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G De Nicolao, D Liberati, JD Veldhuis and A Sartorio

OBJECTIVE: To reconstruct the instantaneous secretion rate (ISR) of LH and FSH after GnRH administration in normal volunteers using non-parametric deconvolution, and to derive a direct integration formula to evaluate the amount of LH and FSH secreted during the first 60 min after the stimulus. DESIGN AND METHODS: First, the deconvolution method was validated in vivo by reconstructing doses ranging from 7.5 IU to 75 IU injected in three healthy adult volunteers whose endogenous LH had previously been downregulated by pretreating them, 3-4 weeks earlier, with 3.75 mg GnRH agonist i.m. Then, 40 healthy adult male volunteers were tested with a single 100 microg GnRH bolus, administered at 0 min. LH and FSH concentrations were determined at -30, 0, 15, 30, 45, 60, 90, and 120 min. RESULTS AND CONCLUSIONS: The validation study, conducted over a 10-fold range of doses, demonstrated that non-parametric deconvolution provided a reasonably accurate estimate of the amount of hormone entering the circulation. Applying deconvolution to the LH and FSH responses to GnRH, the ISRs of both hormones were shown to have a similar pattern, with a clearly delimited pulse after the GnRH bolus. In conjunction with earlier analyses of estimates of GHRH-stimulated GH secretion, we conclude that secretagogues evoke discrete LH, FSH, and GH secretory bursts of about 60 min total duration, despite markedly unequal (glyco-)protein hormone half-lives (18-500 min). With respect to the assessment of total hormone release during the first 60 min after the stimulus, the integration formula provided a reliable approximation of the result obtained by deconvolution, and had a negligible dependence on the samples at times 90 and 120 min.

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RG Veldman, M Frolich, SM Pincus, JD Veldhuis and F Roelfsema

The episodicity of 24 h leptin release was studied in seven women (mean age 39 years, range 22-56 years) with pituitary-dependent hypercortisolism and in seven age- and body mass index (BMI)-matched female controls. Pulsatile leptin release was quantified by model-free cluster analysis and deconvolution, the orderliness of leptin patterns by the approximate entropy statistic (ApEn), and nyctohemeral leptin rhythmicity by cosinor analysis. Blood samples were taken at 10 min intervals for 24 h. Both cluster and deconvolution analysis revealed 2.4-fold increased leptin secretion in patients, caused by combined and equal amplification of basal and pulsatile secretion. Cluster analysis identified 7.1+/-1.5 peaks per 24 h in patients and 6.0+/-0.5 in controls (not significant). The statistical distribution of the individual sample secretory rates was similarly skewed in patients and controls (0.55+/-0.12 vs 0.52+/-0.07). The acrophase (timing of the nyctohemeral leptin peak) in patients occurred at 2314 h (+/-76 min) and at 0058 h (+/-18 min) in controls (not significant). The approximate entropy of leptin release was equivalent in patients and controls (1.67+/-0.03 vs 1.61+/-0.05). The approximate entropy (ApEn) for cortisol in patients was 1.53+/-0.09 and in controls was 0.93+/-0.07 (P<0.0005). Cross-ApEn showed significant pattern synchrony between leptin and cortisol release, which (unexpectedly) was not disrupted by the cortisol excess (patients, 2.02+/-0.04; controls, 1.88+/-0.09; P=0.233). Insulin levels in fasting patients ('fasting insulin') were 27+/-5.7 mU/l vs 14+/-1.6 mU/l in controls (P=0.035). Leptin secretion correlated with fasting insulin levels (R(2)=0.34, P=0.028) and with the cortisol production rate (R(2)=0.33, P=0.033) when patients and controls were combined. In summary, Cushing's disease in women increases leptin production about twofold in an amplitude-specific way. The pulse-generating, nyctohemeral phase-determining, and entropy-control mechanisms that govern the 24 h leptin release are not altered. The increased secretion is not explained by BMI and is probably only partly explained by increased insulin production, suggesting a cortisol-dependent change in adipose leptin secretion.

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AN Moulas, Krieg RJ Jr, JD Veldhuis and JC Chan

OBJECTIVE: To determine the effect of repeated treatments with the growth hormone secretagogue (GHS) L-163,255 on the pulsatile release of GH when administered in meal-fed rats before and after feeding. DESIGN: The first group of rats (AL, n=6) had food available ad libitum. The second (restricted, R, n=6), third (GHSB, n=6), and fourth (GHSA, n=6) groups were fed from 1100 to 1400 h. Groups GHSB and GHSA were given GHS by gavage, 3.0 mg/kg L-163,255, at 1000 h (before feeding, B) and at 1500 h (after feeding, A) respectively. Three weeks after the initiation of the treatment, blood samples were collected at 10-min intervals over 6 h, and GH levels were determined. RESULTS: In group R, the concentrations of GH were higher before feeding (17.6+/-2.4 ng/ml) than during feeding (11.2+/-1.2 ng/ml), P<0.05. The average concentrations of the peak in response to GHS were higher when GHS was administered before (121.70+/-33.68 ng/ml) than after (49.67+/-17.87 ng/ml) feeding. The mass of GH, as calculated by deconvolution analysis was also higher in the GHSB group than in the GHSA group (251.6+/-64.1 ng/ml per min vs 85.3+/-22.9 ng/ml per min respectively, P<0.05). CONCLUSION: L-163,255 is effective in inducing GH release after repeated oral administration in rats. The effectiveness is greater when GHS is administered before rather than after feeding in meal-fed animals.

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T Mulligan, A Iranmanesh, R Kerzner, LW Demers and JD Veldhuis

OBJECTIVE: To examine the possibility that lower serum bioavailable testosterone concentrations, without increased LH release, in healthy older men, reflects hypothalamic GnRH deficiency. DESIGN: We used a randomized, double-blind, placebo-controlled design. METHODS: We treated each of five young (ages 20-34 years) and five older (ages 60-78 years) men with 2 weeks of randomized infusions of saline or pulsatile GnRH (100 ng/kg i.v. every 90 min). RESULTS: At baseline (saline infusion), older men had more LH pulses (young compared with old, 10 +/- 0.6 compared with 15 +/- 1, P = 0.0026) per 24h, reduced fractional LH pulse amplitude (219 +/- 17% compared with 167 +/- 40%, P = 0.0376), and more disorderly hormone release as judged by approximate entropy (ApEn) (LH, P < or = 0.0001; testosterone, P < or = 0.0047). In response to pulsatile i.v. GnRH infusions, serum 24-h LH concentrations (measured by immunoradiometric assay (IRMA)), increased equivalently in young and older men (to 7.3 +/- 1.2 and 7.2 +/- 1.8 IU/l respectively). GnRH treatment also normalized LH pulse frequency and amplitude, ApEn, and plasma biologically active LH (pooled) concentrations. In contrast, 24-h testosterone concentrations failed to increase equivalently in older men (young compared with old, 869 +/- 88 compared with 517 +/- 38 ng/dl, P = 0.0061), reflecting lower testosterone peak maxima (995 +/- 108 compared with 583 +/- 48 ng/dl, P = 0.0083) and interpeak nadirs (750 +/- 87 compared with 427 +/- 26 ng/dl, P = 0.0073). CONCLUSIONS: We have demonstrated that, in older men, successful reconstitution of 24-h pituitary (bioactive) LH output and pulsatile (IRMA) LH release patterns could be achieved by a fixed exogenous GnRH pulse signal, thereby implicating altered endogenous hypothalamic GnRH release in the relative hypogonadotropism of aging. The failure of testosterone concentrations to increase concomitantly points to a simultaneous Leydig cell defect. We conclude that aging in men is marked by a dual defect in the central nervous system-pituitary-Leydig cell axis.

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K Friend, A Iranmanesh, IS Login and JD Veldhuis

Growth hormone (GH) release from the anterior pituitary gland is predominantly regulated by the two antagonistic hypothalamic peptides, growth hormone-releasing hormone (GHRH) and somatostatin. Appraising endogenous GHRH action is thus made difficult by the confounding effects of (variable) hypothalamic somatostatin inhibitory tone. Accordingly, to evaluate endogenous GHRH actions, we used a clinical model of presumptively acute endogenous somatostatin withdrawal with concomitant GHRH release. To this end, we administered in randomized order placebo or the indirect cholinergic agonist, pyridostigmine, for 48 h to 13 healthy men of varying ages (29-77 years) and body mass indices (21-47 kg/m2). We sampled blood at 10-min intervals for 48 h during both placebo and pyridostigmine (60 mg orally every 6 h) administration, and used an ultrasensitive GH chemiluminescence assay (sensitivity 0.0002-0.005 microgram/l) to capture GH pulse profiles. Multiparameter deconvolution analysis was applied to quantitate the number, amplitude, mass, and duration of significant underlying GH secretory bursts, and simultaneously estimate the GH half-life and concurrent basal GH secretion. Approximate entropy was utilized as a novel regularity statistic to quantify the relative orderliness of the hormone release process. All measures of GH secretion/half-life and orderliness were statistically invariant across the two consecutive 24-h placebo sessions. In contrast, pyridostigmine treatment significantly increased the mean serum GH concentration from 0.23 +/- 0.054 microgram/l during placebo to 0.45 +/- 0.072 microgram/l during the first day of treatment (P < 0.01). There was also a significant rise in the calculated 24-h pulsatile GH production rate from 8.9 +/- 1.7 micrograms/l/day on placebo to 27 +/- 5.6 micrograms/l/day during active drug treatment (P < 0.01). Pyridostigmine significantly and selectively amplified GH secretory burst mass to 1.5 +/- 0.35 micrograms/l compared with 0.74 +/- 0.19 microgram/l on placebo (P < 0.01). This was attributable to stimulation of GH secretory burst amplitude (maximal rate of GH secretion attained within the release episode) with no prolongation of estimated burst duration. Basal GH secretion and approximate entropy were not altered by pyridostigmine. However, age was strongly related to more disorderly GH release during both days of pyridostigmine treatment (r = +0.79, P = 0.0013). During the second 24-h of continued pyridostigmine treatment, most GH secretory parameters decreased by 15-50%, but in several instances remained significantly elevated above placebo. Body mass index, but not age, was a significantly negative correlate of the pyridostigmine-stimulated increase in GH secretion (r = -0.65, P = 0.017). In summary, assuming that somatostatin is withdrawn and (rebound) GHRH release is stimulated via pyridostigmine administration, we infer that relatively unopposed GHRH action principally controls GH secretory burst mass and amplitude, rather than apparent GH secretory pulse duration, the basal GH secretion rate, or the serial regularity/orderliness of the GH release process in the human. Moreover, we infer that increasing age is accompanied by greater disorderliness of somatostatin-withdrawn GHRH, and hence rebound GH, release. The strongly negative correlation between pyridostigmine-stimulated GH secretion and body mass index (but not age) further indicates that increased relative adiposity may result in decreased effective (somatostatin-withdrawn) endogenous GHRH stimulus strength.

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R Salvatori, X Fan, JD Veldhuis and R Couch

OBJECTIVE: Inactivating mutations of the GH-releasing hormone receptor (GHRHR) gene (GHRHR) cause familial isolated GH deficiency (IGHD) type IB. The GH response to physical exercise (PE) in patients lacking GHRHR has never been studied. We hypothesized that subjects lacking functional GHRHR may be a model to study GH response to PE. DESIGN: We have analyzed peripheral genomic DNA of a family with two sibs affected by IGHD IB for mutations in the GHRHR, studied the patients' GH response to different GH secretagogues and to PE, and examined the morphology of their pituitary gland by magnetic resonance imaging (MRI). METHODS: The GHRHR was analyzed by direct sequencing of the 13 exons, intron-exon boundaries, and of the proximal 327 bp of the promoter region in the index case. The patients' GH response to GHRH and standardized PE was studied twice, using a GH ultrasensitive assay in the second round of testing. RESULTS: Both subjects were compound heterozygotes for two previously undescribed mutations in the GHRHR that are predicted to cause complete lack of functional GHRHR protein: a nonsense mutation in codon 43 (Q43X), and a splice mutation at the beginning of intron 3 (IVS3+1G-->A). MRI showed hypoplasia of their anterior pituitaries. Both subjects had a small but detectable increase in serum GH after maximal PE. CONCLUSIONS: GHRHR mutations need to be considered in IGHD IB patients even in the absence of parental consanguinity, and patients lacking GHRHR may provide a model to study the mechanism by which PE influences GH secretion.

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R Groote Veldman, G van den Berg, SM Pincus, M Frolich, JD Veldhuis and F Roelfsema

To quantify prolactin (PRL) secretion patterns, ten untreated (female) microprolactinoma patients and six (male) macroprolactinoma patients underwent repetitive blood sampling every 10 min over 24 h. PRL release activity was analyzed from plasma PRL concentration (immunofluorimetric assay) profiles via a model-independent discrete peak detection program (Cluster) and a waveform-independent deconvolution technique (Pulse). Diurnal variations were analyzed by cosinor analysis. The number of distinct PRL pulses (mean +/- S.E.M.) was increased in patients: microprolactinoma 18.6 +/- 0.6/24 h versus female controls 12.4 +/- 0.6 (P = 6.7 x 10-s), and macroprolactinoma 18.0 +/- 0.9 versus male controls 13.5 +/- 0.8/24 h (P = 0.003). In patients, PRL pulse height, amplitude, pulse area and interpeak nadir concentrations were each greatly elevated compared with gender-matched controls. By 2-component deconvolution analysis, the mean nadir PRL secretion rate in microprolactinoma patients was augmented 20-fold at 0.408 +/- 0.089 microgram/l per min versus in female controls 0.019 +/- 0.009 microgram/l per min (P < 0.001); and in macroprolactinoma by 130-fold at 2.067 +/- 0.693 micrograms/l per min versus male controls 0.016 +/- 0.001 microgram/l per min (P = 0.001). Corresponding 24 h mean PRL secretion rates were in women, 0.658 +/- 0.147 and 0.044 +/- 0.018 (P < 0.001), and in men, 3.309 +/- 1.156 and 0.035 +/- 0.010 micrograms/l per min (P = 0.001), being respectively 15- and 94-fold increased in tumors. The estimated PRL production per day was 160 +/- 15 and 187 +/- 20 micrograms in male and female controls respectively. PRL production was 2860 +/- 640 micrograms in female patients with microadenomas (P < 0.001), and 37,800 +/- 5900 micrograms in male macroadenoma patients (P = 0.001). Cosinor analysis of the plasma concentrations revealed a significant rhythm in nine of ten, patients with a microadenoma, and in five of six with a macroadenoma. The same method applied to pulse height and amplitude disclosed a significant rhythm for PRL pulse height, but not for pulse amplitude, suggesting preserved rhythmicity of baseline interpulse nadir PRL concentrations. Approximate entropy (ApEn), a scale- and model-independent regularity statistic, averaged 1.6559 +/- 0.028 in microprolactinoma patients versus 0.8128 +/- 0.079 in female controls (P = 1.7 x 10(-8)); ApEn in macroadenomas was 1.5674 +/- 0.054 versus male controls 0.8773 +/- 0.076 (P = 1.7 x 10(-5), signifying greater secretory irregularity in the patients. Compared with microadenomas, macroadenomas exhibited a higher mean plasma concentration, overall mean PRL secretion rate, nadir secretion rate and pulse area, but similar peak frequency. We conclude that PRL secretion by prolactinomas is characterized by increased plasma PRL episodicity of release, increased total (15- to 100-fold) and basal (20- to 130-fold) secretion rates, and increased disorderlines of minute-to-minute secretion. These abnormalities of secretory control are very similar to those for GH and ACTH identified earlier in acromegaly and Cushing's disease respectively, thus suggesting mechanistic generality of pituitary tumor secretory derangements, independent of the particular hormone.

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A Iranmanesh, S South, AY Liem, D Clemmons, MO Thorner, A Weltman and JD Veldhuis

We here investigate the potential rescue of the relative hyposomatotropism of aging and obesity by 3-day pulsatile GHRH infusions (i.v. bolus 0.33 microg/kg every 90 min) in 19 healthy men of varying ages (18 to 66 years) and body compositions (12 to 37% total body fat). Baseline (control) and GHRH-driven pulsatile GH secretion (in randomly ordered sessions) were quantitated by deconvolution analysis of 24-h (10-min sampling) serum GH concentration profiles measured in an ultrasensitive (threshold 0.005 microg/l) chemiluminescence assay. GHRH infusion significantly increased the mean (24-h) serum GH concentration (0.3 +/- 0.1 basal vs 2.4 +/- 0.4 microg/l treatment; P = 0.0001), total daily pulsatile GH production rate (21 +/- 9.5 vs 97 +/- 17 microg/l/day; P = 0.01), GH secretory burst frequency (11 +/- 0.5 vs 17 +/- 0.3 events/day; P = <0.01), and mass of GH released per burst (1.1 +/- 0.4 vs 5.9 1 microg/l; P < 0.01), as well as serum IGF-I (261 +/- 33 vs 436 +/- 37 microg/l; P = 0.005), insulin (45 +/- 13 vs 79 +/- 17 mU/l; P = 0.0002), and IGF binding protein (IGFBP)-3 (3320 +/- 107 vs 4320 +/- 114 microg/l; P = 0.001) concentrations, while decreasing IGFBP-1 levels (16 +/- 1.2 vs 14 +/- 0.09 microg/l; P = 0.02). Serum total testosterone and estradiol concentrations did not change. GHRH treatment also reduced the half-duration of GH secretory bursts, and increased the GH half-life. GHRH-stimulated 24-h serum GH concentrations and the mass of GH secreted per burst were correlated negatively with age (R[value]:P[value] = -0.67:0.002 and -0.58:0.009 respectively), and percentage body fat (R:P = -0.80:0.0001 and -0.65:0.0005 respectively), but positively with serum testosterone concentrations (R:P = +0.55:0.016 and +0.53:0.019 respectively). GHRH-stimulated plasma IGF-I increments correlated negatively with age and body mass index, and positively with serum testosterone, but not with percentage body fat. Cosinor analysis disclosed persistent nyctohemeral rhythmicity of GH secretory burst mass (with significantly increased 24-h amplitude and mesor values) but unchanged acrophase during fixed pulsatile GHRH infusions, which suggests that both GHRH- and non-GHRH-dependent mechanisms can modulate the magnitude (but only non-GHRH mechanisms can modulate the timing) of somatotrope secretory activity differentially over a 24-h period. In summary, diminished GHRH action and/or non-GHRH-dependent mechanisms (e.g. somatostatin excess, putative endogenous growth hormone-releasing peptide deficiency etc.) probably underlie the hyposomatotropism of aging, (relative) obesity, and/or hypoandrogenemia. Preserved or increased tissue IGF-I responses to GHRH-stimulated GH secretion (albeit absolutely reduced, suggesting GHRH insensitivity in obesity) may distinguish the pathophysiology of adiposity-associated hyposomatotropism from that of healthy aging.

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G Van den Berghe, P Wouters, CY Bowers, F de Zegher, R Bouillon and JD Veldhuis

OBJECTIVE: During prolonged critical illness, nocturnal pulsatile secretion of GH, TSH and prolactin (PRL) is uniformly reduced but remains responsive to the continuous infusion of GH secretagogues and TRH. Whether such (pertinent) secretagogues would synchronize pituitary secretion of GH, TSH and/or PRL is not known. DESIGN AND METHODS: We explored temporal coupling among GH, TSH and PRL release by calculating cross-correlation among GH, TSH and PRL serum concentration profiles in 86 time series obtained from prolonged critically ill patients by nocturnal blood sampling every 20 min for 9 h during 21-h infusions of either placebo (n=22), GHRH (1 microg/kg/h; n=10), GH-releasing peptide-2 (GHRP-2; 1 microg/kg/h; n=28), TRH (1 microg/kg/h; n=8) or combinations of these agonists (n=8). RESULTS: The normal synchrony among GH, TSH and PRL was absent during placebo delivery. Infusion of GHRP-2, but not GHRH or TRH, markedly synchronized serum profiles of GH, TSH and PRL (all P< or =0.007). After addition of GHRH and TRH to the infusion of GHRP-2, only the synchrony between GH and PRL was maintained (P=0.003 for GHRH + GHRP-2 and P=0.006 for TRH + GHRH + GHRP-2), and was more marked than with GHRP-2 infusion alone (P=0.0006 by ANOVA). CONCLUSIONS: The nocturnal GH, TSH and PRL secretory patterns during prolonged critical illness are herewith further characterized to include loss of synchrony among GH, TSH and PRL release. The synchronizing effect of an exogenous GHRP-2 drive, but not of GHRH or TRH, suggests that the presumed endogenous GHRP-like ligand may participate in the orchestration of coordinated anterior pituitary hormone release.