Liu, Peter Y.

Abstract Context Sleep loss in men increases cortisol and decreases testosterone, and sleep restriction by 3 to 4 hours/night induces insulin resistance. Objective We clamped cortisol and testosterone and determined the effect on insulin resistance. Methods This was a randomized double-blind, in-laboratory crossover study in which 34 healthy young men underwent 4 nights of sleep restriction of 4 hours/night under 2 treatment conditions in random order: dual hormone clamp (cortisol and testosterone fixed), or matching placebo (cortisol and testosterone not fixed). Fasting blood samples, and an additional 23 samples for a 3-hour oral glucose tolerance test (OGTT), were collected before and after sleep restriction under both treatment conditions. Cytokines and hormones were measured from the fasting samples. Overall insulin sensitivity was determined from the OGTT by combining complementary measures: homeostasis model assessment of insulin resistance of the fasting state; Matsuda index of the absorptive state; and minimal model of both fasting and absorptive states. Results Sleep restriction alone induced hyperinsulinemia, hyperglycemia, and overall insulin resistance (P < 0.001 for each). Clamping cortisol and testosterone alleviated the development of overall insulin resistance (P = 0.046) and hyperinsulinemia (P = 0.014) by 50%. Interleukin-6, high-sensitivity C-reactive protein, peptide YY, and ghrelin did not change, whereas tumor necrosis factor-α and leptin changed in directions that would have mitigated insulin resistance with sleep restriction alone. Conclusion Fixing cortisol-testosterone exposure mitigates the development of insulin resistance and hyperinsulinemia, but not hyperglycemia, from sustained sleep restriction in men. The interplay between cortisol and testosterone may be important as a mechanism by which sleep restriction impairs metabolic health.

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 the 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 two 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/8 h in young subjects and from 1680 ± 105 to 1960 ± 125 nmol/L/8 h (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 IL-2 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.

Abstract Introduction: In young men, sleep restriction decreases testosterone and increases afternoon cortisol, leading to anabolic-catabolic imbalance, insulin resistance and metabolic, neurocognitive, reproductive, and other adverse effects. Age-related differences in the hypothalamo-pituitary-testicular/adrenal response to sleep restriction could expose older individuals to greater or lesser risk, but this possibility has not been previously studied. Subjects and Methods: Thirty-five healthy young and older men aged 18-30y (n=17) and 60-80y (n=18), underwent blood sampling in the Mayo Clinic Center for Clinical and Translational Science every 10 minutes for 24 hours from 6PM-6PM under two conditions in random order spaced at least 3 weeks apart: awake (no sleep) or sleep (from 10PM to 6AM). Blood was assayed for LH, testosterone (T) and cortisol (F), and then analyzed by automated mathematical deconvolution and with cross approximate entropy statistics to determine hormone secretion and hormone synchrony, respectively. Statistical significance was construed by repeated measures ANOVA using a full factorial model that included age, sleep and the interaction. Results: Sleep deprivation had multiple effects on 24-hour (6PM-6PM) Te secretion with significant reductions in mean concentrations, basal, total and pulsatile secretion, and pulse frequency (each P&lt;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 T pulse frequency (P=0.02). Time-of-day analyses revealed that sleep restriction significantly reduced Te in the morning (6AM-9AM) and afternoon (3PM-6PM), reduced LH in the morning, and increased F in the afternoon, particularly in older men. Cross-approximate entropy statistics showed that sleep restriction enforced greater LH-Te and Te-LH joint synchrony in the morning (P&lt;0.05 for each), but not in the afternoon. Conclusion: Sleep restriction decreases morning LH secretion, morning and afternoon Te secretion, and increases afternoon F secretion, especially in older men. This combination of findings could plausibly cause metabolic and reproductive ill-health when accumulated over decades of life, and may explain how chronic sleep loss contributes to metabolic and reproductive diseases that are more prevalent in older men. These preliminary data also suggest a time-of-day dependent uncoupling of the regulatory control of the testicular axis, and of cortisol secretion. Direct verification by interventions that manipulate hormones during the morning and late afternoon in appropriately matched cohorts of young and older men are now required.

Abstract Context. Interleukin-2 (IL2), a proinflammatory cytokine, is used for treatment of malignancies. Increased cortisol and ACTH were noted, but not investigated in detail. This is the first study in healthy men which uses moderate high IL2 doses as used in cancer treatment. Objective. The goal of this study was to quantify cortisol secretion after a single sc injection of IL2 at 1900 h in young and older healthy men in relation to dose, age and body composition. Design. This was a placebo-controlled, blinded, prospectively randomized cross-over study in 17 young subjects (mean age 24.1 yr, range 19–30 yr) and 18 older subjects (mean age 63.9 yr, range 60–75 yr). The subjects underwent 24 h of blood sampling at 10-min intervals, starting at 1800 h. At 2000 h IL2 (3 or 6 million units per m2 body surface) or saline was injected sc. Lights were off between 2300 and 0700 h. Setting. The study was performed in a Clinical Translational Research Unit. Outcome measures. Mean concentrations of cortisol, deconvolution analysis, and approximate entropy. Abdominal visceral fat (AVF) and total abdominal fat (TAF) were calculated from single slice CT. Results. Cortisol concentrations started to rise at 2300 h. The AUC’s during the lights-on periods were unchanged by IL2. Therefore, most analyses were restricted to the 8 h lights-off period. In young volunteers pulsatile cortisol secretion increased from 52.9±5.8 to 77.0±8.0 µg/L/8h and in older subjects from 60.6±3.8 to 70.6±4.6 µg/L/8h (GLM: treatment P &lt;0.0001, treatment x age: P=0.02, mean ± SEM). Thus, the effect was smaller in older subjects. Increasing the IL2 dose increased cortisol secretion in young subjects (P= 0.001), but not in older subjects (P=0.90). In addition, the slopes (mean ± SE) of the linear part of the concentration curves after IL-2 were steeper than after placebo, pointing to accelerated release (young 1.40±0.13 to 3.76±0.11, P&lt;0.0001, in older 1.27±0.04 to 3.28±0.15, P&lt;0.0001).The incremental nocturnal pulsatile cortisol secretion after IL2 was negatively related to body composition (AVF: R= ̶ 0.41, P=0.019; TAF R= ̶ 0.41, P=0.043). Nocturnal ApEn-cortisol did not increase after low dose IL2 (0.981± 0.099 to 0.991±0.046, P=0.92). After high-dose, ApEn increased from 0.877±0.041 to 1.024±0.049, P=0.008, not correlated with body composition, nor with age. The ApEn increase points to decreased secretory regularity imposed by enhanced CRF signaling, rather than diminished cortisol feedback, which leads to greater secretory regularity. Conclusion. Il2, a paradigm for inflammation, increased pulsatile cortisol secretion, more in young than in older subjects. Higher IL2 doses in young subjects amplified cortisol secretion, but not in older subjects. Cortisol secretion exhibited an advance (earlier) time shift, accompanied by accelerated secretion. Incremental nocturnal cortisol secretion was negatively related to fat mass.

Abstract Context. Aging is associated with diminished testosterone (Te) secretion, which could be attributed to Leydig cell dysfunction, decreased pituitary stimulation and altered Te feedback. Objective. The goal was to quantify all three regulatory nodes of the GnRH-LH-Leydig cell- axis in the same cohort of healthy men, by measuring (1) indirectly the strength of the endogenous GnRH signal on the gonadotrope, (2) the strength of Te feedback on LH by ketoconazole (KTCZ), and (3) the effect of LH infusions on Te secretion, in relation to age and body composition. Design. This was a placebo-controlled, blinded, prospectively randomized cross-over study in 40 men, age 19–73 yr, BMI 20–34.3 kg/m2. A submaximal dose of ganirelix (GnRH antagonist) was used to assess outflow of GnRH, by calculating the difference between LH output during the control and ganirelix arm. Ketoconazole (steroidogenic inhibitor) was used to estimate feedback, by the difference in LH output during ketoconazole and control arm. High-dose ganirelix and repeated 6-min LH (18.75 IU) infusions were used to measure testicular responsivity. Blood sampling was at 10-min intervals. The 4 sessions were concluded with, a single submaximally GnRH stimulus to assess the responsiveness of the gonadotrope during ganirelix inhibition. Setting. The study was performed in a Clinical Translational Research Unit. Interventions. In 3 of the 4 experiments subjects underwent 5 h of blood sampling at 10-min intervals, starting at 0800 h. At 1100 h GnRH was injected and sampling was continued for another 2 h. Admission was at 1700 h the day before. At 2000 h they received KTCZ, dexamethasone or ganirelix and/or placebo. KTCZ and dexamethasone (or placebo) were administered again at 0700 when the IV catheter was placed. High-dose ganirelix was used to test the testicular responsiveness, and 7 LH pulses (90 min intervals) were given., with blood sampling from 1500 till 1300 h next day. Outcome measures. Mean concentrations of LH and (bio)Te, deconvolution analysis, endogenous dose-response LH-bioTe relation, and approximate entropy. Abdominal visceral fat (AVF) was calculated from single slice CT. Results. There were age-, but not body composition-related decreases in estimated endogenous GnRH secretion, Te’s feedback strength on LH, and Leydig cell responsivity to LH, accompanied by changes in approximate entropy. Bioavailable Te levels were negatively related to both age and 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.

Abstract Study Objectives 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. Methods 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. Results 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 &lt; 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. Conclusions 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. Clinical Trial Not applicable.

Abstract 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.

Gonadotrophin-releasing hormone (GnRH) pulsatility is required for optimal luteinizing hormone (LH) secretion, but whether LH pulsatility is required for physiological testosterone (T) secretion is not known. To test the postulate that pulses of recombinant human (rh) LH stimulate greater T secretion than continuous infusion of the same dose, a potent selective GnRH antagonist was administered overnight to 19 healthy men ages 18–49 yr. Subjects then received saline or rhLH intravenously continuously or as 6-min pulses intravenously every 1 or 2 h at the same total dose. Blood was sampled every 10 min for 10 h to quantify T responses. For the four interventions, the descending rank order of mean LH and mean T concentrations was 1-h = 2-h rhLH pulses > continuous rhLH > saline ( P < 10−3). Plateau LH and T concentrations correlated positively ( R2 = 0.943, P = 0.029) as did LH concentrations and LH half-lives ( R2 = 0.962, P = 0.019). Percentage pulsatile T secretion assessed by deconvolution analysis (Keenan DM, Takahashi PY, Liu PY, Roebuck PD, Nehra AX, Iranmanesh A, Veldhuis JD. Endocrinology 147: 2817–2828, 2006) was the highest ( P = 0.019), and half-time to attain peak T concentrations was the shortest ( P < 10−6), for 1-h rhLH pulses. Approximate entropy (a pattern-regularity measure) revealed more orderly T secretion for 1- than 2-h rhLH pulses ( P = 0.0076). Accordingly, a pulsatile LH signal, while not obligatory to maintain mean T concentrations, controls the mean plasma LH concentration and determines quantifiable patterns of T secretion. These data introduce the question whether blood T patterns in turn supervise distinctive target-tissue responses.