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Medical Research Laboratories (U.E., T.K.H., H.Ø., J.O.L.J., J.F.), Clinical Institute and Medical Department M, Aarhus University Hospital, DK-8000 Aarhus C, Denmark; Diabetes Research Centre (K.H., H.B.-N.), Department of Endocrinology, Odense University Hospital, DK-5000 Odense C, Denmark; Department of Assay and Cell Technology (J.T.C.), Research and Development, Novo Nordisk A/S, DK-2880 Bagsværd, Denmark; and Biochemistry and Metabolism (B.S.H.), Novo Nordisk A/S, DK-2760 Måløv, Denmark
Address all correspondence and requests for reprints to: Jan Frystyk, M.D., Ph.D., D.M.Sc., Medical Research Laboratories, Aarhus University Hospital, Aarhus Sygehus, Building #3, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: jan{at}frystyk.dk.
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Abstract |
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Thirty-three young adults, subdivided according to gender and level of obesity, were studied with blood sampling every 3 h from 12–84 h of fasting. Serum ghrelin levels showed a marked diurnal rhythm with a nadir in the morning (0800 h), peak levels in the afternoon, and a gradual decline during the night. This pattern was preserved during the entire fasting period and was independent of gender and obesity. Mean 24-h ghrelin levels exhibited a small but significant decline during the fast (P < 0.001). As expected, GH secretion increased with fasting in lean subjects, and a gradual decline in insulin concentrations was observed in all subjects. Neither GH nor insulin showed any significant relationship to ghrelin. In contrast, serum cortisol exhibited a strong inverse temporal association with ghrelin (r = –0.79; P < 0.0001).
In conclusion, our study yields no evidence that ghrelin stimulates GH release during fasting. As a novel finding, ghrelin appears to be related to cortisol. However, further studies are needed to elucidate the physiological mechanisms behind this relationship.
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Introduction |
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Interestingly, ghrelin levels are significantly altered during acute and chronic aberrations in nutritional status. Thus, ghrelin levels are low in simple obesity but increase after weight loss (11, 12), and in anorexia nervosa, levels are highly elevated (13). In addition, ghrelin levels appear to increase before meal intake followed by low postprandial levels, which has led to the suggestion that ghrelin may play a role in appetite regulation and meal initiation (14). This hypothesis is substantiated by a number of experimental studies in animal models (3, 15, 16), and orexigenic effects of exogenous ghrelin have also been documented in human subjects (17).
Studies reported so far have yielded ambiguous results regarding the possible role of ghrelin in the metabolic and endocrine adaptations to prolonged fasting. Based on single measurements, Nørrelund et al. (6) did not record significant changes in ghrelin concentrations during 36-h fasting in either normal subjects or GH-deficient patients. Muller et al. (18) reported that fasting for 3 d unmasked a diurnal rhythm in ghrelin characterized by low levels in the morning and subsequent elevations in the afternoon and at midnight. The latter study was, however, difficult to evaluate because a synthetic GH secretagogue was administered before and during fasting, and plasma ghrelin levels were only measured every 6 h in a relatively small number of subjects (n = 10) (18).
In the present study, we performed measurements of ghrelin every 3 h during 84 h of fasting in 33 healthy subjects. Our study design enabled a comparison between lean and obese subjects as well as between males and females. Our data clearly suggest that fasting unmasks a hitherto unrecognized, very strong inverse correlation between the circulating levels of ghrelin and cortisol.
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Subjects and Methods |
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The study included 33 healthy Caucasian volunteers with no medical history of chronic illness or malignancy (Tables 1
and 2). Two to three weeks before the study, all participants underwent a thorough medical examination, including blood pressure, electrocardiogram, and biochemical parameters (hemoglobin, creatinine, electrolytes, liver enzymes, and lipid composition). Finally, all subjects had passed an oral glucose tolerance test. No medication was taken except oral contraceptives. The participants were grouped as lean if their body mass index (BMI) was less than 27 kg/m2 for males and less than 25 kg/m2 for females (Table 2). Lean and obese subjects were matched with respect to gender and age. Data from the study focusing on ß-cell polypeptides and counter-regulatory hormones have previously been published (19).
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All subjects underwent 84 h of fasting. The participants started fasting at 2200 h at home. The next morning, they were admitted at the Department of Endocrinology, Odense University Hospital (Odense C, Denmark). During the 72-h stay at the hospital, venous blood samples were collected every 3 h. The participants had free access to tap water and were allowed normal ambulatory activity, but each blood sample was preceded by 30 min of rest. The level of hydration and compliance with the fast were assessed through measurements of serum albumin, free fatty acids, glucose, and insulin. None of the participants experienced any side effects to fasting. The study was approved by the local ethics committee and was performed in accordance with the Helsinki Declaration. Informed written consent was obtained from each participant.
Biochemical analyses
All blood samples were centrifuged immediately at 4 C and serum stored at –20 C before analysis. Care was taken to analyze all samples from each patient within the same run. All samples were analyzed in duplicate; the only exception was GH, which was analyzed in single determination due to a very low intraassay coefficient of variation. Serum insulin and GH were measured by commercial noncompetitive time-resolved immunofluorometric assays (PerkinElmer Life Sciences, Turku, Finland). Serum cortisol was measured by RIA (Orion Diagnostica, Espoo, Finland).
Serum ghrelin was determined by an in-house RIA. Breakable Maxisorb microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 5 C with a polyclonal donkey antirabbit IgG (5 µg/ml, 200 µl per well; Sigma-Aldrich, Copenhagen, Denmark) dissolved in phosphate buffer (40 mM, pH 8.0). After coating, plates were washed once using 50 mM Tris-HCl buffer (pH 8.0) plus 0.9% (wt/vol) NaCl, 0.5% (vol/vol) Tween 20, and 0.05% (wt/vol) NaN3; the plates were then blocked for 2 h at room temperature with 300 µl of 40 mM phosphate buffer plus 1% BSA (Sigma-Aldrich), 0.05% (wt/vol) NaN3, and 0.6% (wt/vol) NaCl. We used 100 µl of standard (a serial dilution of recombinant human octanoylated ghrelin obtained from Novo Nordisk A/S, Bagsværd, Denmark) or undiluted serum. In addition, 50 µl of 125I-labeled octanoylated ghrelin (10,000 cpm per well; Peninsula Laboratories Inc., Division of Bachem, San Carlos, CA) and 50 µl of a polyclonal ghrelin antibody (Novo Nordisk A/S; final dilution, 1:4,800) were added. Standards were dissolved in assay buffer [40 mM phosphate buffer, 0.05% (wt/vol) NaN3, 0.9% (wt/vol) NaCl, and 2% (vol/vol) Tween 20] containing 5% BSA, whereas tracer and specific antibody were dissolved in assay buffer containing 0.2% BSA. All serum samples and standards were analyzed in duplicate except for nonspecific binding and standard zero, which were analyzed in quadruplicate. The plates were incubated for 2 d at 5 C, washed three times, and counted for 5 min in a 
-counter. The assay gave similar results to those obtained with a commercially available RIA kit from Phoenix Pharmaceuticals Inc. (St. Joseph, MO). The lower detection limit was estimated to approximately 0.125 µg/liter, the half-maximal displacement occurred at approximately 2 µg/liter, and the upper standard was 10 µg/liter. The recovery of a serial dilution of recombinant octanoylated ghrelin added to serum was 117 ± 13% (mean ± SD). The intraassay coefficient of variation averaged less than 5%, and all samples were analyzed within the same run. Further evaluation of our in-house ghrelin RIA has been published previously (20).
Statistics
The Kolmogorov-Smirnov test was used to test for normality of data. For continuous variables, comparisons between groups were performed by an unpaired Student’s t test or the Mann-Whitney U test, whereas a 
2 test was used for comparison of noncontinuous variables between groups. Integrated 24-h concentrations of hormones were estimated as the areas under the time-concentration curves using the linear trapezoidal rule, and two-way ANOVA for repeated measurements with time as a within-subject factor was performed to assess changes with time. Cross-correlation analysis was used to investigate possible significant correlations and synchronicities between time series of ghrelin and other hormones. Pearson’s product moment correlation with two-tailed probability values was subsequently used to measure the strength of association between the variables at any given time point. Data are given as the mean ± SEM, and statistical significance was assumed for P < 0.05. All statistical calculations were performed with SPSS for Windows version 11.0 (SPSS, Chicago, IL).
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Results |
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and 4. Males had significantly higher serum glucose levels and lower GH values than females (P < 0.05; Table 3). The obese subjects had normal blood glucose levels but higher insulin levels compared with the lean subjects (P < 0.05; Table 4).
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), with trough values in the mornings (at 0800 h) and maximum values in the afternoon. From early evening, ghrelin declined gradually toward trough values in the morning. This diurnal rhythm was observed in all subjects (Fig. 1A), irrespective of gender (Fig. 2A) and BMI (Fig. 2B).
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). However, serum ghrelin remained significantly higher in women than men throughout the study (P < 0.0001; Fig. 2C). We did not observe any significant differences between lean and obese subjects (Fig. 2, B and D).
Serum levels of cortisol, GH, and insulin have been published previously, but the results are included in the present study for comparative reasons (19). Serum cortisol changed opposite to ghrelin, reaching peak levels every morning at 0800 h and trough values around midnight (Fig. 1). Serum insulin decreased steadily during the fast, with the exception of the last sample. Serum C-peptide, proinsulin, and glucose also increased at the end of the fast (data not shown). This finding is believed to result from increased activity of vagal efferents, activated by the anticipation of food after 84 h of fasting (19). The integrated GH levels increased during fasting but only in lean subjects (34 ± 5 vs. 71 ± 11 vs. 51 ± 6 µg/liter·h, d 1 vs. d 2 vs. d 3; P < 0.01). In obese subjects, integrated 24-h GH levels remained unchanged during the 3 study days (33 ± 9 vs. 44 ± 10 vs. 27 ± 5 µg/liter·h; d 1 vs. d 2 vs. d 3; P = not significant).
Cross-correlation analysis revealed a strong negative correlation between serum ghrelin and serum cortisol with no time lag (i.e. variations occurring in phase, at least with the 3-h sample rate used in this study). Neither GH nor insulin showed any significant cross-correlations with ghrelin. Because leptin shows a known reciprocal rhythm with cortisol (21), we subsequently tested whether a correlation existed between the circulating levels of ghrelin and leptin in the present study. However, no significant correlation was recorded between ghrelin and leptin when measured every 6 h in male participants (r = –0.04; P = 0.9). By contrast, in the same subgroup, the strong correlation between ghrelin and cortisol was reproduced (r = –0.82; P < 0.0001).
Linear regression analyses were subsequently used to assess the relationship between ghrelin and cortisol concentrations in the 25 samples taken in each individual subject. There was a significant negative correlation between ghrelin and cortisol concentrations in 25 subjects, with regression coefficients ranging from –0.40 to –0.79. The regression coefficients were also negative in another seven subjects (range, –0.11 to –0.38), however, without reaching statistical significance. Composite regressions based on individual mean values of the hormones at the 25 different time points also showed a strong inverse relationship between cortisol and ghrelin, whereas neither GH nor insulin correlated with ghrelin (Fig. 1B).
The tight inverse relationship between ghrelin and cortisol prompted us to examine the possible cross-reactivity of ghrelin in the cortisol assay and vice versa. Addition of a serial dilution of ghrelin (up to 10 µg/liter) did not affect the measurement of cortisol. Addition of a serial dilution of cortisol (up to 500 nM) did not yield any detectable signal in the ghrelin RIA, and only at cortisol concentrations of 1000 nM, a low specific signal equivalent to 0.03 µg/liter was detectable in the ghrelin RIA.
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Discussion |
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Based on blood sampling every 8 h, Muller et al. (18) recorded comparable fluctuations in ghrelin levels during 3 d of fasting, which they hypothesized to elicit the concomitant increase in GH secretion. In contrast, Nørrelund et al. (6) detected no major changes in ghrelin levels at the end of a 36-h fast. Taken together with the present findings, the available data indicate that the amplification of GH release during fasting is not caused by ghrelin stimulation.
Purnell et al. (22) failed to show any circadian relationship between circulating ghrelin and cortisol in 31 healthy subjects. In that study, blood samples were collected every 30 min for 24 h, but in contrast to our study, the participants were not fasting, as they received three meals during the daytime. In a newly reported study, Chan et al. (25) described the circadian profiles of ghrelin and cortisol in 72-h fasted healthy subjects (n = 6) who were sampled every 15 min for 24 h. In that study, the 24-h variation in serum cortisol was fully comparable to that observed in the present study (i.e. decreasing levels during the day and increasing levels during the night, reaching peak values at 0800 h). In accordance with our data, serum ghrelin showed an approximately 2-fold diurnal variation. In contrast with our data, serum ghrelin levels started to increase in the evening (at 1900 h) and reached a trough at 0400 h. The reason for this discrepancy remains unexplained. At any rate, the observations that fasting unmasks an inherent circadian rhythm raise important questions about the regulation and action of ghrelin. In the fed state, the time course of plasma ghrelin is reciprocal to insulin (14), whereas no significant relationship between these hormones exists during fasting. Moreover, the fasting-induced fluctuations in ghrelin levels are not associated with blood glucose levels. This indicates that neither insulin nor glucose levels are pivotal regulators of ghrelin secretion. The temporal inverse association between ghrelin and cortisol during fasting has not previously been reported, and the observational nature of our study does not provide insight into the underlying mechanisms. However, very recent data from Otto et al. (26) support our findings. In five patients with Cushing’s syndrome, overnight plasma ghrelin was low preoperatively and increased by 27% after successful tumor removal (postoperative interval ranged from 3–24 months). Furthermore, in healthy subjects, short-term treatment with prednisolone (30 mg daily for 5 d) suppressed overnight plasma ghrelin by 18%. With these results at hand, the authors concluded that both endogenous and exogenous hypercortisolism suppress circulating ghrelin levels (26). This hypothesis is in line with our observations.
From our observational data, it could also be speculated that ghrelin directly regulates (suppresses) cortisol secretion. The GHS-R is expressed in adrenal glands, and a recent in vitro study has demonstrated ghrelin mRNA expression as well as ghrelin binding sites in zona glomerulosa and outer zona fasciculata of human adrenal glands (27). However, the authors could not observe any effect of ghrelin on either basal or ACTH-stimulated steroid hormone synthesis from adrenocortical slices (27).
Our obese cohort showed a blunted GH response to fasting, and they had higher levels of glucose, insulin, proinsulin, and C-peptide at baseline as well as during the fast when compared with lean participants. Still, we did not find any significant differences in serum ghrelin between lean and obese participants, which contrasts with previous findings. However, as judged from the BMI values, our obese cohort suffered from mild overweight only, whereas previous studies have included obese study groups with mean BMI way above 35 kg/m2 (12, 28).
Women showed higher ghrelin levels than men throughout this fasting study. This gender difference is in accordance with previous studies (8, 29), whereas others have failed to observe any relationship between serum ghrelin and either postmenopausal status or estrogen replacement therapy (22). The reason for this discrepancy remains to be settled.
In conclusion, during 3 d of fasting, serum ghrelin exhibited a marked circadian rhythm, which was inversely related to changes in circulating cortisol levels, whereas GH, glucose, insulin, proinsulin, or C-peptide appeared not to be involved. Furthermore, serum ghrelin was significantly higher in females than in males. Our study indicates a novel regulator mechanism of ghrelin. However, our findings were observational, and therefore, further investigations are needed to elucidate the relationship between cortisol and ghrelin.
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Acknowledgments |
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Footnotes |
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First Published Online November 2, 2004
Abbreviations: BMI, Body mass index; GHS-R, GH secretagogue receptor.
Received March 30, 2004.
Accepted October 22, 2004.
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