1836, 2007. First published February 20, 2007; doi:10.1152/ajpendo.00682.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/E1829    most recent
00682.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vestergaard, E. T.
Right arrow Articles by Jorgensen, J. O. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vestergaard, E. T.
Right arrow Articles by Jorgensen, J. O. L.

Constant intravenous ghrelin infusion in healthy young men: clinical pharmacokinetics and metabolic effects

Esben Thyssen Vestergaard,1 Troels Krarup Hansen,1 Lars Christian Gormsen,1 Preben Jakobsen,2 Niels Moller,1 Jens Sandahl Christiansen,1 and Jens Otto Lunde Jorgensen1

1Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital; and 2Department of Pharmacology, University of Aarhus, Aarhus, Denmark

Submitted 13 December 2006 ; accepted in final form 31 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ghrelin levels fluctuate rapidly and dynamically with surges before meal times and postprandial troughs, and ghrelin increases appetite and food intake. Circulating ghrelin correlates negatively with body mass index (BMI), but obese individuals have a reduced postprandial decrease in ghrelin levels. Whether this reflects changes in secretion or clearance of ghrelin is uncertain. We therefore studied the pharmacokinetics of ghrelin in relation to anthropometric and biochemical measures. We also studied the effects of ghrelin on hormones and metabolites. In fasting humans, we used a constant infusion rate of ghrelin lasting 180 min at 5 pmol·kg body wt–1·min–1 in a randomized, double-blind, placebo-controlled crossover study. Serum ghrelin (s-ghrelin; total levels) was distributed and eliminated according to a two-compartment model. s-Ghrelin initial half-life was 24 ± 2 min and terminal half-life 146 ± 36 min, respectively. Mean residence time (MRT) of ghrelin was 93 ± 16 min. MRT correlated positively with both BMI (r = 0.51, P < 0.001) and high-density cholesterol (HDL) levels (r = 0.75, P < 0.001). Serum insulin levels remained constant during ghrelin infusion, whereas plasma glucose increased 0.3 ± 0.1 mmol/l (P < 0.01) and free fatty acid levels more than doubled (to 1.03 ± 0.08 mmol/l, P < 0.001), translating into a significant reduction of insulin sensitivity (P < 0.001). In conclusion, 1) we describe novel pharmacokinetics of ghrelin that are useful when tailoring ghrelin infusion rates in clinical experiments, 2) BMI and HDL correlate positively with MRT of infused ghrelin, and 3) supraphysiological ghrelin levels impair insulin sensitivity.

mean residence time; insulin sensitivity

THE DISCOVERY OF GHRELIN as an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) was a long awaited scientific breakthrough (18) considering that synthetic ligands had been known for decades and that the GHS-R was identified in 1996 (16). The GHS-R is present not only in the hypothalamus and the pituitary gland but also in several peripheral tissues (15, 28), suggesting pleiotropic effects of ghrelin.

Ghrelin is an acylated 28-amino acid peptide produced primarily in the gastric mucosa and the hypothalamic arcuate nucleus (18) but also in other tissues (15). As expected, administration of ghrelin is associated with pronounced stimulation of GH secretion. Additional endocrine effects of ghrelin include stimulation of adrenocorticotropic hormone (ACTH) and prolactin secretion (3, 33). There is accumulating evidence to support the hypothesis that ghrelin plays an important role in the regulation of food intake (8, 10, 26, 27, 32, 38), energy metabolism (2, 5, 36), and gastric motility (23), and, when administered to humans, ghrelin increases left ventricular function and improves vasodilation (12, 24, 25, 35). Moreover, in rodents, ghrelin administration induces adiposity (36), whereas absence of ghrelin or the GHS-R prevents the development of obesity (37, 40).

Circulating ghrelin levels increase before feeding followed by suppressed postprandial levels (8). In patients with anorexia nervosa, a compensatory increase in ghrelin levels are reported (34), but hyperphagia secondary to Prader-Willi syndrome seems to be driven by increased ghrelin secretion (7).

In the literature, ghrelin has so far been administered to ~300 healthy subjects and patients. Ghrelin bolus doses between 1 and 10 µg/kg body wt, administered subcutaneously (12), and ≤10 µg/kg body wt, administered intravenously (24), have resulted in systemic ghrelin increments from 2- to 61-fold. When ghrelin is infused at a rate of 5 pmol·kg body wt–1·min–1 (corresponding to 16.9 ng·kg body wt–1·min–1), ghrelin levels are usually reported to reach steady state after 60–90 min (9, 23, 27, 38).

To our knowledge only two articles have reported pharmacokinetic data (1, 24), and both describe first-order elimination in a one-compartment model with a half-life between 9 and 31 min.

Ghrelin levels are low in obesity (11, 19, 22). It remains to be studied whether this decrease is caused by increased ghrelin degradation or decreased secretion and if any biochemical variables predict ghrelin turnover. In this regard it is noteworthy that acylated ghrelin binds to high-density lipoproteins (HDL) in vitro (4).

In view of the potential therapeutic applications of ghrelin it is relevant to evaluate in more detail the pharmacokinetics and pharmacodynamics of exogenous ghrelin. In the present double-blind crossover study, 17 healthy young men received a 3-h constant intravenous infusion of ghrelin and placebo. This enabled calculation of mean steady-state levels, exponential coefficients, half-lives, rate of elimination, mean residence time (MRT), and distribution volume (VSS) of ghrelin. The pharmacodynamic measurements included the secretion of GH, prolactin, ACTH, insulin levels, and glucose homeostasis, as well as satiety and hunger.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study subjects. Seventeen healthy men aged 23.1 ± 0.4 yr with body mass indexes (BMIs) of 23.0 ± 0.3 kg/m2 volunteered in this study. None of the study participants was smoking or abusing alcohol or taking any medication. All had a normal physical examination. Fasting blood glucose, triglyceride, and cholesterol levels and hematological, renal, and hepatic functions assessed by biochemical screening were normal in all participants. Baseline characteristics are provided in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. Baseline characteristics of the subjects

 
The study was conducted in accordance with the Helsinki Declaration, and all subjects gave their oral and written informed consent to participate in the trial. The study protocol was approved by the local Ethics Committee of Aarhus County, the Danish Medicines Agency, and the Good Clinical Practice (GCP) Unit of Aarhus University Hospital. According to the International Committee of Medical Journal Editors, the protocol was registered (Clinicaltrials.gov; ID NCT00116025 [ClinicalTrials.gov] ) before the onset of enrolment.

Preparation of synthetic ghrelin. Synthetic human acylated ghrelin (NeoMPS, Strasbourg, France) was dissolved in isotonic saline and sterilized by double passage through a 0.8/0.2 µm pore size filter (Super Acrodisc; Gelman Sciences, Ann Arbor, MI) by the local hospital pharmacy.

Protocol. Subjects were studied at 0800 in a quiet, thermoneutral indoor environment following a minimum of 9 h of fasting. The subjects fasted during the trial. Two intravenous cannulae were inserted in the antecubital region, one for infusion and one for blood sampling. All subjects were examined on two occasions (ghrelin or placebo) in random order, separated by a minimum of 3 wk. In a randomized, double-blind, crossover design we used a constant infusion of human acylated ghrelin lasting 180 min (infusion period t = 0–180 min) at 5 pmol·kg–1·min–1 to investigate pharmacokinetic parameters. The intravenous line was kept patent during both ghrelin and placebo treatment by infusion of an isovolumetric load of isotonic saline at a rate of 1.69 ml·kg body wt–1·h–1. Blood samples were drawn every 20 min during the infusion period (t = 0–180 min). After termination of the infusion period, blood samples were drawn every 5 min for the first hour (t = 180–240 min) and at 10-min intervals for the last 2 h (t = 240–360 min).

To investigate the effects of 180 min of ghrelin infusion on appetite we used the same visual analog questionnaire (VAS), as previously reported (38). The subjects completed VAS ratings of 1) hunger, 2) satiety, 3) desire to eat, and 4) prospective food consumption. Hunger was defined as "the subjective driving force for the search for, choice of, and ingestion of food." Satiety was defined as "the sensation after eating so that a person does not feel the need to eat for some time afterward." The desire to eat was defined as "the person's subjective feeling of the need to consume a meal." The prospective food consumption was defined "as the meal size the subject imagined he/she could eat at the moment." Subjects were instructed to make a single vertical mark on a horizontal line (possible scores 0–100 mm) to indicate their current feelings, ranging between 1) "not hungry at all" and "maximal hunger," 2) between "not full at all" and "full," 3) between "no desire to eat" and "extreme desire to eat," and 4) "nothing" and "as much as possible." Baseline evaluations were collected prior to the infusion period (t = 0) and at termination of the infusion period (t = 180 min). A blinded observer measured the scores.

Analyses. Serum ghrelin (s-ghrelin; total levels) was measured in duplicate by an in-house assay, as described previously (13). The assay measures immunoreactive levels of ghrelin using 125I-labeled bioactive ghrelin tracer and rabbit polyclonal antibodies raised against octanoylated human ghrelin. The assay recognizes the COOH terminal of ghrelin and as such determines acylated as well as desacylated ghrelin. The intra-assay coefficient of variation averaged 2.8%, and samples from each individual were analyzed in one assay. A double commercial monoclonal immunofluorometric assay (DELFIA; PerkinElmer, Wallac, Turku, Finland) was used to measure serum growth hormone (s-GH), cortisol (s-cortisol), and insulin (s-insulin). Plasma glucose (p-glucose) levels were measured in duplicate on a glucose analyzer (Beckman Instruments, Palo Alto, CA). Levels of serum free fatty acids (s-FFA) were determined using a commercial kit (Wako Chemicals, Neuss, Germany). Plasma ACTH was determined using a commercial method (Immulite Diagnostic Product Scandinavia). Plasma albumin (p-albumin), total cholesterol, and HDL cholesterol levels were determined by commercial methods (Cobas Integra 800) using an immunoturbidimetric measurement, an enzymatic colorimetric, and a homogeneous enzymatic colorimetric method, respectively.

Pharmacokinetic parameters. The postinfusion serum ghrelin concentration time data from each subject were analyzed by nonlinear least-squares regression analysis using a monoexponential and a biexponential decay model. Pharmacokinetic analyses were done by means of the computer program GraphPad Prism v4.00 for Windows (GraphPad Software, San Diego, CA). The preferred model was chosen on the basis of visual inspection of the concentration time data and the extra sum-of-squares F-test. To correct the s-ghrelin values for naturally occurring ghrelin, the mean of the s-ghrelin measurements during placebo treatment were subtracted from each serum value.

A biexponential equation

Formula
representing a two-compartment open model with first-order elimination gave the best fit.

As ghrelin infusion was given over a 180-min period, the intercepts A' and B' were corrected according to the following equations:

Formula
and

Formula
where T is the infusion period (20).

The pharmacokinetic variables were then calculated using the corrected intercepts A and B and slopes {lambda}1 and {lambda}2.

The distribution half-life t1/2({lambda}1) and the elimination (terminal) half-life t1/2({lambda}2) were calculated as

Formula
and

Formula
respectively. The area under the serum concentration vs. time curve, AUC{infty}, was calculated as

Formula

The area under the first moment curve (AUMC) was calculated as

Formula

Total clearance (Cl), the volume of distribution of the central compartment (Vc), the volume of distribution during the terminal phase [V({lambda}2)], and the volume of distribution at steady state (Vss) were calculated as follows:

Formula

Formula

Formula
and

Formula

MRT was calculated as

Formula

Micro constants k12, k21, and k01 were calculated from

Formula

Formula
and

Formula

Simulation of the serum concentration during the infusion was done according to the following equation (31):

Formula
where R0 is the rate of infusion and f1 and f2 denominate the fraction of elimination associated with the first and last exponential term, respectively:

Formula
and

Formula

Serum concentration at steady state (CSS) was calculated as

Formula

Statistical analyses. Results are expressed as means ± SE. Systemic levels of FFA, glucose, insulin, GH, prolactin, ACTH, and cortisol were analyzed by two-way analysis of variance (ANOVA). The interaction between time and treatment ("time x treatment") was considered the term of interest. The Bonferroni correction was used to account for multiple comparisons. Data were examined by Student's two-tailed paired t-test when appropriate. Appetite scores were analyzed by comparing the changes in VAS by a paired t-test. In backward regressions analyses, BMI and total and HDL cholesterol levels were included as independent variables, and AUC, half-lives, k01, or Cl were included as dependent variables, respectively. A P value <0.05 was considered significant. Statistical analysis was performed using SPSS version 13.0 for Windows.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ghrelin pharmacokinetics. The mean total ghrelin serum concentrations (s-ghrelin) vs. time profile are shown in Fig. 1. According to comparison of two nested nonlinear models (1- and 2-phase exponential decay) by the extra sum-of-squares F-test, the pharmacokinetics of infused ghrelin was best fitted to the latter two-compartment model (F = 256.9, P < 0.0001).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Levels of total serum ghrelin above baseline vs. time. Data are means ± SE. Ghrelin was infused from t = 0 to 180 min. The curve from 180 to 360 min is the best-fit curve from nonlinear regression analysis, resulting in a 2-compartment model. The curve from 0 to 180 min is the infusion curve derived from the best-fit postinfusion pharmacokinetic parameters.

 
Summary pharmacokinetic parameters are presented in Table 2. Linear regression analysis revealed a significant increase in s-ghrelin levels after 100 min of ghrelin infusion (r = 0.94, P = 0.02). During this period we attained s-ghrelin Cmax (4.41 ± 0.29 µg/l) above baseline levels. The observed s-ghrelin Cmax level was not different from the level predicted by the model (expected C180 = 4.35 ± 0.28 µg/l, P = 0.45), but we did not reach a true steady-state level because the measured Cmax was significantly smaller than the predicted CSS (5.00 ± 0.35 µg/l, P = 0.003). The initial disposition rate constant, {lambda}1, was 0.033 ± 0.003/min, and the terminal rate constant, {lambda}2, was 0.007 ± 0.003/min. After termination of the ghrelin infusion, s-ghrelin was eliminated with Cl = 3.68 ± 0.30 ml·kg–1·min–1. The initial and terminal s-ghrelin half-lives, t1/2({lambda}1) and t1/2({lambda}2), were 24.2 ± 2.5 and 146.0 ± 35.6 min, respectively. The fraction of ghrelin eliminated from the terminal phase (f2) was 33 ± 5%. MRT was 92.7 ± 16.3 min and VSS was 0.35 ± 0.06 l/kg.


View this table:
[in this window]
[in a new window]

  		Table 2. Pharmacokinetic parameters of infused ghrelin

 
Hormones and metabolites. Circulating levels of FFA, GH, glucose, prolactin, insulin, ACTH, and cortisol are depicted in Fig. 2. FFA increased after 40 min of ghrelin infusion (0.57 ± 0.06 vs. 0.37 ± 0.06 mmol/l, P = 0.04) to levels almost twice as high as those after placebo at t = 200 min (0.97 ± 0.08 vs. 0.56 ± 0.07 mmol/l, P < 0.001) and were normalized by termination of the postinfusion period (t = 360 min). As expected, serum GH increased rapidly in response to ghrelin infusion and reached the peak levels after 60 min of infusion (36.8 ± 4.7 vs. 1.5 ± 0.6 ng/ml, P < 0.001). Eighty minutes after termination of ghrelin infusion and throughout the study we observed a rebound decrease of GH levels. Plasma glucose increased ~0.3 ± 0.1 mmol/l after 120 min of ghrelin infusion (P < 0.01) and returned to baseline levels shortly after termination of ghrelin infusion. Serum prolactin increased after 60 min (9.8 ± 0.6 vs. 4.3 ± 0.2 µg/l, P < 0.001) and remained elevated throughout the infusion period. Serum ACTH more than doubled after 60 min of ghrelin infusion compared with placebo (25.8 ± 3.5 vs. 11.5 ± 1.4 ng/ml, P = 0.002), followed by a decline toward baseline after 180 min. Serum cortisol increased after 60 min of ghrelin infusion (336.3 ± 28.9 vs. 212.9 ± 20.6 nmol/l, P < 0.01), and the elevation persisted throughout the infusion period. Cortisol levels returned to baseline 80 min after termination of the ghrelin infusion, whereafter a further decrease was recorded at t = 360 min (150.1 ± 20.3 vs. 198.6 ± 15.0 nmol/l, P = 0.02). Serum insulin levels remained constant during ghrelin infusion despite elevation in p-glucose levels. At t = 180 min, insulin levels increased (24.1 ± 2.4 vs. 17.3 ± 2.1 pmol/l, P = 0.004), and the elevation was sustained throughout the postinfusion period. Overall, ANOVA for repeated measurements uncovered a highly significant increase in insulin levels.


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 2. The hormonal and metabolite responses to ghrelin and placebo infusion. All measured variables revealed a significant interaction of time and treatment [P (time x treatment interaction) < 0.001; A–H]. Asterisks in A–H refer to post hoc comparison between treatment groups (ghrelin vs. placebo) at different time points. A: serum levels of free fatty acids (FFA); *P < 0.05. B: serum growth hormone (GH); *P <0.05. C: plasma glucose; *P < 0.05. D: serum prolactin; *P < 0.01. E: serum insulin; *P < 0.01. F: serum ACTH; *comparison at t = 60 min, P = 0.002. G: insulin sensitivity as determined as revised QUICKI; *P < 0.05. H: serum cortisol; *P < 0.05.

 
Insulin sensitivity. Insulin sensitivity as determined by the revised QUICKI method (30) decreased after 40 min of ghrelin infusion and outlasted both the infusion period and the postinfusion interval [P (interaction of ghrelin) < 0.001; Fig. 2G].

Correlations. MRT correlated positively with both BMI (r = 0.51, P < 0.001; Fig. 3A) and HDL cholesterol (r = 0.75, P < 0.001; Fig. 3B). The correlation between MRT and BMI was to a large extent carried by the two observations with the highest MRTs. If those observations were removed, the relationship was still significant (r = 0.53, P = 0.04). However, backward regressions analyses revealed no correlation between total cholesterol levels, HDL cholesterol levels, and BMI as independent variables and AUC, half-lives, k01, or Cl as dependent variables, respectively.


Figure 3
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 3. A: correlation analysis between mean residence time and body mass index (BMI). B: correlation analysis between mean residence time and high-density lipoprotein (HDL) cholesterol levels.

 
Appetite. The VAS scores at baseline were comparable between the two study days. The VAS scores increased during both placebo and ghrelin infusion and only reached significantly higher levels in the response to ghrelin with regard to "lack of satiety" (P = 0.03; Fig. 4).


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 4. Visual analog questionnaire (VAS) scores. The vertical bars depict the increase in appetite scores after 180 min of ghrelin and placebo infusion, respectively. The VAS score was increased only with regard to the lack of satiety (VAS 2) but not to hunger (VAS 1), desire to eat (VAS 3), or prospective food consumption (VAS 4).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that the distribution kinetics of constantly infused ghrelin follows a two-compartment model with a biexponential decay curve after termination of the infusion. Of note, we recorded significant positive correlations between the mean residence time of total ghrelin levels and both BMI and HDL.

Endogenous ghrelin exhibits a distinct ultradian rhythm; in fact, frequent blood sampling has revealed up to 22 pulses/24 h (39). The rapid and dynamic ghrelin fluctuations suggest that the half-life of ghrelin is short. Indeed, Nagaya et al. (24) reported that total ghrelin levels disappeared from plasma with a half-life of 10 min after a bolus injection. The pharmacokinetics of ghrelin were further analyzed by Akamizu et al. (1), who reported half-lives of both total (t1/2 27 to 31 min) and acylated plasma ghrelin (t1/2 9 to 13 min) in a one-compartment model following two different doses of ghrelin bolus injections and blood samples taken every 15 min. These previously reported half-lives of total ghrelin are in line with the initial half-life (t1/2 24.2 min) in our present study.

We compared a mono- and a biexponential description of the decay curve of total ghrelin levels after termination of a 180-min infusion period. By statistical means we found a significantly better description using a biexponential equation. This contrasts former pharmacokinetic ghrelin studies favoring the monoexponential model (1, 24). One explanation for this apparent discrepancy can be attributed to the frequency of blood sampling, since the concentration-time curve appears monophasic when the fraction of a substance eliminated by the last exponential term (f2) is relatively large (in this study 33%) and blood is drawn less frequently. To circumvent this problem, we collected blood samples every 5 min for the first hour after termination of the ghrelin infusion to detect the two distinct slopes of the decay curve.

Based on the estimated pharmacokinetic parameters from the biexponential decay curve, we simulated the expected concentration-time curve during the infusion period. The concentration-time curve even tended to overestimate the experimentally obtained ghrelin levels, indicating that simplifying distribution kinetics of ghrelin to a one-compartment model is inadequate. However, by means of the estimated pharmacokinetic parameters and the two-compartment model, the currently presented mathematical model adequately explains our experimental observations. Peak ghrelin concentration observed was very similar to the mathematically predicted level, but a steady-state level was not reached. Our observations contrast previous ghrelin infusion studies reporting steady state within 60–90 min (9, 23, 27, 38). However, none of the previous reports provides the pharmacokinetic approach, making it difficult to draw direct comparisons with our results.

We measured total ghrelin levels only. Whether acylated ghrelin degrades to desacylated ghrelin remains to be convincingly demonstrated. If so, desacylated ghrelin levels should increase when the fatty acid side chain of acylated ghrelin is cleaved off during breakdown. With respect to this, divergent observations exist; one in vitro study revealed no increase in desacylated ghrelin levels along the degradation of acylated ghrelin (17), whereas the study by Akamizu et al. (1) indicated that acylated ghrelin accounted for only ~50% of the increase in total ghrelin levels after administration of acylated ghrelin. Gauna et al. (14) detected higher total serum ghrelin levels after administration of acylated ghrelin than after an equal dose of desacylated ghrelin. They suggested that the apparent increase in desacylated ghrelin following administration of acylated ghrelin was not solely explained by degradation of acylated to desacylated ghrelin but rather stemmed from endogenous release of desacylated ghrelin.

In the present study, we revealed a positive correlation between BMI and the MRT of ghrelin. Thus, the counterregulatory decline in ghrelin levels that presumably serves to compensate for a positive energy expenditure in obese individuals (11, 19, 22) is probably caused by a decrease in ghrelin secretion rather than an increase in ghrelin degradation. This is also in accord with the observation that a constant ghrelin infusion increases circulating ghrelin levels more in obese than in lean subjects (10), and it is compatible with previous studies (11, 19, 22) reporting that obese individuals have a reduced postprandial decrease in ghrelin levels compared with lean subjects.

We also revealed a positive correlation between MRT and HDL cholesterol levels. Although correlation does not imply causality, it has previously been shown that the majority of circulating acylated ghrelin is bound to larger molecules (29), and the HDL fraction has been demonstrated to bind acylated ghrelin in vitro (4). HDL cholesterol levels could thus be an independent biological determinant of ghrelin bioavailability in humans.

After 180 min of sustained supraphysiological ghrelin levels we detected a significant decrease in the sensation of "satiety" but no effects on either hunger, the desire to consume a meal, or an individual expected amount of food to be consumed if an ad libitum meal was served. This contrasts the more pronounced orexigenic effects of ghrelin reported previously (10, 27, 32, 38). Temporal and concentration differences between the earlier reports and the present results may help to explain these discrepancies. Wren et al. (38) studied the orexigenic and appetite effects of more physiological increments (~2-fold elevations from baseline), significantly less than the 6.5-fold increase we obtained. High ghrelin levels may entail compensatory mechanisms, such as internalization of the GHS-R from the cell surface, to desensitize the cell responsiveness. The internalization is maximal after ~20 min and the receptor level rises to basal levels after 360 min following removal of its agonist (6). The acute orexigenic effects of ghrelin administration were demonstrated by Druce et al. (10) and Schmid et al. (32). They observed significant orexigenic effects after 45 and 60 min following initiation of ghrelin administration, respectively. Moreover, 90 min of ghrelin infusion caused increased energy intake and meal appreciation in cancer patients suffering cachexia (27).

As expected, we observed significant elevations in circulating levels of GH, ACTH, cortisol, prolactin, glucose, and FFA, in accord with most previous reports (1, 3, 9, 10, 12, 14, 18, 21, 24, 25, 27, 32, 33, 38). We also recorded a small but significant increase in plasma glucose levels, but in contrast to one previous study (5) this was associated with an increase rather than a decrease in insulin levels. This translated into a decrease in insulin sensitivity following ghrelin infusion, as estimated by the revised QUICKI model (30). The decrease was evident after only 40 min and lasted throughout the study period. This effect may partly be attributable to the increase in GH levels. Interestingly, the reduced insulin sensitivity remained after normalization of both GH and glucose levels, suggesting that the effect could be caused by the ghrelin infusion per se. The present results are in accord with earlier observations (14) reporting ghrelin-induced insulin resistance. In that study, concomitant GH administration surprisingly ameliorated the impairment of metabolic control induced by ghrelin. This apparent discrepancy may be attributable to the differences in ghrelin doses and thus differences in GH responses and intrinsic effects of ghrelin. At first glance, the increase in cortisol levels contrasts the previous established inverse correlation between ghrelin and cortisol observed during fasting (13). However, the ACTH concentration-time curve reveals a transient increase in ACTH levels after 60 min of ghrelin infusion only, whereafter ACTH levels return to baseline levels. The present ghrelin infusion period is not sufficient to disclose any effect of ghrelin on cortisol levels after normalization of ACTH levels, but the decrease in cortisol levels at t = 360 min indicates that the long-term effect of ghrelin may actually be to inhibit cortisol levels.

An important limitation applies to the present results. We measured total ghrelin levels only, and as such we report the resulting levels of both acylated and desacylated ghrelin probably along with other molecules possessing ghrelin-like immunoreactivity.

In conclusion, the kinetics of infused ghrelin follow a two-compartment model, and the MRT of infused ghrelin correlates positively with both HDL cholesterol and BMI. Supraphysiological ghrelin levels decrease insulin sensitivity. It remains to be convincingly demonstrated whether the reduced insulin sensitivity is caused by ghrelin per se or by the concomitant increase in GH levels. The present data are useful when tailoring ghrelin infusion rates in clinical experiments.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by grants from the World Anti-Doping Agency, Research Initiative of Aarhus University Hospital, an unrestricted educational grant from Novo Nordisk, and The John and Birthe Meyer Foundation.


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Susanne Sorensen and Merete Moller was highly appreciated. The GCP Unit of Aarhus University Hospital is acknowledged for monitoring that GCP guidelines were followed.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. T. Vestergaard, Medical Dept. M (Endocrinology and Diabetes), Aarhus University Hospital, Dk-8000 Aarhus C, Denmark (e-mail: e.t.vestergaard{at}ki.au.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akamizu T, Takaya K, Irako T, Hosoda H, Teramukai S, Matsuyama A, Tada H, Miura K, Shimizu A, Fukushima M, Yokode M, Tanaka K, Kangawa K. Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects. Eur J Endocrinol 150: 447–455, 2004.[Abstract]
  2. Arosio M, Ronchi CL, Gebbia C, Cappiello V, Beck-Peccoz P, Peracchi M. Stimulatory effects of ghrelin on circulating somatostatin and pancreatic polypeptide levels. J Clin Endocrinol Metab 88: 701–704, 2003.[Abstract/Free Full Text]
  3. Arvat E, Maccario M, Di Vito L, Broglio F, Benso A, Gottero C, Papotti M, Muccioli G, Dieguez C, Casanueva FF, Deghenghi R, Camanni F, Ghigo E. Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin Endocrinol Metab 86: 1169–1174, 2001.[Abstract/Free Full Text]
  4. Beaumont NJ, Skinner VO, Tan TM, Ramesh BS, Byrne DJ, MacColl GS, Keen JN, Bouloux PM, Mikhailidis DP, Bruckdorfer KR, Vanderpump MP, Srai KS. Ghrelin can bind to a species of high density lipoprotein associated with paraoxonase. J Biol Chem 278: 8877–8880, 2003.[Abstract/Free Full Text]
  5. Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, Van der Lely AJ, Deghenghi R, Ghigo E. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 86: 5083–5086, 2001.[Abstract/Free Full Text]
  6. Camina JP. Cell biology of the ghrelin receptor. J Neuroendocrinol 18: 65–76, 2006.[CrossRef][Web of Science][Medline]
  7. Cummings DE, Clement K, Purnell JQ, Vaisse C, Foster KE, Frayo RS, Schwartz MW, Basdevant A, Weigle DS. Elevated plasma ghrelin levels in Prader Willi syndrome. Nat Med 8: 643–644, 2002.[CrossRef][Web of Science][Medline]
  8. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50: 1714–1719, 2001.[Abstract/Free Full Text]
  9. Damjanovic SS, Lalic NM, Pesko PM, Petakov MS, Jotic A, Miljic D, Lalic KS, Lukic L, Djurovic M, Djukic VB. Acute effects of ghrelin on insulin secretion and glucose disposal rate in gastrectomized patients. J Clin Endocrinol Metab 91: 2574–2581, 2006.[Abstract/Free Full Text]
  10. Druce MR, Wren AM, Park AJ, Milton JE, Patterson M, Frost G, Ghatei MA, Small C, Bloom SR. Ghrelin increases food intake in obese as well as lean subjects. Int J Obes Relat Metab Disord 29: 1130–1136, 2005.[CrossRef][Web of Science][Medline]
  11. English PJ, Ghatei MA, Malik IA, Bloom SR, Wilding JP. Food fails to suppress ghrelin levels in obese humans. J Clin Endocrinol Metab 87: 2984, 2002.[Abstract/Free Full Text]
  12. Enomoto M, Nagaya N, Uematsu M, Okumura H, Nakagawa E, Ono F, Hosoda H, Oya H, Kojima M, Kanmatsuse K, Kangawa K. Cardiovascular and hormonal effects of subcutaneous administration of ghrelin, a novel growth hormone-releasing peptide, in healthy humans. Clin Sci (Lond) 105: 431–435, 2003.[Medline]
  13. Espelund U, Hansen TK, Hojlund K, Beck-Nielsen H, Clausen JT, Hansen BS, Orskov H, Jorgensen JO, Frystyk J. Fasting unmasks a strong inverse association between ghrelin and cortisol in serum: studies in obese and normal-weight subjects. J Clin Endocrinol Metab 90: 741–746, 2005.[Abstract/Free Full Text]
  14. Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, Van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, Van der Lely AJ. Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 89: 5035–5042, 2004.[Abstract/Free Full Text]
  15. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87: 2988–2991, 2002.[Abstract/Free Full Text]
  16. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Van der Ploeg LH. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273: 974–977, 1996.[Abstract]
  17. Kanamoto N, Akamizu T, Hosoda H, Hataya Y, Ariyasu H, Takaya K, Hosoda K, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K. Substantial production of ghrelin by a human medullary thyroid carcinoma cell line. J Clin Endocrinol Metab 86: 4984–4990, 2001.[Abstract/Free Full Text]
  18. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656–660, 1999.[CrossRef][Medline]
  19. le Roux C, Patterson M, Vincent R, Hunt C, Ghatei M, Bloom S. Postprandial plasma ghrelin is suppressed proportional to meal calorie content in normal weight but not obese subjects. J Clin Endocrinol Metab 90: 1068–1071, 2004.[CrossRef][Web of Science][Medline]
  20. Loo JC, Riegelman S. Assessment of pharmacokinetic constants from postinfusion blood curves obtained after I.V. infusion. J Pharm Sci 59: 53–55, 1970.[CrossRef][Web of Science][Medline]
  21. Lucidi P, Murdolo G, Di Loreto C, Parlanti N, De Cicco A, Fatone C, Taglioni C, Fanelli C, Broglio F, Ghigo E, Bolli GB, Santeusanio F, De Feo P. Metabolic and endocrine effects of physiological increments in plasma ghrelin concentrations. Nutr Metab Cardiovasc Dis 15: 410–417, 2005.[CrossRef][Web of Science][Medline]
  22. Marzullo P, Caumo A, Savia G, Verti B, Walker GE, Maestrini S, Tagliaferri A, Di Blasio AM, Liuzzi A. Predictors of postabsorptive ghrelin secretion after intake of different macronutrients. J Clin Endocrinol Metab 91: 4124–4130, 2006.[Abstract/Free Full Text]
  23. Murray CD, Martin NM, Patterson M, Taylor S, Ghatei MA, Kamm MA, Johnston C, Bloom SR, Emmanuel AV. Ghrelin enhances gastric emptying in diabetic gastroparesis: a double-blind, placebo-controlled, cross- over study. Gut 54: 1693–1698, 2005.[Abstract/Free Full Text]
  24. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, Kangawa K. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol 280: R1483–R1487, 2001.[Abstract/Free Full Text]
  25. Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda H, Kojima M, Nakanishi N, Mori H, Kangawa K. Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol Metab 86: 5854–5859, 2001.[Abstract/Free Full Text]
  26. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 409: 194–198, 2001.[CrossRef][Medline]
  27. Neary NM, Small CJ, Wren AM, Lee JL, Druce MR, Palmieri C, Frost GS, Ghatei MA, Coombes RC, Bloom SR. Ghrelin increases energy intake in cancer patients with impaired appetite: acute, randomized, placebo-controlled trial. J Clin Endocrinol Metab 89: 2832–2836, 2004.[Abstract/Free Full Text]
  28. Papotti M, Ghe C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G. Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab 85: 3803–3807, 2000.[Abstract/Free Full Text]
  29. Patterson M, Murphy KG, le Roux CW, Ghatei MA, Bloom SR. Characterization of ghrelin-like immunoreactivity in human plasma. J Clin Endocrinol Metab 90: 2205–2211, 2005.[Abstract/Free Full Text]
  30. Perseghin G, Caumo A, Caloni M, Testolin G, Luzi L. Incorporation of the fasting plasma FFA concentration into QUICKI improves its association with insulin sensitivity in nonobese individuals. J Clin Endocrinol Metab 86: 4776–4781, 2001.[Abstract/Free Full Text]
  31. Rowland M, Tozer TN. Clinical Pharmacokinetics: Concepts and Applications. Philadelphia, PA: Lippincott Williams and Wilkins, 1995, p. 313–335.
  32. Schmid DA, Held K, Ising M, Uhr M, Weikel JC, Steiger A. Ghrelin stimulates appetite, imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology 30: 1187–1192, 2005.[CrossRef][Web of Science][Medline]
  33. Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab 85: 4908–4911, 2000.[Abstract/Free Full Text]
  34. Tanaka M, Naruo T, Yasuhara D, Tatebe Y, Nagai N, Shiiya T, Nakazato M, Matsukura S, Nozoe S. Fasting plasma ghrelin levels in subtypes of anorexia nervosa. Psychoneuroendocrinology 28: 829–835, 2003.[CrossRef][Web of Science][Medline]
  35. Tesauro M, Schinzari F, Iantorno M, Rizza S, Melina D, Lauro D, Cardillo C. Ghrelin improves endothelial function in patients with metabolic syndrome. Circulation 12: 2986–2992, 2005.
  36. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 407: 908–913, 2000.[CrossRef][Medline]
  37. Wortley KE, Del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, Sleeman MW. Absence of ghrelin protects against early-onset obesity. J Clin Invest 115: 3573–3578, 2005.[CrossRef][Web of Science][Medline]
  38. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86: 5992–5995, 2001.[Abstract/Free Full Text]
  39. Yildiz BO, Suchard MA, Wong ML, McCann SM, Licinio J. Alterations in the dynamics of circulating ghrelin, adiponectin, and leptin in human obesity. Proc Natl Acad Sci USA 101: 10434–10439, 2004.[Abstract/Free Full Text]
  40. Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB, Elmquist JK. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest 115: 3564–3572, 2005.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 DiabetesHome page
E. T. Vestergaard, L. C. Gormsen, N. Jessen, S. Lund, T. K. Hansen, N. Moller, and J. O. L. Jorgensen
Ghrelin Infusion in Humans Induces Acute Insulin Resistance and Lipolysis Independent of Growth Hormone Signaling
Diabetes, December 1, 2008; 57(12): 3205 - 3210.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
E. T. Vestergaard, N. H. Andersen, T. K. Hansen, L. M. Rasmussen, N. Moller, K. E. Sorensen, E. Sloth, and J. O. L. Jorgensen
Cardiovascular effects of intravenous ghrelin infusion in healthy young men
Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3020 - H3026.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/E1829    most recent
00682.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vestergaard, E. T.
Right arrow Articles by Jorgensen, J. O. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vestergaard, E. T.
Right arrow Articles by Jorgensen, J. O. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.