Johannes D. Veldhuis

Context Interleukin-2 (IL-2), a proinflammatory cytokine, has been used to treat malignancies. Increased cortisol and adrenocorticotropin (ACTH) were noted, but growth hormone (GH) secretion was not investigated in detail. Objective We quantified GH secretion after a single subcutaneous injection of IL-2 in 17 young and 18 older healthy men in relation to dose, age, and body composition. Methods This was a placebo-controlled, blinded, prospectively randomized, crossover study. At 20:00 hours IL-2 (3 or 6 million units/m²) or saline was injected subcutaneously. Lights were off between 23:00 and 07:00 hours. Blood was sampled at 10-minute intervals for 24 hours. Outcome measures included convolution analysis of GH secretion. Results GH profiles were pulsatile under both experimental conditions and lower in older than young volunteers. Since the effect of IL-2 might be time limited, GH analyses were performed on the complete 24-hour series and the 6 hours after IL-2 administration. Total and pulsatile 24-hour GH secretion decreased nonsignificantly. Pulsatile secretion fell over the first 6 hours after IL-2 (P = .03), with visceral fat as a covariate (P = .003), but not age (P = .10). Plots of cumulative 2-hour bins of GH pulse mass showed a distinction by treatment and age groups: A temporary GH decrease of 32% and 28% occurred in the first 2-hour bins after midnight (P = .02 and .04) in young participants, whereas in older individuals no differences were present at any time point. Conclusion This study demonstrates that IL-2 temporarily diminishes GH secretion in young, but not older, men.

Background: Interleukin-2 (IL-2), one of the proinflammatory cytokines, is used in the treatment of certain malignancies. In some studies, transient increases in cortisol and ACTH secretion occurred. Thus, this agent may be used as an experimental probe of adrenal cortisol secretion. Objective: This study quantifies effects of low and moderate doses of IL-2 on cortisol secretion, and assesses the modulation by age, dose and body composition. Site: Mayo Clinical Translational Research Unit. Subjects: Study comprised 35 healthy men, 17 young and 18 older. Methods: Randomized prospective double-blind saline-controlled study of IL-2 administration in 2 doses with concurrent 10-min blood sampling for 24 h. Outcome measures: Deconvolution analysis and Approximate Entropy of cortisol secretion. Results: Low dose IL-2 administration increased nocturnal pulsatile cortisol secretion from 1460±160 to 2120±220 nmol/L/8h in young subjects and from 1680±105 to 1960±125 nmol/L/8h (treatment P<0.0001, but more in young than older, P=0.02). Comparable results were obtained for total cortisol secretion (P treatment <0.0001, age effect P=0.005). The higher IL2 dose caused a large increase in young (P<0.0001), but not in older (P=0.90) subjects. This dose also increased approximate entropy from 0.877±0.041 to 1.024±0.049 (P=0.008), pointing to reduced secretory orderliness. Incremental cortisol (nocturnal) secretion correlated negatively with visceral fat mass (R=-0.41, P=0.019). Conclusion: In healthy men, IL-2 injection drives pulsatile cortisol secretion in a dose-dependent way in young, but not older, individuals, and erodes cortisol secretory orderliness at a higher dose in young subjects. Cortisol responses are diminished with increasing abdominal visceral fat mass.

Neuroendocrine data are particularly challenging to analyze quantitatively in view of their unique characteristics, such as the tendency of observations to be relatively 182infrequent (i.e., short or coarse data series), noisy, confounded by variable half-lives and nonuniform experimental uncertainty, and composed of unknown true signal (e.g., theoretical waveform of secretion over time), with or without concomitant basal hormone release.^1,2 Since neuroendocrinology is concerned with quantal signaling and interglandular communication, quantification of short-term variations and longer-term trends in neurohormone measurements are often critical to investigating and understanding mechanisms of in vivo or in vitro neurohormone regulation, pathophysiological disturbance, gender differences, etc. Thus, a primary task of neuroendocrine investigation is accurate, reproducible, valid and vivid quantitation of contrasts between and among neurohormone time series. This goal is made more difficult by potential background drift in the data mean or variance (technically termed, nonstationarity), differences in some circumstances between decay rates of exogenously injected and endogenously secreted hormone, unequal distribution spaces for various neurohormones (e.g., testosterone approximately 32 liters in a 70-kilogram adult, whereas luteinizing hormone (LH) released within the male reproductive axis is confined largely to the 3.5 liters of plasma volume), as well as the necessity to obtain precise, sensitive, specific, reliable, and valid neurohormone measurements. Indeed, the last mentioned issue, while beyond the scope of this review, represents a critical step in quantitative neuroendocrinology.³ Last, neurohormone rhythms commonly occur on multiple time scales concurrently, e.g., ultradian with events occurring multiple times over any given 24-hr period, circadian (approximating a 24-hr rhythm), and sometimes longer-term trends that extend over weeks or a month (e.g., menstrual cycle). Even more extended trends exist that are seasonal or circannual. Given these issues in neuroendocrine data acquisition, processing, and analysis, pulsatile or episodic neurohormone release cannot be evaluated adequately without thoughtful, pertinent, and intensive attention to a variety of primary issues. Here, I will review several distinct issues in quantitative neuroendocrinology, including the specific quantification of pulsatile neurohormone data, evaluation of subordinate (non-pulsatile) patterns within the measurements, the notion of feedback and feed-forward control (network function of a neuroendocrine axis), the potential concordance or synchrony in pulse trains, and approaches to establishing reproducibility of biological data. Of necessity, some issues remain incompletely resolved, and these will be noted.

Background: Interleukin?2 (IL?2), one of the proinflammatory cytokines, is used in the treatment of renal cell cancer, melanoma and hematological malignancies. In some studies, temporary cortisol and ACTH increases were noted. Thus, this agent may be used as an experimental probe of adrenal cortisol secretion. Objective: This study quantifies effects of low and moderate doses of IL?2 on cortisol secretion, and assesses the modulation by age, dose and body composition. Site: Mayo Clinical Translational Research Unit. Subjects: Study comprised 35healthy men, 17young and 18older. Methods: Randomized prospective double?blind saline?controlled study of IL?2 administration in 2 doses with concurrent 10?min blood sampling for 24h. Outcome measures: Deconvolution analysis and Approximate Entropy of cortisol secretion. Results: Low dose IL?2 administration increased nocturnal pulsatile cortisol secretion from 53±5.8 to 77±8.0µg/dL/8h in young subjects and from 61±3.8 to 71±4.6µg/dL/8h (treatment P <0.0001, but more in young than older, P=0.02). Comparable results were obtained for total cortisol secretion (P treatment <0.0001, age effect P=0.005). The higher dose caused a large increase in young (P<0.0001), but not in older subjects (P=0.90). This dose also increased approximate entropy from 0.877±0.041 to 1.024±0.049, P=0.008, pointing to reduced secretory orderliness. Incremental cortisol (nocturnal) secretion correlated negatively with visceral fat mass (R=?0.41, P=0.019). Conclusion: In healthy subjects, IL?2 increases cortisol secretion in a dose?dependent way in young, but not in older subjects. Loss of cortisol secretory orderliness occurred at a higher IL?2 dose in young subjects. The cortisol response diminished with increasing abdominal visceral fat mass.

In young men, sleep restriction decreases testosterone (Te) and increases afternoon cortisol (F), leading to anabolic–catabolic imbalance, insulin resistance, and other andrological health consequences. Age-related differences in the hypothalamo–pituitary–testicular/adrenal response to sleep restriction could expose older individuals to greater or lesser risk. We aimed to evaluate and compare the 24-h and time-of-day effect of sleep restriction on F, luteinizing hormone (LH), and Te in young and older men. Thirty-five healthy men, aged 18–30 (n = 17) and 60–80 (n =18) years, underwent overnight sleep deprivation (complete nighttime wakefulness) or nighttime sleep (10 pm to 6 am) with concurrent 10-min blood sampling in a prospectively randomized crossover study. F, LH, and Te secretion were calculated by deconvolution analysis. Sleep deprivation had multiple effects on 24-h Te secretion with significant reductions in mean concentrations, basal, total and pulsatile secretion, and pulse frequency (each p < 0.05), in the absence of detectable changes in LH. These effects were most apparent in older men and differed according to age for some parameters: pulsatile Te secretion (p = 0.03) and Te pulse frequency (p = 0.02). Time-of-day analyses revealed that sleep restriction significantly reduced Te in the morning and afternoon, reduced LH in the morning in both age groups, and increased F in the afternoon in older men. These data suggest a time-of-day dependent uncoupling of the regulatory control of the testicular axis and of F secretion. Future studies will need to directly verify these regulatory possibilities specifically and separately in young and older men. Not applicable.

Background Aging is associated with diminished testosterone (Te) secretion, which may be attributed to Leydig cell dysfunction, decreased pituitary stimulation, and altered Te feedback. Objective To study all regulatory nodes—gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and Leydig cell—in the same cohort of healthy men. Study Design This was a placebo-controlled, blinded, prospectively randomized cross-over study in 40 men, age range 19 to 73 years, and body mass index (BMI) range 20 to 34.3 kg/m². A submaximal dose of the GnRH antagonist ganirelix was used to assess outflow of GnRH, by calculating the difference between LH output during the control arm and ganirelix arm. Ketoconazole (a steroidogenic inhibitor) was used to estimate feedback, by the difference in LH output during the ketoconazole and control arm. High-dose ganirelix and repeated LH infusions were used to measure testicular responsivity. Blood sampling was performed at 10-minute intervals. Results There were age-related, but not body composition–related decreases in estimated GnRH secretion, the feedback strength of Te on LH, and Leydig cell responsivity to LH, accompanied by changes in approximate entropy. Bioavailable Te levels were negatively related to both age and computed tomography (CT)–estimated abdominal visceral mass (AVF), without interaction between these variables. The LH response to a submaximal dose of GnRH was independent of age and AVF. Conclusion Advancing age is associated with (1) attenuated bioavailable Te secretion caused by diminished GnRH outflow and not by decreased GnRH responsivity of the gonadotrope, (2) diminished testicular responsivity to infused LH pulses, and (3) partial compensation by diminished Te feedback on central gonadotropic regulation.

Background Oral administration of estradiol (E₂) generally increases GH secretion in postmenopausal women, associated with decrease in IGF-I, whereas parenteral or transdermally administered E₂ may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E₂ levels stimulate GH, and that P4 modulates E₂-stimulated GH secretion. Study design Subjects: 4 parallel groups of randomly assigned postmenopausal women (total 40). Treatments: (1) saline placebo and oral placebo, (2) saline placebo im and oral micronized P4 (3x200 mg/day), (3) E₂ im (5 mg) and oral placebo, and (4) E₂ im and oral micronized P4. Outcome measures: overnight GH secretion (10 h) and stimulated (ghrelin, 0.3 µg/kg iv bolus) GH secretion. CT-estimated visceral fat. Results Intramuscular E₂ administration did not alter nocturnal and ghrelin-stimulated GH secretion. Nocturnal GH secretion was not changed by P4 administration. However, P4 diminished ghrelin-stimulated pulsatile GH release with or without E₂: average 7.20±2.14 and 9.58±1.97 µg/L/2h (P=0.045), respectively. Respective outcomes for mean GH concentrations and GH peak amplitudes were 0.97±0.31 and 1.52±0.29 (P=0.025), and 2.76±1.04 and 3.95±0.90 (P=0.031). Ghrelin-stimulated GH secretion correlated negatively with progesterone concentration, with or without correction for visceral fat area in the regression equation (R=0.49, P=0.04, β=-0.040±0.016). Conclusions Low-range physiological E₂ concentrations do not impact spontaneous or ghrelin-stimulated pulsatile GH secretion. Conversely, P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion. The mechanistic aspects and physiological significance of natural P4’s regulation of ghrelin-evoked GH secretion require further studies.

Context Quantitative studies of testosterone’s (T) short-term feedback on LH secretion in healthy men are relatively rare. Such studies require shutting down endogenous T secretion, and imposing experimentally controlled iv T addback. Objective To evaluate whether pulsatile and continuous T delivery confer equivalent negative feedback on LH secretion. Design This was a placebo-controlled, blinded, and prospectively randomized crossover study comprising 16 healthy men (age range 23-54 yr and BMI between 22.334.2 kg/m²).Subjects received ketoconazole to block endogenous T secretion, and received continuous or 90-min pulses of iv T addback. Setting The study was performed in a Clinical Translational Research Unit. Interventions. Subjects underwent 14 h of blood sampling at 10-min intervals, with a bolus iv injection of 33 ng/kg GnRH. Main outcome measures Log-transformed LH and T concentration ratios before and after GnRH administration. Results Despite higher T concentrations during pulsatile T feedback, LH concentrations and secretion rates, whether driven by endogenous or exogenous GnRH, were similar to those during continuous T infusion, indicating diminished pulsatile T feedback. Feedback correlated negatively with BMI. Under controlled T feedback, basal but not pulsatile LH secretion correlated negatively with CT-estimated visceral fat mass. Conclusion Feedback by pulsatile T delivery has diminished inhibitory strength compared with continuous infusion. Feedback is negatively correlated with BMI.