Veldhuis, Johannes D.

The age‐associated decline in growth hormone (GH) secretion may be a consequence of the reduction in the number of GH‐releasing hormone (GHRH) positive neurones. However, it remains unclear whether an alteration in the number or distribution of somatostatin (SST) neurones contributes to this change. In the present study, we characterised the role of SST in modulating the change in pulsatile GH secretion in male C57Bl/6J mice throughout puberty and into early adulthood. We assessed pulsatile GH secretion in mice at 4, 8 and 16 weeks of age. These ages correspond to early pubertal, early adulthood and adulthood, respectively. We show an elevation in peak, total and pulsatile GH secretion coinciding with periods of rapid linear growth. Using in situ hybridisation and morphometric methods, we mapped the distribution of Sst mRNA expression within the mouse brain relative to this change in pulsatile GH secretion. The results obtained show that altered pulsatile GH secretion in male mice from 4–16 weeks of age does not coincide with a significant change in the number of Sst mRNA expressing neurones or an abundance of Sst mRNA expression throughout the arcuate nucleus (ARC) and periventricular nucleus (PeV). Rather, we observed a progressive decline in Sst mRNA expressing neurones within subnuclei of the paraventricular nucleus at this time. We conclude that structural changes in Sst expression within the PeV and ARC may not reflect the observed decline in pulsatile GH secretion in mice from puberty into early adulthood.

Pulsatile growth hormone (GH) secretion putatively reflects integrated regulation by GH-releasing hormone (GHRH), somatostatin (SST), and GH-releasing peptide (GHRP). GHRH and SST secretion is itself pulsatile. However, how GHRH and SST pulses act along with GHRP to jointly determine pulsatile GH secretion is unclear. Moreover, how testosterone (T) modulates such interactions is unknown. These queries were assessed in a prospectively randomized, placebo-controlled double-blind cohort comprising 26 healthy older men randomized to testosterone (T) vs. placebo supplementation. Pulses of GHRH, SST, or saline were infused intravenously at 90-min intervals for 13 h, along with either continuous saline or ghrelin analog (GHRP-2). The train of pulses was followed by a triple stimulus (combined l-arginine, GHRH, and GHRP-2) to estimate near-maximal GH secretion over a final 3 h. Testosterone vs. placebo supplementation doubled pulsatile GH secretion during GHRH pulses combined with continuous saline (GHRH/saline) ( P < 0.01). Pulsatile GH secretion correlated positively with T concentrations (270–1,170 ng/dl) in the 26 men during saline pulses/saline ( P = 0.015, R2 = 0.24), GHRH pulses/saline ( P = 0.020, R2 = 0.22), and combined GHRH pulses/GHRP-2 ( P = 0.016, R2 = 0.25) infusions. Basal nonpulsatile GH secretion correlated with T during saline pulses/GHRP-2 drive ( P = 0.020, R2 = 0.16). By regression analysis, pulsatile GH secretion varied negatively with body mass index (BMI) during saline/GHRP-2 infusion ( P = 0.001, R2 = 0.36), as well as after the triple stimulus preceded by GHRH/GHRP-2 ( P = 0.013, R2 = 0.23). Mean (10-h) GH concentrations under GHRP-2 were predicted jointly by estradiol (positively) and BMI (negatively) ( P < 0.001, R2 = 0.520). These data indicate that estradiol, T, and BMI control pulsatile secretagogue-specific GH-regulatory mechanisms in older men.

SummaryBackgroundKisspeptin is a novel hypothalamic peptide which stimulates endogenous gonadotrophin releasing hormone (GnRH) secretion. A single subcutaneous bolus injection of kisspeptin‐54 increases circulating luteinizing hormone (LH) levels in women, but its acute effects on LH pulsatility are not known.AimsTo investigate the effects of a single subcutaneous (sc) injection of kisspeptin‐54 administration on LH pulsatility in healthy female volunteers.MethodsSix healthy female adult volunteers underwent 10‐minute blood sampling for serum LH measurement for 8 h during the follicular phase of menstrual cycle. Sc bolus injection of saline or kisspeptin‐54 (0·15, 0·30 or 0·60 nmol/kg) was administered 4 h after commencing the study. A previously described, blinded deconvolution method was used to detect LH pulses.ResultsMean number of LH pulses was increased significantly following 0·30 and 0·60 nmol/kg kisspeptin‐54 when compared with saline (mean increase in number of LH pulses per 4 h, following injection: −0·17 ± 0·54, saline; +2·33 ± 0·56, 0·30 nmol/kg kisspeptin‐54, P < 0·05 vs saline; +2·33 ± 0·80, 0·60 nmol/kg kisspeptin‐54, P < 0·05 vs saline). LH pulse secretory mass increased following injection of 0·60 nmol/kg in five of six subjects, but the mean change in all subjects was non‐significant when compared with saline (mean increase in pulse secretory mass in IU/l following injection: +0·35 ± 0·40, saline; +2·61 ± 1·17, 0·60 nmol/kg kisspeptin‐54, P = 0·10 vs saline).ConclusionsA single injection of kisspeptin‐54 temporarily stimulates the number of LH pulses in healthy women. Further studies are required to investigate the therapeutic potential of kisspeptin‐54 injection to restore LH pulsatility in patients with reproductive disorders caused by impaired GnRH secretion.

AbstractRationaleLow serum testosterone is commonly observed in men with type 2 diabetes (T2DM), but the neuroendocrine pathophysiology remains to be elucidated.ObjectivesThe hypothalamic neuropeptide kisspeptin integrates metabolic signals with the reproductive axis in animal models. We hypothesized that administration of exogenous kisspeptin‐10 will restore luteinizing hormone (LH) and testosterone secretion in hypotestosteronaemic men with T2DM.ParticipantsFive hypotestosteronaemic men with T2DM (age 33·6 ± 3 years, BMI 40·6 ± 6·3, total testosterone 8·5 ± 1·0 nmol/l, LH 4·7 ± 0·7 IU/l, HbA1c 7·4±2%, duration of diabetes <5 years) and seven age‐matched healthy men.Experiment 1Mean LH increased in response to intravenous administration of kisspeptin‐10 (0·3 mcg/kg bolus) both in healthy men (5·5 ± 0·8 to 13·9 ± 1·7 IU/l P < 0·001) and in men with T2DM (4·7 ± 0·7 to 10·7 ± 1·2 IU/l P = 0·02) with comparable ΔLH (P = 0·18).Experiment 2Baseline 10‐min serum sampling for LH and hourly testosterone measurements were performed in four T2DM men over 12 h. An intravenous infusion of kisspeptin‐10 (4 mcg/kg/h) was administered for 11 h, 5 days later. There were increases in LH (3·9 ± 0·1 IU/l to 20·7 ± 1·1 IU/l P = 0·03) and testosterone (8·5 ± 1·0 to 11·4 ± 0·9 nmol/l, P = 0·002). LH pulse frequency increased from 0·6 ± 0·1 to 0·9 ± 0 pulses/h (P = 0·05) and pulsatile component of LH secretion from 32·1 ± 8·0 IU/l to 140·2 ± 23·0 IU/l (P = 0·007).ConclusionsKisspeptin‐10 administration increased LH pulse frequency and LH secretion in hypotestosteronaemic men with T2DM in this proof‐of‐concept study, with associated increases in serum testosterone. These data suggest a potential novel therapeutic role for kisspeptin agonists in enhancing endogenous testosterone secretion in men with T2DM and central hypogonadism.

Aim Postprandial insulin pulsatility is impaired in patients with type 2 diabetes, but the effects of exogenous insulin therapy on pulsatile insulin secretion are not known. We addressed, whether pulsatile insulin secretion is related to glycaemic control, whether basal insulin supplementation increases postprandial insulin secretion, and if so, is this accomplished by a specific improvement in pulsatile insulin secretion? Methods Fourteen patients with type 2 diabetes underwent a mixed meal test before and after an 8‐week treatment period with insulin glargine. Glucose, insulin and C‐peptide levels were measured, and insulin pulsatility was determined by deconvolution analysis. Results Insulin treatment lowered fasting glycaemia from 179.6 ± 7.5 mg/dl to 117.6 ± 6.5 mg/dl (p < 0.001). Postprandial insulin and C‐peptide levels increased significantly after the treatment period (p < 0.0001). The total calculated insulin secretion rate increased with insulin treatment (p = 0.0039), with non‐significant increases in both pulsatile and non‐pulsatile insulin secretion. Insulin pulse frequency was unchanged by the intervention. There was an inverse relationship between fasting and postprandial glycaemia and insulin pulse mass ( r 2 = 0.51 and 0.56, respectively), whereas non‐pulsatile insulin secretion was unrelated to either fasting or postprandial glucose concentrations ( r 2 = 0.0073 and 0.031). Conclusions Hyperglycaemia in type 2 diabetes is associated with a reduction in postprandial insulin secretion, specifically through a reduction in insulin pulsatility. Reducing chronic hyperglycaemia by basal insulin therapy enhances endogenous β‐cell function in the postprandial state. These data support the use of basal insulin regimens in the pharmacotherapy of overtly hyperglycaemic patients with type 2 diabetes.

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.

The long-lived glycoprotein hormone, human chorionic gonadotropin (hCG), downregulates testosterone (T) biosynthesis in vitro and in vivo in animals and humans. The degree to which short-lived pulses of pituitary luteinizing hormone (LH) do so, particularly at physiological concentrations, is not known. We test the hypothesis that continuous LH infusion compared with bolus injections of LH every 1 h or every 2 h overnight downregulates T secretory responses to a subsequent fixed template of three consecutive intravenous pulses of a physiological amount of recombinant human (rh) LH (triple stimulus). Nineteen healthy men ages 18–49 yr each underwent four separate randomly ordered overnight gonadotropin-releasing hormone-receptor antagonist treatments with superimposed intravenous infusions of saline or rhLH (1-h pulses, 2-h pulses, or continuously). Each 12-h infusion protocol was followed by the triple rhLH-pulse stimulus the next morning. During the triple stimulus, basal (nonpulsatile) as well as total (basal plus pulsatile) T secretion was higher after overnight 2- and 1-h rhLH pulses than after continuous rhLH or saline delivery. Approximate entropy, a probabilistic measure of feedforward-induced irregularity of T concentration time series, was higher after 1-h rhLH pulses than after continuous rhLH. Analytical estimation of pulsatile rhLH-T dose-response measures revealed higher T secretory sensitivity and greater rhLH potency (lower EC50) after exposure to 1-h than 2-h rhLH pulses. Collectively, these data indicate that in vivo dynamics of LH-stimulated T secretion under standardized conditions in men depend on the prior time mode of LH delivery in the bloodstream.

The transition between puberty and adulthood is accompanied by a slowing in linear growth. Although GH is a key factor that drives somatic development into adulthood, early adulthood coincides with a reduction in circulating levels of GH. To this extent, a pathological decline in postpubertal GH secretion is detrimental to attainment of peak lean muscle mass and bone mass and promotes adiposity and increases susceptibility to the development of obesity in adulthood. Here we characterized pulsatile GH secretion in C57BL/6J mice at 12 and 16 wk of age. Deconvolution analysis of these measures reveals a reduction in pulsatile GH secretion between 12 and 16 wk of age. Dietary intervention with high-fat feeding at 8 wk of age results in a significant increase in adiposity, the development of glucose intolerance, and hyperinsulinemia. We show the exacerbation of the age-associated decline in pulsatile GH secretion in high-fat-fed mice after 4 wk of dietary intervention (at 12 wk of age), and a further suppression of pulsatile GH secretion by 8 wk of dietary intervention (at 16 wk of age). Suppressed pulsatile secretion of GH did not coincide with an elevation in circulating free fatty acids. Rather, we observed increased hepatic triglyceride content and an eventual decrease in circulating levels of IGF-I. Given the established role of GH in maintaining healthy aging, we anticipate that an advancing of the age-associated decline in pulsatile GH secretion as a consequence of dietary-induced weight gain may have long-term ramifications on adult health.