GH Pulse Research Articles

9318 Growth Response To Oral Growth Hormone Secretagogue LUM-201 In Children With Moderate GH Deficiency (GHD) Is Dependent On The Pattern Of Pulsatile GH Secretion Induced By LUM-201

Abstract Disclosure: A. Roslan: None. R. Roman: None. A. Avila: None. D. Said: None. I. Baier: None. E. Brincks: Employee; Self; Lumos Pharma, Inc.. Stock Owner; Self; Lumos Pharma, Inc. A. Bruchey: Employee; Self; Lumos Pharma, Inc.. Stock Owner; Self; Lumos Pharma, Inc. J. McKew: Employee; Self; Lumos Pharma, Inc.. Stock Owner; Self; Lumos Pharma, Inc. P. Pitukcheewanont: Employee; Self; Lumos Pharma, Inc.. Stock Owner; Self; Lumos Pharma, Inc.. M. Johnson: None. T. Garner: Consulting Fee; Self; Lumos Pharma, Inc.. Grant Recipient; Self; Novo Nordisk. M.O. Thorner: Consulting Fee; Self; Lumos Pharma, Inc.. Stock Owner; Self; Lumos Pharma, Inc. P.E. Clayton: Advisory Board Member; Self; Lumos Pharma, Inc.. Consulting Fee; Self; Lumos Pharma, Inc.. Grant Recipient; Self; Novo Nordisk. A. Stevens: Consulting Fee; Self; Lumos Pharma, Inc. F. Cassorla: Consulting Fee; Self; Lumos Pharma, Inc.. Background: Oral GH secretagogue, LUM-201, increases growth rates in children with moderate GHD in Phase 2 trials (OraGrowtH210 and 212) [ENDO 2023, OR21-06]. GH pulse profiles at baseline (D1) and 6 months (6M) were assessed in OraGrowtH212. This study aims to understand relationships between pulse profiles and growth response to LUM-201. Methods: The OraGrowtH212 trial is being conducted at a single site (Chile). 22 prepubertal children (M 14: F 8) with moderate GHD (peak GH level on two stimulation tests >3 and <10ng/ml), who had basal IGF-I >30ng/ml and an acute GH response to 0.8mg/kg LUM-201 of ≥5ng/ml, were randomised to receive LUM-201 1.6 or 3.2 mg/kg/day. 10min samples over 12hrs (08.00-20.00) were collected at D1 and M6 for measurement of GH concentration (ng/ml), with GH secretion rates (ng/ml/min) derived by deconvolution analysis. A univariate Spearman’s rank correlation matrix was used to screen for relationships (significant at p<0.05) between D1 characteristics, D1 height velocity, 6M annualised height velocity (AHV) and interpulse, pulsatile and total GH secretion at D1 and M6.The pattern of pulsatile GH secretion in the 12hr profile was characterised using Functional Principal Component Analysis (FPCA) - used to explore the dominant modes of variation in functional data, in this case the GH time series. Subjects were grouped into tertiles based on 6M AHV (High [mean 8.9cm/yr], Medium [7.7], and Low [6.7]), and the 12hr profiles were divided into three 4hr intervals. The interquartile range (IQR) for mean GH secretion (representing variation in amount) and principal components [PCs] (variation in pattern) for all subjects in each AHV tertile at D1 and M6 were generated. Results: All GH secretion parameters increased significantly from D1 to M6 (p<0.01). Age was negatively associated with 6m AHV (rs= -0.46), and D1 pulsatile GH secretion was positively associated with D1 AHV (rs = +0.51). However, GH secretion at D1 and M6 was not correlated with 6m AHV. In the FPCA, the difference in IQR for mean GH secretion from D1 to 6M was highest over 0-4hrs for subjects in the high and medium AHV tertiles (5- and 3.3-fold greater respectively compared to the other time intervals), while subjects with low AHV had the highest difference over 8-12hrs (42-fold greater). Using the first PC (which accounts for ∼50% of total variation), the change in IQR was highest over 0-4 and 4-8hrs in the high AHV tertile (3-fold greater compared to other time intervals), over 0-4hrs in the medium tertile (46-fold greater) and over 8-12hrs in the low tertile (3.4-fold greater). Conclusions: Oral LUM-201 stimulates significant increases in GH secretion over 6 months in children with moderate GH deficiency. Complex relationships exist between growth response and both the amount and pattern of GH secretion, with the highest growth responses to LUM-201 associated with the greatest pulsatile activity early in the profile. Presentation: 6/3/2024

OR21-03 Dose Responsiveness Of LUM-201 As Measured By Acute GH Response And IGF-1 And Annualized Height Velocity (AHV) Measured At 6 Months In The Interim Analysis Of The OraGrowtH212 Study In Idiopathic Pediatric Growth Hormone Deficiency (iPGHD)

Abstract Disclosure: F. Cassorla: Research Investigator; Self; Lumos Pharma, Inc. Speaker; Self; Lumos Pharma, Inc. R. Román: Research Investigator; Self; Lumos Pharma, Inc. M.L. Johnson: Consulting Fee; Self; Lumos Pharma, Inc. A. Avila: Research Investigator; Self; Lumos Pharma, Inc. G. Iñiguez: Research Investigator; Self; Lumos Pharma, Inc. I. Baier: Research Investigator; Self; Lumos Pharma, Inc. D. Said: Research Investigator; Self; Lumos Pharma, Inc. A. Bruchey: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. C. Smith: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. E.L. Brincks: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. J.C. McKew: None. D.B. Karpf: Employee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. M.O. Thorner: Consulting Fee; Self; Lumos Pharma, Inc. Stock Owner; Self; Lumos Pharma, Inc. Objective: To evaluate acute GH, IGF-1, AHV at 6 mos of treatment of iPGHD subjects. Background: LUM-201 is an investigational oral GH secretagogue (GHS) currently in 3 Phase 2 iPGHD trials. The LUM-201 predictive enrichment marker (PEM) may be used to identify patients previously diagnosed with iPGHD who are likely to respond to LUM-201. PEM positivity is defined as a baseline IGF-1 level >30 ng/mL and a peak GH of ≥5 ng/mL in response to a single 0.8 mg/kg LUM-201 dose. LUM-201, a potent, durable oral GHS agonist, acts on the GHS Receptor 1a in the hypothalamus and anterior pituitary. LUM-201 enhances the amplitude of endogenous GH pulsatile release over 24 hrs, while also acting as a functional antagonist of the somatostatin receptor. The increased IGF-1 and GH reach the growth plates and promote linear growth. Three advantages of LUM-201 as a potential therapeutic for iPGHD are that it is a small oral tablet, it enhances natural GH pulses over 24 hr, and GH release is subject to the endogenous negative feedback mechanisms, so serum IGF-1 is expected to remain within the normal range. Method: Each subject is screened with the PEM test, which includes determining the max GH concentration in the first 90 min after a single 0.8 mg/kg dose of LUM-201. Additionally, each of these PEM positive subjects was assessed for increasing doses of LUM-201 to induce further acute GH secretion on their first day of therapeutic dosing with either a 1.6 or 3.2 mg/kg dose. The first 10 subjects with 6 months of growth on LUM-201 (5 subjects on each dose) were evaluated on their IGF-1 levels and AHVs. Results: The mean (+SD, n) peak GH for the 0.8 mg/kg dose was 26.04 ng/mL (+10.6, 22), at the 1.6 mg/kg dose 35.50 ng/mL (+17.5, 9), and at a 3.2 mg/kg dose 38.34 ng/mL (+10.4, 8). Serum IGF-1 levels of the 5 subjects in each cohort were monitored at baseline and at 6 months of treatment. The mean (+ SD) serum IGF-1 levels showed a change from baseline of 84.6 ng/mL (+30.1) at 6 months for the 1.6 mg/kg cohort and 113.8 (+49.9) at the 3.2 mg/kg dose. The pretreatment HV were 4.9 (+ 0.4) at 1.6 mg/kg and 4.3 (+ 0.9) cm/year at 3.2 mg/kg respectively and the respective mean AHVs (+ SD) at 6 months were 7.14 cm/year (+0.65) and 8.60 cm/year (+1.22). The acute GH response demonstrated a dose response between the 0.8 and 1.6 mg/kg doses, but the 1.6 and 3.2 mg/kg doses produced similar GH responses. These two doses of LUM-201 induced comparable changes from baseline values of IGF-1 after 6 months of treatment. In addition to these pharmacodynamic changes, the AHVs generated from each dose also appear similar. Conclusion: LUM-201 generated the expected AHV in this naive iPGHD population. The dose-response for GH secretion after an initial dose of LUM-201 and increases from baseline of serum IGF-1 after 6 months of treatment suggest that 1.6 mg/kg/day is the optimum dose for efficacy and durability. The results from this small study are corroborated by the larger OraGrowtH210 study data. Presentation: Saturday, June 17, 2023

THU043 Single-cell Transcriptomics Show Sex-specific Responses To A High Fat Diet, Under Thermoneutral Conditions, Pointing To Reduced Pituitary Plasticity

Abstract Disclosure: T.K. Miles: None. A.N. Lagasse: None. A.K. Odle: None. S.S. Lira: None. M.M. Syed: None. N. Akhter: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. Hyperinsulinemia caused by a high fat diet (HFD) directly suppressed pituitary growth hormone (Gh) and growth hormone releasing hormone receptor (Ghrhr) mRNAs (Luque and Kineman, 2006). To study the broader impact of a HFD, we exposed 3 groups of FVB.129P mice to 60% HFD for 10 or 15 wks at thermoneutrality (TN) (30 C), or 15 wks at room temperature (RT). Pituitary transcript expression was analyzed with single-cell RNA-seq. HFD-fed mice gained >20 g by 12 wks, showing increased serum leptin (1.8-2X), insulin (2-8X) and IL-6 (4-6X), decreased glucose clearance after a glucose challenge and decreased respiratory quotient indicating fat burning. Activity was unaffected by HFD. Rank plot analyses showed reduced amplitude GH pulses in HFD females fed at RT (15 wks), but not in mice fed at TN. Serum levels of other pituitary hormones were unaffected in HFD females. In HFD males, only serum TSH was elevated significantly (1.8X, 15 wks, TN). Analysis of the scRNA-seq data detected separate clusters representing each cell type. Two multihormonal cell clusters evident in control females mostly disappeared following HFD (15 wks, RT). At the threshold cutoff of avg Log2FC±0.5 with adj p<0.05, female somatotrope clusters showed reduced Pomc, Cga, and ribosomal proteins. Female lactotrope clusters exhibited decreased Gh mRNA (avg Log2FC -1.8 or -2.5) and galanin at 10 wks (TN). At 15 wks (TN), lactotrope clusters showed significant changes in 46 transcripts including reductions in Prl, 29 ribosomal proteins, galanin, and 3 transcriptional regulators. Notably, this cluster showed an increase in synaptotagmin-4 (Syt4), which is also elevated in oxytocin neurons following a HFD. Stem cell clusters in females were strongly impacted by HFD after 15 weeks (TN) with 94 reduced transcripts of which 48 were involved in cytoplasmic translation and ribosome biogenesis and >20 were involved with signaling and cell differentiation. In males, whereas changes in most clusters fell below the threshold cutoffs, stem cell clusters had 13 reduced transcripts after 10 or 15 weeks, most of which involved immune system processes including defense responses and antigen processing and presentation. The GH deficiency in mice fed under thermoneutral conditions was not as severe as that reported by Luque and Kineman, perhaps due to differences in strain, sex, and/or the environmental temperature. The reduced expression of multihormonal clusters, Gh by lactotropes and corticotropes, and Cga and Pomc by somatotropes suggests that HFD may reduce plasticity and activity of progenitor cells in the pituitary. The changes in lactotropes and somatotropes also indicate that HFD may compromise ribosome function and cytoplasmic translation. The reduced transcriptome in stem cells in both males and females suggests HFD interference with cell differentiation and pituitary plasticity as well as dysfunctional immune defense responses. Presentation: Thursday, June 15, 2023

Increased GH Secretion and Body Growth in Mice Carrying Ablation of IGF-1 Receptor in GH-releasing Hormone Cells

Growth hormone (GH) secretion is controlled by short and long negative feedback loops. In this regard, both GH (short-loop feedback) and insulin-like growth factor 1 (IGF-1; long-loop feedback) can target somatotropic cells of the pituitary gland and neuroendocrine hypothalamic neurons to regulate the GH/IGF-1 axis. GH-releasing hormone (GHRH)-expressing neurons play a fundamental role in stimulating pituitary GH secretion. However, it is currently unknown whether IGF-1 action on GHRH-expressing cells is required for the control of the GH/IGF-1/growth axis. In the present study, we investigated the phenotype of male and female mice carrying ablation of IGF-1 receptor (IGF1R) exclusively in GHRH cells. After weaning, both male and female GHRHΔIGF1R mice exhibited increases in body weight, lean body mass, linear growth, and length of long bones (tibia, femur, humerus, and radius). In contrast, the percentage of body fat was similar between control and GHRHΔIGF1R mice. The higher body growth of GHRHΔIGF1R mice can be explained by increases in mean GH levels, GH pulse amplitude, and pulse frequency, calculated from 36 blood samples collected from each animal at 10-minute intervals. GHRHΔIGF1R mice also showed increased hypothalamic Ghrh mRNA levels, pituitary Gh mRNA expression, hepatic Igf1 expression, and serum IGF-1 levels compared with control animals. Furthermore, GHRHΔIGF1R mice displayed significant alterations in the sexually dimorphic hepatic gene expression profile, with a prevailing feminization in most genes analyzed. In conclusion, our findings indicate that GHRH neurons represent a key and necessary site for the long-loop negative feedback that controls the GH/IGF-1 axis and body growth.

Growth Hormone Secretion Does Not Increase After Interleukin-2 Administration in Healthy Men

Context: Interleukin-2 (IL2), a proinflammatory cytokine, is used for treatment of malignancies. Increased cortisol and ACTH were noted, but GH secretion was 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 GH secretion after a single sc injection of IL2 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) and 18 older subjects (mean age 63.9 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. Outcome Measures: Deconvolution analysis of GH. Abdominal visceral fat (AVF) was calculated from single slice CT. Results: GH secretion (pulsatile and total) was negatively related to AVF (R=0.67, P<0.0001) and to age (R=0.56, P=0.001). These relations were maintained after IL2 administration. GH profiles were pulsatile under both experimental conditions. Since the effect of IL2 might be limited in time, GH deconvolution analyses were performed on the complete 24 h data series, the period with lights off and 6 h after IL2 administration. Total 24 h GH secretion decreased non-significantly from 74.6 ±10.2 to 67.5±7.4 µg/L (P=0.25) and pulsatile secretion from 69.4±9.8 to 62.2±7.4 µg/L (P=0.23). In the GLM procedure the effect of IL2 treatment was borderline significant (P=0.08), no interaction with age (P=0.28), but borderline with IL2 dose (P=0.07), and caused by decreased GH secretion (88 to 67 µg/L) at a low dose IL2. In a sub-analysis of the two age groups no effect on GH secretion in the older group was noted, in contrast to young subjects, especially on low dose IL2 (P=0.07). The analysis was refined further by calculating cumulative pulse masses in 2-h bins. By this approach it could be demonstrated that the cumulative GH pulse mass in older subjects across the 24 h was unchanged, while that in young subjects was temporarily inhibited in the bins between midnight and 0400 h (P=0.019 and 0.038). Conclusion: This study demonstrated that IL2 temporarily diminished GH secretion in young but not elderly volunteers. Pituitary hormone secretion by IL2 has not been studied as extensively as other cytokines, including IL1, IL6 and TNF. IL2 stimulates ACTH and TSH, inhibits LH and has no effect on GH secretion by rat pituitary (PNAS 58:185,1993). In patients with renal cancer IL2 infusion has no effect on GH levels (Anti-Cancer Res 10:753,1990. The present findings extend these observations to a large group of healthy volunteers, in whom we demonstrated previously suppression of the gonadal axis and activation of the HPA-axis (JCEM 101:539,2016, Endocr Connect 9:637,2010).

Effects of transdermal testosterone gel or an aromatase inhibitor on serum concentration and pulsatility of growth hormone in older men with age-related low testosterone

This randomized controlled proof-of-concept trial investigated the relative effects of testosterone and estradiol on GH pulsatility and IGF-1 in older men with low testosterone. Thirty-seven men, ≥65years with total testosterone <350ng/dL were randomized to 5g transdermal testosterone gel (TT), 1mg oral aromatase inhibitor (AI) or placebo daily for 12months. Primary outcome was deconvolution and approximate entropy analyses of pulsatile including basal and entropic modes of secretion performed at baseline and 3months. Secondary outcomes included IGF-1 evaluated at baseline, 3 and 6months. At 3months, mean GH and in IGF-1 were similar between the three groups. At 6months, IGF-1 significantly increased by Δ 15.3±10.3ng/ml in the TT-group compared to placebo (P=0.03). Both intervention groups significantly increased GH pulse frequency (TT-group, P=0.04; AI-group, P=0.05) compared to placebo. The GH secretory-burst mode (duration) significantly decreased in the TT-group (P=0.0018) compared to placebo while it remained unchanged in the AI-group (P=0.059). In older men, testosterone increases GH pulse frequency while the aromatization to estradiol is involved in the rise of IGF-1 levels.

Endogenous Estrogen Regulates Somatostatin-Induced Rebound GH Secretion in Postmenopausal Women.

Systemic concentrations of T, estradiol (E2), GH, IGF-1, and IGF binding protein-3 decline in healthy aging individuals. Conversely, T and E2 stimulate GH and IGF-1 production in hypogonadal patients. Because E2 stimulates GH secretion, putatively via the nuclear estrogen receptor-α and E2 and GH fall with menopause, we postulated that diminished endogenous E2 contributes to low GH output in older women. The study was conducted at the Mayo Center for Clinical and Translational Science. This was a randomized, double-blind, controlled study in 60 healthy postmenopausal women treated with the following: 1) double placebo; 2) anastrozole, a potent inhibitor of aromatase-enzyme activity, which mediates E2 synthesis from T; and/or 3) fulvestrant, a selective estrogen receptor-α antagonist. GH pulse generation was quantified by frequent GH sampling before and after short-term iv somatostatin infusion, thought to induce hypothalamic GHRH-mediated rebound-like GH secretion. On anastrozole, E2 fell from 3.1 ± 0.35 pg/mL to 0.36 ± 0.04 pg/mL, and estrone from 13 ± 1.4 pg/mL to 1.9 ± 0.01 pg/mL (P < .001) by mass spectrometry. Estrogen values were unchanged by fulvestrant. T concentrations did not change. One-hour peak GH rebound after somatostatin infusion declined markedly during both estrogen-deprivation schedules (P < .001). Mean (150 min) maximal GH rebound decreased comparably (P < .001). Measures of GH rebound correlated negatively with computed tomography-estimated abdominal visceral fat (all P < .05). These data suggest a previously unrecognized dependence of hypothalamo-pituitary GH regulation on low levels of endogenous estrogen after menopause.

Cyp1b1 affects external control of mouse hepatocytes, fatty acid homeostasis and signaling involving HNF4α and PPARα

Cytochrome P450 1b1 (Cyp1b1) is expressed in endothelia, stellate cells and pre-adipocytes, but not hepatocytes. Deletion alters liver fatty acid metabolism and prevents obesity and hepatic steatosis. This suggests a novel extra-hepatocyte regulation directed from cells that express Cyp1b1. To characterize these mechanisms, microarray gene expression was analyzed in livers of normal and congenic Cyp1b1-ko C57BL/6 J mice fed either low or high fat diets. Cyp1b1-ko gene responses indicate suppression of endogenous PPARα activity, a switch from triglyceride storage to mitochondrial fatty acid oxidation and decreased oxidative stress. Many gene responses in Cyp1b1-ko are sexually dimorphic and correspond to increased activity of growth hormone mediated by HNF4α. Male responses stimulated by GH pulses are enhanced, whereas responses that decline exhibit further suppression, including Cyp regulation by PPARα, CAR and PXR. These effects of Cyp1b1 deletion overlap with effects caused by deletion of the small heterodimeric partner, a suppressor of these nuclear factors. Redirection of gene expression associated with liver fat homeostasis in Cyp1b1-ko mice that directs hypothalamic control of GH and leptin. Cyp1b1-ko suppresses neonatal Scd1 and delays adult maturation of dimorphic GH/HNF4α signaling. Alternatively, deletion may diminish hypothalamic metabolism of estradiol, which establishes adult GH regulation.

A Reevaluation of the Question: Is the Pubertal Resurgence in Pulsatile GnRH Release in the Male Rhesus Monkey (Macaca mulatta) Associated With a Gonad-Independent Augmentation of GH Secretion?

A somatic signal has been posited to trigger the pubertal resurgence in pulsatile GnRH secretion that initiates puberty in highly evolved primates. That GH might provide such a signal emerged in 2000 as a result of a study reporting that circulating nocturnal GH concentrations in castrated juvenile male monkeys increased in a 3-week period immediately preceding the pubertal resurgence of LH secretion. The present study was conducted to reexamine this intriguing relationship, again in an agonadal model. Four castrated juvenile male monkeys were implanted with indwelling jugular catheters, housed in remote sampling cages, and subjected to 24 hours of sequential blood sampling (every 30 min) every 2 weeks from 19.5 to 22 months of age. Twenty-four-hour profiles of circulating GH concentrations were analyzed using the pulse detection algorithm, PULSAR, and developmental changes in pulsatile GH release with respect to the initiation of the pubertal rise of LH secretion (week 0; observed between 22.5 and 32 mo of age) were examined for significance by a repeated-measures ANOVA. Changes in the parameters of pulsatile GH secretion, including mean 24-hour GH concentration and GH pulse frequency and pulse amplitude for 3 (n = 4) and 6 (n = 3) months before week 0 were unremarkable and nonsignificant. These findings fail to confirm those of the earlier study and lead us to conclude that the timing of the pubertal resurgence of GnRH release in the male monkey is not dictated by GH. Reasons for the discrepancy between the two studies are unclear.

Permanent uncoupling of male-specific CYP2C11 transcription/translation by perinatal glutamate

Perinatal exposure of rats and mice to the typically reported 4mg/g bd wt dose of monosodium glutamate (MSG) results in a complete block in GH secretion as well as obesity, growth retardation and a profound suppression of several cytochrome P450s, including CYP2C11, the predominant male-specific isoform — all irreversible effects. In contrast, we have found that a lower dose of the food additive, 2mg/g bd wt on alternate days for the first 9days of life results in a transient neonatal depletion of plasma GH, a subsequent permanent overexpression of CYP2C11 as well as subnormal (mini) GH pulse amplitudes in an otherwise normal adult masculine episodic GH profile. The overexpressed CYP2C11 was characterized by a 250% increase in mRNA, but only a 40 to 50% increase in CYP2C11 protein and its catalytic activity. Using freshly isolated hepatocytes as well as primary cultures exposed to the masculine-like episodic GH profile, we observed normal induction, activation, nuclear translocation and binding to the CYP2C11 promoter of the GH-dependent signal transducers required for CYP2C11 transcription. The disproportionately lower expression levels of CYP2C11 protein were associated with dramatically high expression levels of an aberrant, presumably nontranslated CYP2C11 mRNA, a 200% increase in CYP2C11 ubiquitination and a 70–80% decline in miRNAs associated, at normal levels, with a suppression of CYP2C expression. Whereas the GH-responsiveness of CYP2C7 and CYP2C6 as well as albumin was normal in the MSG-derived hepatocytes, the abnormal expression of CYP2C11 was permanent and irreversible.

Somatostatin Is Essential for the Sexual Dimorphism of GH Secretion, Corticosteroid-Binding Globulin Production, and Corticosterone Levels in Mice

Distinct male and female patterns of pituitary GH secretion produce sexually differentiated hepatic gene expression profiles, thereby influencing steroid and xenobiotic metabolism. We used a fully automated system to obtain serial nocturnal blood samples every 15 minutes from cannulated wild-type (WT) and somatostatin knockout (Sst-KO) mice to determine the role of SST, the principal inhibitor of GH release, in the generation of sexually dimorphic GH pulsatility. WT males had lower mean and median GH values, less random GH secretory bursts, and longer trough periods between GH pulses than WT females. Each of these parameters was feminized in male Sst-KO mice, whereas female Sst-KO mice had higher GH levels than all other groups, but GH pulsatility was unaffected. We next performed hepatic mRNA profiling with high-density microarrays. Male Sst-KO mice exhibited a globally feminized pattern of GH-dependent mRNA levels, but female Sst-KO mice were largely unaffected. Among the differentially expressed female-predominant genes was Serpina6, which encodes corticosteroid-binding globulin (CBG). Increased CBG was associated with elevated diurnal peak plasma corticosterone in unstressed WT females and both sexes of Sst-KO mice compared with WT males. Sst-KO mice also had exaggerated ACTH and corticosterone responses to acute restraint stress. However, consistent with their lack of phenotypic signs of excess glucocorticoids, cerebrospinal fluid concentrations of free corticosterone in Sst-KO mice were not elevated. In summary, SST is necessary for the prolonged interpulse troughs that define masculinized pituitary GH secretion. SST also contributes to sexual dimorphism of the hypothalamic-pituitary-adrenal axis via GH-dependent regulation of hepatic CBG production.

OR3-6: Loss of acyl-ghrelin signalling in male ghrelin-O-acyltransferase knock-out mice results in reduced pulsatile growth hormone secretion and a derangement of GH pulse pattern

OR3-6: Loss of acyl-ghrelin signalling in male ghrelin-O-acyltransferase knock-out mice results in reduced pulsatile growth hormone secretion and a derangement of GH pulse pattern

An early reduction in GH peak amplitude in preproghrelin-deficient male mice has a minor impact on linear growth.

Ghrelin is a gut hormone processed from the proghrelin peptide acting as the endogenous ligand of the GH secretagogue receptor 1a. The regulatory role of endogenous ghrelin on pulsatile GH secretion and linear growth had to be established. The aim of the present study was to delineate the endogenous actions of preproghrelin on peripheral and central components of the GH axis. Accordingly, the ultradian pattern of GH secretion was measured in young and old preproghrelin-deficient males. Blood samples were collected by tail bleeding every 10 minutes over a period of 6 hours. Analysis of the GH pulsatile pattern by deconvolution showed that GH was secreted in an ultradian manner in all genotypes, with major secretory peaks occurring at about 3-hour intervals. In older mice, the peak number was reduced and secretion was less irregular compared with younger animals. Remarkably, in young Ghrl(-/-) mice, the amplitude of GH secretory bursts was significantly reduced. In older mice, however, genotype differences were less significant. Changes in GH pulsatility in young Ghrl(-/-) mice were associated with a tendency for reduced GH pituitary contents and plasma IGF-I concentrations, but with only a minor impact on linear growth. In Ghrl(+/-) mice, despite reduced Acyl ghrelin to des-acyl ghrelin ratio, GH secretion was not impaired. Ghrelin deficiency was not associated with a reduction in hypothalamic GHRH content or altered response to GHRH stimulation. Therefore, reduction in GHRH production and/or sensitivity do not primarily account for the altered GH pulsatile secretion of young Ghrl(-/-) mice. Instead, GHRH expression was elevated in young but not old Ghrl(-/-) mice, suggesting that differential compensatory responses resulting from the absence of endogenous ghrelin is occurring according to age. These results show that endogenous ghrelin is a regulator of GH pulse amplitude in growing mice but does not significantly modulate linear growth.

The Role of Prolactin Receptor in GH Signaling in Breast Cancer Cells

GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Furthermore, GHR and PRLR associate in a fashion augmented acutely by GH, even though GH primarily activates PRLR, rather than GHR, in these cells. To better understand PRLR's impact, we examined the effects of PRLR knockdown on GHR availability and GH sensitivity in T47D cells. T47D-ShPRLR cells, in which PRLR expression was reduced by stable short hairpin RNA (shRNA) expression, were compared with T47D-SCR control cells. PRLR knockdown decreased the rate of GHR proteolytic turnover, yielding GHR protein increase and ensuing sensitization of these cells to GHR signaling events including phosphorylation of GHR, Janus kinase 2, and signal transducer and activator of transcription 5 (STAT5). Unlike in T47D-SCR cells, acute GH signaling in T47D-ShPRLR cells was not blocked by the PRLR antagonist G129R but was inhibited by the GHR-specific antagonist, anti-GHR(ext-mAb). Thus, GH's use of GHR rather than PRLR was manifested when PRLR was reduced. In contrast to acute effects, GH incubation for 2 h or longer yielded diminished STAT5 phosphorylation in T47D-ShPRLR cells compared with T47D-SCR, a finding perhaps explained by markedly greater GH-induced GHR down-regulation in cells with diminished PRLR. However, when stimulated with repeated 1-h pulses of GH separated by 3-h washout periods to more faithfully mimic physiological GH pulsatility, T47D-ShPRLR cells exhibited greater transactivation of a STAT5-responsive luciferase reporter than did T47D-SCR cells. Our data suggest that PRLR's presence meaningfully affects GHR use in breast cancer cells.

The association of macro- and micronutrient intake with growth hormone secretion

ContextGrowth hormone (GH) is known to be nutritionally regulated, but the effect of dietary composition on detailed GH secretion parameters has not previously been comprehensively evaluated. ObjectiveThe objective of the study was to determine whether specific macro- and micronutrients are associated with discrete parameters of GH secretion among subjects with wide ranges of body mass index. DesignDetailed macro- and micronutrient intake was assessed by 4-day food records while GH secretion was assessed by standard stimulation testing in 108 men and women in one study (Study 1), and by overnight frequent blood sampling in 12 men in another study (Study 2). ResultsPeak stimulated GH was positively associated with vitamin C (r=+0.29; P=0.003), dietary fiber (r=+0.27; P=0.004), arachidic acid (r=+0.25; P=0.008), and behenic acid (r=+0.30; P=0.002) intake in univariate analysis. Controlling for age, gender, race/ethnicity, visceral fat, HOMA-IR, total caloric intake and these four dietary factors in step-wise multivariate modeling, peak GH remained significantly associated with vitamin C and visceral fat (both P<0.05). In addition, vitamin C intake was associated with various parameters of endogenous GH secretion including basal GH secretion (r=+0.95; P<0.0001), GH half-life (r=+.75; P=0.005), total GH production (r=+0.76; P=0.004), GH area-under-the-curve (r=+0.89; P=0.0001), mean log10 GH pulse area (r=+0.67; P=0.02), and overnight maximum (r=+0.62; P=0.03), nadir (r=+0.97; P<0.0001), and mean GH secretion (r=+0.89; P=0.0001). ConclusionsThese results suggest that certain micronutrients such as vitamin C intake are strongly and uniquely associated with stimulated and endogenous spontaneous GH secretion.

Differential Ontogenetic Patterns of in vitro Desensitization to GHRH in Fetal and Neonatal Anterior Pituitary

The aim of this study was to assess the ontogenetic changes in vitro in both the responsiveness of anterior pituitary tissue to growth hormone-releasing hormone (GHRH) and the critical role of GHRH in the long-term regulation of pulsatile GH secretion during perinatal porcine life. A superfusion system was used to apply three consecutive 10-min pulses of GHRH (the first of 1 nM and the other two of 10 nM) for 3 consecutive days in pituitary glands isolated from fetal (95- and 110-day) and neonatal (12-day) male pigs. In fetuses, total GHRH-induced GH release decreased progressively over the 3 days. However, in neonates, GH did not decrease until day 3, but remained higher than in fetuses. When each GH pulse was assessed individually, fetuses showed a similar pattern. GH secretion induced by the first GHRH pulse on days 1 and 2 was lower than that induced by the second and third pulses. By day 3, GH release lowered dramatically after all pulses. In contrast, in neonates no differences were observed among the GH levels induced by the three GHRH pulses at any day, although day 3 showed lower GH rates. In conclusion, during perinatal development, a desensitizing effect to long-term repetitive GHRH pulses was observed in both fetuses and neonates, but this effect was delayed in neonates. Thus, the capacity of somatotrope cells to maintain GH response to GHRH seems to be developmentally regulated during perinatal stages. Furthermore, the frequency of GHRH pulses, rather than the concentrations, might be a key factor to elicit desensitization.

Influence of testicular hormones on the somatostatin-GH system during the growth promoted transition to puberty in sheep

The aim of the present study was to investigate whether the growth promoted transition to puberty in lambs involved changes in the effects of testicular hormones on somatostatin in hypothalamic neurons and GH secretion. The study was performed in infants (9-week-old) testis-intact (TEI) and orchidectomized (ORCHX) at the sixth week of age, and pubertal lambs (16-week-old) TEI and ORCHX at the 12th week of age (n = 20). In TEI lambs, the changes included a pubertal increase in immunoreactive somatostatin in the periventricular nucleus and median eminence with simultaneous neuropeptide depletion in the median eminence, and a decrease in the percentage of the hypophyseal area (PA) occupied by GH-immunoreactive cells (P < 0.05). The mean concentration of GH in the peripheral blood plasma was greater (P < 0.001) in early infancy (5 wk), because of the greater (P < 0.0001) pulse amplitude, and then uniformly low until puberty. The postnatal increase in the body weight (BW) was prominent (P < 0.01) in middle-late infancy (9–12 wk) because of the large daily live-weight gain. After orchidectomy somatostatin was abundant. This effect on nerve terminals in the median eminence was greater (P < 0.01) in infancy and lesser (P < 0.05) in puberty. Conversely, the PA occupied by GH cells was lower in the ORCHX pubertal lambs compared to TEI lambs (P < 0.05). The GH concentration and pulse characteristics were less (P < 0.05) in the infantile and pubertal ORCHX lambs compared to the TEI lambs. However, this effect was weak (P < 0.05) until middle infancy because of no influence on the GH basal concentration, and strong (P < 0.001) after late infancy. The BW did not differ (P > 0.05) between TEI and ORCHX lambs. Findings suggest activation of GH negative autofeedback loop in middle infancy. Testicular factors may play an inhibitory role in regulating somatostatin accumulation and a stimulatory role in GH secretion until puberty. The start of puberty is related to an attenuation in the stimulatory role of gonadal factors in regulating somatostatin depletion in nerve terminals associated with an intensification of the stimulatory role of gonadal factors in regulating GH secretion. From a somatic perspective of growth rate, these mechanisms do not seem to be important. Thus, testicular factors modulate mechanisms within the somatostatin-GH system to integrate somatotropic and gonadotropic functions at the time of growth-promoted sexual maturation in sheep.

Gender, Sex-Steroid, and Secretagogue-Selective Recovery from Growth Hormone-Induced Feedback in Older Women and Men

GH negatively regulates its own secretion. How gender, sex steroids, and secretagogues modulate GH autofeedback is not known. Supplementation with sex steroids and/or a peptidyl secretagogue will enhance the escape of GH from autoinhibition, thus framing a mechanism for amplifying pulsatile GH secretion. Ten healthy postmenopausal women and 10 comparably aged men participated at the Clinical-Translational Science Unit. Randomly ordered, double-blind, prospective crossover treatment with placebo vs. testosterone (men) or placebo vs. estradiol (women). Autofeedback was imposed by an iv pulse of GH. Recovery of feedback inhibition was quantified during constant infusion of saline, GHRH, or GH-releasing peptide-2 (three peptide categories). During negative feedback, total (integrated) GH recovery depended upon gender (P = 0.017), sex hormone (P < 0.001), and peptide category (P < 0.001). Mechanistic analysis revealed that feedback-suppressed nadir GH concentrations were determined by sex-steroid treatment (P = 0.018) but not by gender (P = 0.444). Peak GH escape was controlled by both treatment (P = 0.004) and gender (P = 0.003). Nadir GH and peak GH during feedback were enhanced by GHRH or GHRP-2 (P < 0.001 for both). Gender × peptide (P = 0.012 for nadir GH), treatment × peptide (P < 0.001 total and peak GH), and gender × treatment (P = 0.017 nadir GH) regulated GH recovery interactively. Gender, sex-steroid supplementation, and secretagogue type confer distinct feedback-rescuing effects, introducing a new level of complexity in the control of pulsatile GH regulation.

Complex regulation of GH autofeedback under dual-peptide drive: studies under a pharmacological GH and sex steroid clamp

To test the postulate that sex difference, sex steroids, and peptidyl secretagogues control GH autofeedback, 11 healthy postmenopausal women and 14 older men were each given 1) a single iv pulse of GH to enforce negative feedback and 2) continuous iv infusion of saline vs. combined GHRH/GHRP-2 to drive feedback escape during pharmacological estradiol (E(2); women) or testosterone (T; men) supplementation vs. placebo in a double-blind, prospectively randomized crossover design. By three-way ANCOVA, sex difference, sex hormone treatment, peptide stimulation, and placebo/saline responses (covariate) controlled total (integrated) GH recovery during feedback (each P < 0.001). Both sex steroid milieu (P = 0.019) and dual-peptide stimulation (P < 0.001) determined nadir (maximally feedback-suppressed) GH concentrations. E(2)/T exposure elevated nadir GH concentrations during saline infusion (P = 0.003), whereas dual-peptide infusion did so independently of T/E(2) and sex difference (P = 0.001). All three of sex difference (P = 0.001), sex steroid treatment (P = 0.005), and double-peptide stimulation (P < 0.001) augmented recovery of peak (maximally feedback-escaped) GH concentrations. Peak GH responses to dual-peptidyl agonists were greater in women than in men (P = 0.016). E(2)/T augmented peak GH recovery during saline infusion (P < 0.001). Approximate entropy analysis corroborated independent effects of sex steroid treatment (P = 0.012) and peptide infusion (P < 0.001) on GH regularity. In summary, sex difference, sex steroid supplementation, and combined peptide drive influence nadir, peak, and entropic measurements of GH release under controlled negative feedback. To the degree that the pharmacological sex steroid, GH, and dual-peptide clamps provide prephysiological regulatory insights, these outcomes suggest major determinants of pulsatile GH secretion in the feedback domain.

Reverse Feeding Suppresses the Activity of the GH Axis in Rats and Induces a Preobesogenic State

Reversed feeding (RF) is known to disrupt hormone rhythmicity and metabolism. Although these effects may be mediated in part by phase inversion of glucocorticoid secretion, the precise mechanism is incompletely characterized. In this study, we demonstrate that acute nocturnal food deprivation in male rats suppressed the amplitude of spontaneous GH secretion during the dark phase by 62% (P < 0.001), without affecting baseline secretion. Prolonged RF, which reduced pituitary weight (by 22%; P < 0.05), also suppressed GH pulse height sufficiently to reduce skeletal growth (by 4-5%; P < 0.01) and terminal liver weight (by 11%; P < 0.001). Despite this suppression of the GH axis, proportionate adiposity was not elevated, probably due to the accompanying 16% reduction in cumulative food intake (P < 0.01). We demonstrate that RF also resulted in phase inversion of core clock gene expression in liver, abdominal white adipose tissue (WAT) and skeletal muscle, without affecting their expression patterns in the suprachiasmatic nucleus. In addition, RF resulted in phase inversion of hepatic peroxisome proliferator-activated receptor γ2 mRNA expression, a 3- to 5-fold elevation in fatty acid synthase mRNA in WAT in both light- and dark-phase samples (P < 0.01) and an elevation in muscle uncoupling protein 3 mRNA expression at the beginning of the light phase (P < 0.01). Consumption of a high-fat diet increased inguinal (by 36%; P < 0.05) and retroperitoneal WAT weight (by 72%; P < 0.01) only in RF-maintained rats, doubling the efficiency of lipid accumulation (P < 0.05). Thus, RF not only desynchronizes central and peripheral circadian clocks, and suppresses nocturnal GH secretion, but induces a preobesogenic state.