1Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands; and 2Division of Endocrinology and Metabolism, Mayo Medical and Graduate Schools of Medicine, Mayo Clinic, Rochester, Minnesota
Submitted 19 July 2004 ; accepted in final form 21 August 2004
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ABSTRACT |
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cortisol; entropy; adrenal adenoma
Because obesity, frequently a prominent feature of hypercortisolism, is accompanied by a decreased GH response to stimuli and diminished spontaneous GH secretion, it is mandatory that any comparison between the hypercortisolemic state and healthy subjects must include body mass index (BMI)-matched controls. In a previous study in patients with pituitary-dependent hypercortisolism, the 24-h GH secretion was negatively correlated to urinary cortisol excretion, and the GH secretion regularity was significantly decreased (17). Hypothetically, the GH secretory abnormalities could be the result of the presence of the pituitary adenoma itself, a tumoral product acting as a paracrine signal on the somatotrope, or the result of cortisol excess per se on the somatotropic axis.
The present study aimed to explore the dynamics of spontaneous diurnal GH secretion in patients with Cushing's syndrome, since these patients lack a pituitary adenoma but otherwise suffer from chronic endogenous cortisol excess. The prime issue is whether such patients display low-amplitude and/or disorderly GH secretion compared with BMI-matched controls, as we previously found in pituitary-dependent hypercortisolism (17).
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SUBJECTS AND METHODS |
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Patients and control subjects were admitted to the hospital on the day of the study. An indwelling intravenous cannula was inserted in a forearm vein at least 60 min before sampling began. Blood samples were withdrawn at 10-min intervals for 24 h, starting at 0900. A slow infusion of 0.9% NaCl and heparin (1 U/ml) was used to keep the line open. The subjects were free to ambulate but not to sleep during the daytime. Meals were served at 0900, 1230, and 1730. Lights were turned off between 2200 and 2400. No sleep monitoring by EEG was used. Plasma for GH and cortisol measurements was collected, centrifuged at 4°C for 10 min, and stored at 20°C until later analysis. The results of the cortisol data are not shown; here, we use only the 24-h secretion rates in regression analyses.
Assays
Plasma GH concentrations were measured in duplicate with the use of a sensitive time-resolved immunofluorometric assay (Wallac, Turku, Finland) specific for the 22-kDa GH protein. Human biosynthetic GH (Pharmacia & Upjohn, Uppsala, Sweden) was used as standard calibrated against WHO-IRP 80-505, with a detection limit of 0.03 mU/l and an intra-assay variation coefficient of 1.68.4% at plasma values between 0.25 and 40 mU/l (to convert mU/l to µg/l, divide by 2.6). All samples from any subject were run in the same assay.
The serum IGF-I was determined by RIA (Incstar, Stillwater, MN) with a detection limit of 1.5 nmol/l and an interassay variation coefficient of <11%. Plasma cortisol concentrations were measured by RIA (Sorin Biomedica, Milan, Italy). The detection limit of the assay was 25 nmol/l. The interassay variation varied from 2 to 4% at the concentrations obtained in this study.
Calculations and Statistics
Deconvolution analysis. A multiparameter deconvolution technique was used to estimate relevant measures of GH secretion from the 24-h serum GH concentration profiles, as described previously (53). Initial estimates of basal GH secretion rate were calculated to approximate the lowest 5% of all plasma GH concentrations in the time series. Peak detection entailed application of 95% statistical confidence intervals to two-thirds of all GH secretory peaks considered jointly and individual 95% statistical confidence intervals to the remaining one-third smaller pulses, as validated in simulations (12). The following four secretory and clearance measures of interest were estimated: 1) the number and locations of secretory events, 2) the amplitudes of secretory bursts, 3) the durations of randomly dispersed GH secretory bursts, and 4) the endogenous single-component subject-specific plasma half-life of GH. It was assumed that the GH distribution volume and half-life were time and concentration invariant. The following parameters were calculated: half-duration of secretory bursts (duration of the secretory burst at half-maximal amplitude), hormone half-life, burst frequency, amplitude of the secretory burst (maximal secretory rate attained within a burst), mass secreted per burst, basal secretion rate, pulsatile secretion rate (product of burst frequency and mean burst mass), and total secretion (sum of basal and pulsatile).
Approximate entropy. The univariate approximate entropy (ApEn) statistic was developed to quantify the degree of irregularity, or disorderliness, of a time series (42). Technically, ApEn quantifies the summed logarithmic likelihood that templates (of length m) of patterns in the data that are similar (within r) remain similar (within the same tolerance r) on the next incremental comparison and has been formally defined elsewhere (43). The ApEn calculation provides a single nonnegative number that is an ensemble estimate of relative process randomness, wherein larger ApEn values denote greater irregularity, as observed for ACTH in Cushing's disease, GH in acromegaly, and prolactin in prolactinomas (43, 51, 52). Cross-ApEn (X-ApEn) quantifies joint pattern synchrony between two separate but parallel time series after standardization (z-score transformation) (44, 45). In the present analysis, we calculated X-ApEn between cortisol (leading) and GH, with r = 20% of the standard deviation of the individual time series and m = 1. This parameter choice affords sensitive, valid, and statistically well-replicated ApEn and X-ApEn metrics for assessing hormone time series of this length (44). ApEn and X-ApEn results are reported as absolute values and as the ratio of the absolute value to that of the mean of 1,000 randomly shuffled data series. Ratio values that approach 1.0 thus denote mean empirical randomness.
Copulsatility. Copulsatility between the cortisol and GH time series was quantified by hypergeometric (joint binomial) distribution (54). This program calculates the probability that hormone pulses in time series occur randomly. We used a time window of 40 min, with cortisol as leading hormone series. The position (time of maximal secretion rate within a pulse) and number of pulses were derived from the deconvolution analyses.
Statistical analysis. Results are expressed as means ± SE. Comparison between groups was done with one-way ANOVA, followed post hoc by Tukey's honestly significantly different test to contrast means. Derived measures (deconvolution and ApEn) were transformed logarithmically before analysis to limit dispersion of variance. In addition, (stepwise) linear regression was applied to evaluate the relation between relevant variables. Cross-correlation analysis was applied to test for significant time-lagged (linear) synchrony between successive serum concentrations of cortisol and GH, considered pairwise, as described previously (54). Calculations were carried out with Systat (release 11; Systat Software, Richmond, CA). Differences were considered significant for P < 0.05.
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RESULTS |
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Secretion profiles of the 24-h plasma GH concentration series of the patients are shown in Fig. 1. Deconvolution of the GH profiles revealed no differences in basal GH secretion rate, secretory burst half-duration, burst amplitude, burst mass, half-life, basal secretion, pulsatile secretion, and total secretion between the patients and the BMI-matched controls (Table 2). GH was secreted in a predominantly pulsatile fashion in patients and in BMI-matched controls, as displayed in Fig. 1. In healthy lean controls, GH secretion was 2-fold higher than in patients and was accomplished by a 2.5-fold increase in burst mass (P = 0.001) at similar pulse frequency. Total serum IGF-I concentrations were similar in the groups: patients, 16.5 ± 3.4; BMI-matched controls, 16.8 ± 0.8; and lean controls, 20.1 ± 2.2 nmol/l (ANOVA, P = 0.44).
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The influence of meals on GH concentrations was analyzed by comparing the mean of 10 serial samples preceding lunch and dinner in patients and body weight-matched controls and mean GH in the samples after the start of lunch and dinner during 90 min. In patients, the mean GH decrease after lunch was 0.48 mU/l (P = 0.03) and in controls 1.16 mU/l (P = 0.006). The mean GH decrease after dinner was 1.51 mU/l in patients (P = 0.04) and 2.19 mU/l in controls (P = 0.02). The mean GH decreases in patients and controls were statistically similar.
ApEn
ApEn in patients was increased, denoting an irregular secretion pattern: patients 0.7386 ± 0.044 vs. BMI-matched controls 0.5271 ± 0.0455 (P = 0.04) and vs. lean controls 0.4492 ± 0.050 (P = 0.001). The ApEn ratio was 0.5102 ± 0.015 in patients, 0.4250 ± 0.021 in body weight-matched controls (P = 0.016), and 0.3820 ± 0.024 in lean controls (P = 0.0002).
Factors Influencing GH Secretion
In a stepwise linear regression analysis, the 24-h GH secretion in patients and BMI-matched controls was significantly negatively correlated with BMI (R = 0.55, P = 0.005), as displayed in Fig. 2. However, other parameters, including cortisol secretion rate, free urinary cortisol excretion, age, estradiol, gender, and duration of cortisol excess (in patients only), were nonsignificant predictors. Thus the variation in total GH secretion was explained by BMI for 30%. In addition, ApEn was significantly and positively correlated (R = 0.77, P = 0.003) with the cortisol secretion rate, as displayed in Fig 3, but not with BMI (R = 0.03).
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Pattern synchrony between cortisol and GH was quantified by X-ApEn in patients and BMI-matched controls. X-ApEn in patients was 1.648 ± 0.113 and in controls was 1.004 ± 0.050 (P < 0.0001). The ApEn ratios were 0.8682 ± 0.054 and 0.6134 ± 0.026, respectively (P < 0.0001), denoting diminished pattern synchrony in patients. Conventional linear cross-correlation between cortisol (leading) and GH concentrations revealed a negative correlation in control subjects [median 0.30, 95% confidence interval (CI) 0.15 to 0.39] and a mean time lag of 30 min (95% CI 065 min), indicating opposite changes in cortisol concentrations, followed by those of GH. Five of the patients had a positive correlation. Median correlation coefficient was 0.09, with a 95% CI of 0.15 to +0.16. The mean time lag was 75 min, with a 95% CI of 37100 min. Copulsatility of cortisol and GH pulses was statistically highly significant in all patients and in 10 of 12 control subjects (P values between 103 and 1013).
Unilateral Versus Bilateral Adrenal Pathology
BMI, IGF-I, and age were comparable in these subgroups. No differences were found in GH secretion parameters, as estimated by deconvolution, ApEn, and synchrony estimates of GH and cortisol.
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DISCUSSION |
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Stimulated GH release is severely restricted in Cushing's syndrome, and either no increase or only a small increment is noted after administration of GHRH, GH-releasing peptide (GHRP; hexarelin and GHRP-2), and ghrelin (2, 20, 30, 33). Because most of the GH stimulation studies in Cushing's syndrome lack body weight-matched controls, the specificity of this finding might be questioned. GH release after reduction of the endogenous somatostatin tonus is also greatly diminished in hypercortisolism, e.g., by pretreatment with pyridostigmine or arginine infusion or after abrupt cessation of an intravenous infusion with somatostatin (13, 29, 32). Collectively, these results could point to a (reversible) defect of the pituitary gland, i.e., the somatotropic cell. Indeed, repeated GHRH administration in the hypercortisolemic state leads to potentiation to this hormone (31). Furthermore, administration of acipimox caused a sevenfold increase in GH release after GHRH administration, accompanied by a threefold decrease in circulating free fatty acids, and almost doubling of spontaneous 24-h GH secretion (28). Finally a hypocaloric diet for 3 days resulted in a fourfold GH increase after GHRH injection (34).
Similarities with experimental results in obesity are distinct, since it is well established that GHRH-stimulated GH release is diminished in obesity and increases during caloric restriction and after weight loss (14). Spontaneous 24-h GH secretion is severely restricted in the overweight human and increases or normalizes after weight reduction and during acipimox treatment (23, 41). In other studies, both BMI and abdominal visceral fat mass predict irregular (disorderly) GH release (12, 50). The basis for this inferred feedback alteration in GH secretion is not known (14).
Reports on spontaneous GH secretion in Cushing's syndrome, as studied with 24-h blood sampling protocols, are scarce. In one such contribution, Magiakou et al. (35) studied 15 patients with hypercortisolism (14 pituitary-dependent patients and 1 with primary bilateral pigmented nodular hyperplasia), of whom 6 were prepubertal. They described severely depressed GH secretion compared with normal-weight controls, mainly caused by decreased pulse amplitude, but with unchanged pulse frequency (35). The intriguing observation was that the expected restoration of GH secretion after curative pituitary surgery failed to occur, notwithstanding significant weight loss and normalization of BMI in 50% of the patients who had preoperatively increased values. These observations suggest that (visceral) obesity is an important determinant of GH secretion in Cushing's syndrome, irrespective of its etiology; apparently, however, after pituitary surgery, other factors play (or still play) a role in the diminished GH secretion.
We established a significant negative relationship between BMI and GH secretion in pituitary-independent hypercortisolism and in the matched controls. BMI, however, explained only 30% of the variability in GH, suggesting that other mechanisms likely contribute to the observed hyposomatotropism, as discussed above. It is unfortunate that we had no data on visceral fat mass in our patients and controls, because most likely, a higher correlation coefficient would have been found. Nevertheless, we did not find a relation between the degree of cortisol excess and GH secretion rate, as was previously found in our laboratory (17) for pituitary-dependent hypercortisolism. A conspicuous difference in clinical presentation between the two forms of the syndrome was the very high cortisol secretion rate in some of the (male) Cushing's disease patients, which could explain the divergent results.
Compared with lean controls, our patients had a 50% reduction in pulsatile GH secretion, exclusively caused by secretory burst amplitude decrement. In the absence of a significant change in basal (nonpulsatile) secretion, this observation is compatible with heightened somatostatin inhibition (3), decreased hypothalamic GHRH secretion, a defect in the GHRH/GH secretagogue receptor signaling, or direct nonreceptor-related GH inhibition. Experimental evidence, mainly obtained in the rat, has demonstrated that high doses of glucocorticoids decrease the expression of hypothalamic GHRH mRNA and increase that of somatostatin (11, 14, 27). On the other hand, dexamethasone increased mRNA of the GHRH receptor and the GH secretagogue receptor, which certainly explains the dexamethasone potentiation of GH release after GHRH in the human and in the rat (26, 37, 49) but not the diminished GH response to GHRH/GHS during chronic glucocorticoid excess. Accordingly, the amount and duration of cortisol excess appear to be important.
Other mechanisms might limit GH secretion in chronic hypercortisolism. For instance, in the rat, dexamethasone administration decreased mitosis and increased apoptosis of pituitary cells (38). If such a mechanism is also present in the human somatotrope, this might (partly) explain the extended time (1 year or more) it takes for restoration of GH secretion in most of the (adult) patients after surgical cure of Cushing's syndrome (16, 21, 49). Nonetheless, permanent damage to the somatotrope appears to be the rule rather than exception in childhood-onset Cushing's disease after surgery and radiation treatment (4). Another mechanism potentially relevant for the inhibitory effect of glucocorticoids on GH secretion is via the action of annexin 1. This peptide is a mediator of the anti-inflammatory actions of glucocorticoids and has significant effects on cell growth, differentiation, apoptosis, membrane fusion, endocytosis, and exocytosis (22). This peptide, widely distributed in the body, is also present in the folliculostellate cells in the pituitary gland, but not in the pituicytes, and exerts its GH-suppressing effect on the somatotrope via a paracrine mechanism at a point distal to the formation of cAMP and Ca ion entry (48). However, the same mediator also has a centrally stimulatory effect on GH (40). Finally, leptin might also be involved in the GH regulation. Circulating leptin concentrations in Cushing's syndrome are disproportionately increased compared with BMI-matched healthy controls (7, 15, 36). Short-term fasting in Cushing's syndrome did not restore normal leptin levels, and GH secretion remained blunted (19). However, several recent clinical studies suggest that a direct role for leptin in GH regulation is rather limited. In morbidly obese patients treated by biliopancreatic diversion, changes in insulin levels predicted changes in leptin levels and the somatotropic axis (8). Also, observations in patients with homozygous and heterozygous leptin gene mutations indicate that GH secretion is correlated with adiposity (39). Finally, recombinant methionyl human leptin administration in healthy lean men did not prevent fasting-induced augmentation of GH pulsatility or a decline in free IGF-I levels but restored, in part, total IGF-I levels (5).
GH concentration fell after meals in patients and in controls. Theoretically, one might expect a diminished inhibitory action in patients because of decreased hypothalamic GHRH expression and increased somatostatin expression, as discussed above (11, 14, 27). The differences between patients and controls were not significant (P values 0.60), suggesting that lack of power was not responsible.
A conspicuous and specific observation was the decreased regularity of GH secretion measured using ApEn, as previously described in patients with ACTH-producing pituitary adenomas (17). The degree of irregularity of GH release in patients with adrenal cortisol excess was significantly greater than that estimated in obese controls. The ApEn statistic quantitates the relative orderliness or reproducibility of subordinate (nonpulsatile) secretory patterns in neurohormone time series, which in turn mirrors feedforward and feedback adjustments driven by (patho)physiological changes in interglandular communication. The validity of ApEn to this end has been established in theoretical and experimental contexts (9, 55, 56). In view of the unchanged IGF-I feedback signal in the patients, decreased regularity of GH secretion could reflect impaired coordinate control of GH secretion by somatostatin, GHRH, and ghrelin and/or altered pituitary responsiveness to these peptides (9, 10). Available data do not address the reversibility of disorderly GH release due to endogenous adrenal cortisol excess with presumptively normal premorbid hypothalamo-pituitary function.
The 24-h concentration profiles allowed an appraisal of possible coordinate secretion of cortisol with GH. In normal subjects, we found a reciprocal relationship between these two hormones, as previously demonstrated in midluteal-phase women and in children (1, 6). The inverse relationship might be explained by the known ability of glucocorticoids to suppress GH secretion, possibly via heightened somatostatinergic tone (14). In patients, the correlation between the two hormones was smaller and even positive in five subjects. Indeed, abolishment of the cortisol-GH correlation can be induced by fasting in adult healthy women, while a positive correlation is seen in children with congenital adrenal hyperplasia under glucocorticoid substitution therapy (1, 6). Changes of cortisol patterns as observed during the stress of caloric deprivation and, by definition, nonphysiological glucocorticoid substitution therapy lead to desynchronization of hormone secretion patterns, as we now also described for endogenous primary adrenal hypercorticism. The loss of interaxis synchrony in our patients is corroborated by (lag-independent) cross-ApEn analysis. Disruption of pattern synchrony of GH and cortisol is also seen during fasting in adult women. Interesting, and not previously reported, was the loss of synchrony between GH and cortisol in 15 patients with ACTH-dependent hypercorticism (45, 47). In these patients, cross-ApEn was 1.640 ± 0.068, greatly elevated to a similar degree as the adrenal form of hypercorticism (P = 0.000013 vs. controls, and P = 0.99 vs. adrenal hypercorticism). Collectively, these results indicate that endogenous hypercorticism leads to disruption of cortisol-GH synchrony, irrespective of its cause. Notwithstanding the obvious loss in synchrony, copulsatility of cortisol and GH remained strong. This finding is somewhat surprising, since tumoral cortisol secretion in patients with adrenal adenoma is ACTH independent, and could therefore indicate that cortisol feedback is involved in the temporal timing of GH pulses. At present, no other data in literature are available to support this hypothetical view.
In summary, patients with primary adrenal Cushing's syndrome exhibit moderate hyposomatotropism, as demonstrated by decreased GH pulsatile secretion, which is only partly (30%) explained by adiposity. This observation in combination with disruption of GH pattern regularity and synchrony points to impaired net peptidyl drive of orderly somatotrope secretion.
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FOOTNOTES |
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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.
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REFERENCES |
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