Growth Hormone Receptor Research Articles

Food deprivation reduces sensitivity of liver Igf1 synthesis pathways to growth hormone in juvenile gopher rockfish (Sebastes carnatus)

Growth hormone (Gh) regulates growth in part by stimulating the liver to synthesize and release insulin-like growth factor-1 (Igf1), which then promotes somatic growth. However, for fish experiencing food limitation, elevated blood Gh can occur even with low circulating Igf1 and slow growth, suggesting that nutritional stress can alter the sensitivity of liver Igf1 synthesis pathways to Gh. Here, we examined how recent feeding experience affected Gh regulation of liver Igf1 synthesis pathways in juvenile gopher rockfish (Sebastes carnatus) to illuminate mechanisms underlying the nutritional modulation of Igf1 production. Juvenile gopher rockfish were maintained under conditions of feeding or complete food deprivation (fasting) for 14 d and then treated with recombinant sea bream (Sparus aurata) Gh or saline control. Gh upregulated hepatic igf1 mRNA levels in fed fish but not in fasted fish. The liver of fasted rockfish also showed a lower relative abundance of gene transcripts encoding teleost Gh receptors 1 (ghr1) and 2 (ghr2), as well as reduced protein levels of phosphorylated janus tyrosine kinase 2 (pJak2) and signal transducer and activator of transcription 5 (pStat5), which function to induce igf1 gene transcription following Gh binding to Gh receptors. Relative hepatic mRNA levels for suppressors of cytokine signaling (Socs) genes socs2, socs3a, and socs3b were also lower in fasted rockfish. Socs2 can suppress Gh activation of Jak2/Stat5, and fasting-related variation in socs expression may reflect modulated inhibitory control of igf1 gene transcription. Fasted rockfish also had elevated liver mRNA abundances for lipolytic hormone-sensitive lipase 1 (hsl1) and Igf binding proteins igfbp1a, -1b and –3a, reduced liver mRNAs encoding igfbp2b and an Igfbp acid labile subunit-like (igfals) gene, and higher transcript abundances for Igf1 receptors igf1ra and igf1rb in skeletal muscle. Together, these findings suggest that food deprivation impacts liver Igf1 responsiveness to Gh via multiple mechanisms that include a downregulation of hepatic Gh receptors, modulation of the intracellular Jak2/Stat5 transduction pathway, and possible shifts in Socs-inhibitory control of igf1 gene transcription, while also demonstrating that these changes occur in concert with shifts in liver Igfbp expression and muscle Gh/Igf1 signaling pathway components.

Anomalies du récepteur de l’hormone de croissance hypophysaire et de son effecteur de l’axe somatotrope, l’Insulin-Like Growth Factor I

Postnatal statural growth is under the hormonal control of the somatotropic axis: hypothalamus-pituitary-liver-bone. Numerous gene abnormalities have been described, located at the hypothalamus and especially at the pituitary levels. Rarer damages are located downstream at the level of the pituitary growth hormone (GH) receptor, as in Laron's syndrome or in post-receptor signaling, Stat5b or the terminal effector of growth, the Insulin-Like Growth Factor I (IGF I). For several decades now, these severe statural growth retardations can benefit from recombinant IGF I treatment, with variable efficacy depending on the genetic abnormality.

Genetic Engineering of Donor Pig for the First Human Cardiac Xenotransplantation: Combatting Rejection, Coagulopathy, Inflammation, and Excessive Growth.

The first successful pig to human cardiac xenotransplantation in January 2022 represented a major step forward in the fields of heart failure, immunology, and applied genetic engineering, using a 10-gene edited (GE) pig. This review summarizes the evolution of preclinical modelling data which informed the use of each of the 10 genes modified in the 10-GE pig: GGTA1, Β4GalNT2, CMAH, CD46, CD55, TBM, EPCR, CD47, HO-1, and growth hormone receptor. The translation of the 10-GE pig from preclinical modelling to clinical compassionate xenotransplant use was the culmination of decades of research combating rejection, coagulopathy, inflammation, and excessive xenograft growth. Understanding these 10 genes with a view to their combinatorial effects will be useful in anticipated xenotransplant clinical trials.

Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

Effects of feeding diets with zinc-l-selenomethionine on growth performance of broilers subjected to cyclic heat stress.

Limited information exists on the use of zinc-l-selenomethionine (Zn-L-SeMet) in broiler diets and its effects on the growth performance, body temperature, mortality rates, blood profile, and gene expression, especially when animals are reared under cyclic heat stress conditions. This study aimed to investigate the impact of Zn-L-SeMet in broiler diets from 1 to 42days of age reared under cyclic heat stress and its effects on growth performance, cloacal temperatures, mortality rate, blood parameters, and insulin-like growth factor 1 (IGF-1) and growth hormone receptor (GHR) gene expression in the breast muscle. A total of 1000 male Cobb 500® broiler chicks were randomly assigned to five treatments: 0, 0.15, 0.23, 0.47, and 1.30mg/kg of Zn-L-SeMet. Each treatment consisted of 10 replicates with 20 birds each. No statistically significant differences in growth performance were observed from 1 to 21days of age (P > 0.05). However, from 1 to 42days, feed intake (FI) and feed conversion ratio (FCR) decreased linearly (P < 0.05). Cloacal temperatures showed no significant effects (P > 0.05), while overall mortality rate exhibited a quadratic response (P < 0.05), with the optimal inclusion level predicted to reduce broiler mortality at 0.71mg/kg. Triglyceride (TRG) levels increased with 0.97mg/kg (P < 0.05), and gama-glutamil transferase (GGT) levels decreased with the inclusion of 1.19mg/kg (P < 0.05). No significant effects on IGF-1 and GHR gene expression were found (P > 0.05). In conclusion, the inclusion of 1.30mg/kg of Zn-L-SeMet in diets of heat-stressed broilers improved growth performance from 1 to 42days of age. An inclusion of 0.71mg/kg reduced mortality rate, while 0.97mg and 1.19mg increased and reduced TRG and GGT levels, respectively.

An investigation of the assumed efficacy of methionine hydroxy analogue compared to DL-methionine by measuring growth performance, carcass traits, and GHR and IGF-I expression in broilers

This experiment was conducted to investigate the assumed efficacy of 65% on the product basis of methionine hydroxy analog (OH-Met, 88% aqueous solution of DL-2-hydroxy-4-(methylthio) butanoic acid) relative to DL-methionine (DL-Met) in broiler chickens. A total of 792-day-old male chickens were randomly allotted to 7 dietary treatments consisting of a methionine deficient-basal diet based on corn-soybean meal and 6 different diets obtained by supplying the basal diet with methionine at 3 levels (25, 100 and 125%; levels of addition relative to the required amount of additional Met to meet SID Met + Cys requirements) from either DL-Met or OH-Met at a ratio of 65:100 (DL-Met to OH-Met on a product basis) in the corresponding treatments. Regardless of the source, methionine supplementation enhanced (p < .05) growth performance, carcase and breast meat yield. Supplemental level and the source of Met had no significant effect on the proximate chemical composition of breast meat (p > .05). Comparison of effects of OH-Met and DL-Met supplemented at 100–65% weight ratio revealed no differences at any Met + Cys level and for performance criterion confirming the applied concept. Expression of growth hormone receptor (GHR) mRNA, which showed a positive correlation with body weight gain, carcase yield and breast percentage, in broiler liver significantly increased with 100% and 125% relative addition of DL-Met, while only 100% of OH-Met addition did (p < .05) when compared to the basal diet. The expression of the insulin-like growth factor-I (IGF-I) gene was not significantly affected by either Met source or supplementation level. In conclusion, our data indicated that DL-Met can be substituted with a 1.54 times higher amount of OH-Met in corn-soybean meal based broiler diets.

Relative expression of hormone receptors by endothelial and smooth muscle cells in proliferative and non-proliferative areas of congenital arteriovenous malformations

BackgroundEpisodic growth due to microvascular proliferations (MVP) has been reported in congenital arteriovenous malformations (AVM), which are normally quiescent lesions composed of mature malformed vessels. Since AVM also may worsen under conditions of hormonal dysregulation, we hypothesized that hormonal influences may stimulate this process of vasoproliferative growth through potential interactions with hormone receptors (HR).Methods13 Cases of AVM tissue with histologically documented vasoproliferative growth were analyzed quantitatively for the presence and tissue localization of estrogen receptor (ER), progesterone receptor (PGR), growth hormone receptor (GHR) and follicle-stimulating hormone receptor (FSHR) in relation to resident cells of interest (endothelial cells (EC), smooth muscle cells (SMC) and mast cells (MC)) by applying multiplex immunohistochemistry (IHC) staining. Expression patterns in lesions with MVP and mature vessels were quantified and compared. Available fresh frozen tissues of 3 AVM samples were used to confirm the presence of HR using Reverse-Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR).ResultsAll four HR studied were expressed in all cases within EC and SMC in areas of MVP and mature vessels, but not in normal skin tissue. ER, GHR, and FSHR showed more expression in EC of MVP and in SMC of mature vessels. RT-qPCR confirmed presence of all 4 HR in both areas.ConclusionExpression of ER, PGR, GHR, and FSHR in vasoproliferative areas of congenital AVM could explain onset of sudden symptomatic growth, as has observed in a subpopulation of patients. These findings may have implications for eventual anti-hormonal targeted therapy in the lesions involved.

A cluster of X-linked miRNAs are de-repressed with age in mouse liver and target growth hormone signaling.

Growth hormone (GH) signaling influences lifespan in a wide variety of mammalian species. We previously reported that a cluster of miRNAs located on the X-chromosome are de-repressed with age in male mouse liver, and a subset, the mir-465 family, can directly attenuate expression of the growth hormone receptor (GHR) in vitro leading to a reduction in GH signaling. Here we show that this cluster of miRNAs is also upregulated in the liver with age in females, and that calorie restriction and the Ames dwarf genotype, both known to delay aging, attenuate the upregulation of the miRNA cluster. Upregulation of mir-465 in vivo leads to a reduction in GHR mRNA in the liver and an attenuation of GH signaling, indicated by a reduction in GHR, IGF-1, IGFBP3, and ALS mRNA expression. There is a corresponding reduction in IGF-1 protein levels in the liver and plasma. These results suggest that the age-associated upregulation of the X-chromosomal cluster of miRNAs could influence lifespan.

Clinical-genetic analysis of selected genes involved in the development of the human skeleton in 128 Czech patients with suspected congenital skeletal abnormalities

BackgroundCongenital skeletal abnormalities are a heterogeneous group of diseases most commonly associated with small or disproportionate growth, cranial and facial dysmorphisms, delayed bone maturation, etc. Nonetheless, no detailed genotype-phenotype correlation in patients with specific genetic variants is readily available. Ergo, this study focuses on the analysis of patient phenotypes with candidate variants in genes involved in bone growth as detected by molecular genetic analysis. MethodsIn this study we used molecular genetic methods to analyse the ACAN, COL2A1, FGFR3, IGFALS, IGF1, IGF1R, GHR, NPR2, STAT5B and SHOX genes in 128 Czech children with suspected congenital skeletal abnormalities. Pathogenic variants and variants of unclear clinical significance were identified and we compared their frequency in this study cohort to the European non-Finnish population. Furthermore, a prediction tool was utilised to determine their possible impact on the final protein. All clinical patient data was obtained during pre-test genetic counselling. ResultsPathogenic variants were identified in the FGFR3, GHR, COL2A1 and SHOX genes in a total of six patients. Furthermore, we identified 23 variants with unclear clinical significance and high allelic frequency in this cohort of patients with skeletal abnormalities. Five of them have not yet been reported in the scientific literature. ConclusionCongenital skeletal abnormalities may lead to a number of musculoskeletal, neurological, cardiovascular problems. Knowledge of specific pathogenic variants may help us in therapeutic procedures.

OR01-04 The Growth Hormone Receptor Interacts With Transcriptional Regulator, HMGN1, Upon GH-induced Nuclear Translocation

Abstract Disclosure: L. Jain: None. M.H. Vickers: None. B. Jacob: None. M.J. Middleditch: None. D.A. Chudakova: None. A.R. Ganley: None. J.M. O’Sullivan: None. J.K. Perry: None. Numerous classical cell-surface receptors that were previously thought to signal exclusively at the cell-surface can also translocate to the nucleus upon ligand binding, with increasing evidence supporting a transcriptional role for examples such as the epidermal growth factor receptor. In addition to classic cell-surface growth hormone receptor (GHR)-mediated signaling, the GHR is rapidly imported into the nucleus of cells upon stimulation with growth hormone (GH). Nuclear GHR localization has been associated with increased cancer cell proliferation. However, whether there are transcriptional changes associated with nuclear GHR import remains to be determined. To investigate whether the GHR interacts with transcription regulators in the nucleus following nuclear translocation we used immunoprecipitation combined with mass spectrometry. GH-dependent phosphorylation of STAT5 was confirmed by western blotting in the human cell-lines RL95-2 (endometrial cancer cell-line), and MCF-10A (mammary epithelial cell-line). The GHR was found to rapidly translocate to the nucleus in both cell lines following GH treatment by immunofluorescence staining, with maximal localisation at 5-10 minutes (p&amp;lt;0.001; RL95-2). Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates following GH stimulation (500 ng/mL, 10 mins) identified 40 novel GHR binding partners, including 3 transcriptional regulators (HMGN1, SUMO1 and DDX21). Gene expression analysis identified 416 genes that were differentially expressed following GH treatment (500 ng/mL, 90 mins). Intersection of differentially expressed genes with known gene targets of HMGN1 (ChIP-Atlas) identified an enrichment of HMGN1 target genes (p&amp;lt;0.001). Co-immunoprecipitation combined with western blot analysis confirmed that HMGN1 associated with the GHR in nuclear extracts. Our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs. Presentation: Thursday, June 15, 2023

THU545 Growth Hormone Receptor Antagonist As A Novel Combination Therapy Against Triple Negative Breast Cancer

Abstract Disclosure: P. Mora-Criollo: None. R. Basu: None. J.J. Kopchick: None. Triple Negative breast (TNBC) cancer is classified based on the absence of ER-, PR-, and HER2- receptors and is associated with high metastatic potential. TNBC accounts for approximately 15-20% of new breast cancer diagnoses and is responsible for most breast cancer-related deaths. First-line treatment of anticancer drugs can be effective in 30-70% of patients with TNBC, but during treatment, more than half of the patients develop resistance and progress into metastatic disease. Previous studies have demonstrated the link between growth hormone receptor (GHR) expression and mammary carcinogenesis and established that GHR signaling promotes breast cancer development and progression. Conversely, inhibition of GHR expression has been shown to arrest tumor initiation and reduce metastasis of mammary carcinoma cells. Here we present the feasibility studies for employing GHR as a target for novel combination therapeutic approaches in TNBC. We performed retrospective analysis of GHR transcript expression and survival in TNBC patients, followed by experimental data in cellular models using combinations of Pegvisomant (an FDA-approved GHR antagonist) with chemotherapeutic agents, as a potential therapeutic option in TNBC. Survival probability plots from the TCGA dataset of 405 TNBC samples showed that patients expressing high levels of the GHR showed a significantly poor survival (27 months, p=0.038) compared to patients expressing low GHRs (40.37 months). Additionally, human breast cancer MDA-MB-453 cells, when treated with human (hGH)(2.5 nM) increased activation of JAK2, STAT5, SRC, ERK1/2, and AKT, associated with cancer progression. Importantly, treatment of the cells with Paclitaxel (1 nM) + (hGH)(2.5 nM) increased ABCG2 expression and the epithelial-to-mesenchymal transition (EMT) makers (SLUG, SNAIL, N-cad) associated with drug resistance and tumor progression. On the contrary, when cells were treated with Pegvisomant (500 nM) in combination with Paclitaxel + GH, a significant decrease in protein and RNA expression of ABCG2 as well as EMT markers was observed. Next, serum collected from GH antagonist (GHA) mice and WT as control, were used to treat PY8119 mouse cells in combination with Paclitaxel (1 nM) + bovine (bGH)(2.5 nM). Downregulation of protein and RNA expression levels of ABCG2 as well EMT makers in GHA serum treatment compared to WT was also found. This preliminary data shows that the GHR antagonist, Pegvisomant, in combination with anticancer drugs, can be effective in attacking drug resistance in TNBC. Key words: Pegvisomant, growth hormone receptor Presentation: Thursday, June 15, 2023

THU048 Determination Of Receptor Binding Affinity Of Growth Hormone Analogues To The Growth Hormone Receptor In A Drug Screening Context

Abstract Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023

THU061 Treatment Patterns In Acromegaly: Analysis Of Real-World US Insurance Claims From The MarketScan® Database

Abstract Disclosure: M. Fleseriu: Advisory Board Member; Self; Amryt, Ipsen, Recordati Rare Diseases, Pfizer, Inc. Grant Recipient; Self; Amryt, Crinetics, Ionis Pharmaceuticals Inc. A.L. Barkan: None. T.C. Brue: Advisory Board Member; Self; Advanz Pharma, Debio Pharm, Recordati Rare Diseases. Consulting Fee; Self; Ipsen, Recordati Rare Diseases, Novartis Pharmaceuticals, Pfizer, Inc. Grant Recipient; Self; Pfizer, Inc. Other; Self; Ipsen, Recordati Rare Diseases, Novartis Pharmaceuticals, Pfizer, Inc. E. Duquesne: Employee; Self; Ipsen. A. Houchard: Employee; Self; Ipsen. Stock Owner; Self; Ipsen. M. del Pilar Schneider: Employee; Self; Ipsen. A. Ribeiro-Oliveira Jr.: Employee; Self; Ipsen. Stock Owner; Self; Ipsen. S. Melmed: Advisory Board Member; Self; Ionis Pharmaceuticals Inc., Ipsen, Novo Nordisk, Recordati Rare Diseases. Grant Recipient; Self; Pfizer, Inc. Background: Medical treatment for acromegaly (characterized by excess growth hormone [GH] production) includes somatostatin receptor ligands (SRLs), dopamine agonists (DAs), and GH receptor antagonists (GHRAs). However, there are few real-world treatment evaluations in the US. We present treatment patterns for patients receiving medications for acromegaly (1/1/2010-7/31/2022). Methods: De-identified data were extracted from MarketScan®, a US health insurance claims database. Eligible patients: those receiving monotherapy or combination therapy (≥2 treatments overlapping for &amp;gt;3 months [m]) for ≥90 days without treatment gaps; ≥2 claims associated with acromegaly; data ≥3 m before and ≥6 m after diagnosis/first treatment claim (earlier date); and ≥18 years (y) old at diagnosis. Outcomes were: demographic characteristics; treatment frequency by line of treatment (LOT) and changes between LOTs (Sankey plot); treatment persistence for first LOT (LOT 1) monotherapies (Kaplan-Meier estimator); and treatment up-/downtitration (≥30% dose change, evaluated for octreotide long-acting release [OCT] and lanreotide depot [LAN]). Biochemical control values were unavailable in the database. Results: Of 882 patients, 50.1% were female; mean age at diagnosis was 48.6 y (standard deviation 13.6 y). In the cohort, 59.4% had exactly 1 LOT, 23.1% had exactly 2 LOTs, and 17.5% had exactly 3 LOTs. The most common LOT 1 medication class was first-generation SRLs: OCT (27.7%) and LAN (21.1%). Other LOT 1 classes were Das (cabergoline; 34.5%) and GHRAs (pegvisomant; 10.5%). Only 7 patients received second-generation SRL pasireotide and no patients received oral octreotide in LOT 1. Most patients initiated treatment with monotherapies (94.6%). In monotherapy LOT 1, GHRAs had the longest median persistence (pegvisomant; 24.8 m; 95% confidence interval [CI]: 16.59-32.49 m; n=93), followed by OCT and LAN (20.0 m; 95% CI: 16.98-23.87 m; n=430). Das had the shortest persistence (cabergoline; 14.4 m; 95% CI: 12.23-16.82 m; n=304). Among patients receiving OCT or LAN, 67.6% had ≥1 dose increase and 45.0% had ≥1 dose reduction. Conclusions: Despite guidelines recommending DA monotherapy for few select patients with acromegaly,1 our real-world analysis found that approximately one-third of patients initiated treatment with this class with the shortest treatment persistence. OCT and LAN monotherapies were also commonly used as LOT 1 and had longer persistence. GHRAs, the class with the longest persistence, were not common monotherapy in LOT 1, possibly because they do not target pituitary adenomas directly. Interestingly, only 5% of patients initiated treatment with combination therapies. Recommendations for individualized therapy2 should consider medication persistence and real-world treatment patterns. 1Giustina et al. Rev Endocr Metab Disord. 2020 2Fleseriu et al. Lancet Diabetes Endocrinol. 2022 Presentation: Thursday, June 15, 2023

THU046 Repeated Treatment With AZP-3813, A Bicyclic, 16-Amino Acid Peptide Antagonist Of The Human Growth Hormone Receptor, Induces Enhanced Suppression Of IGF1 In Beagle Dogs

Abstract Disclosure: G. Ravel: None. C. Chalmey: None. C. Berardet: None. D. Duracher: None. H. Kurasaki: None. T. Tomiyama: None. P. Reid: None. M.D. Culler: None. Medical treatment of acromegaly is based on either suppressing pituitary growth hormone (GH) secretion or inhibiting GH action by preventing interaction with its receptor in order to suppress the elevated levels of insulin-like growth factor 1 (IGF1). AZP-3813 is a 16-amino acid, bicyclic peptide antagonist of the GH receptor (GHR) derived from peptide sequences discovered using a unique, cell-free in vitro transcription-translation system screened against the human GHR, and optimized by rational design to increase binding affinity, solubility and half-life. The KD of AZP-3813 for the human GHR is 1.9nM. In previous studies, AZP-3813 was demonstrated to suppress IGF1 secretion in juvenile rats in a dose-related manner, and to maintain IGF1 suppression when given daily for extended periods. Recently, we demonstrated that AZP-3813 is also very effective in suppressing IGF1 in normal Beagle dogs. Dogs receiving a single 0.1, 1 and 10 mg/kg subcutaneous injection of AZP-3813 responded with a dose-related 2.2 + 7.1, 21.4 + 2.7 and 27.8 + 1.5% decrease in IGF1 after 24 hours, respectively, and the decrease in IGF1 attained with a single 10 mg/kg dose was maintained for over 72 hours. The relatively small further increase in IGF1 suppression with 10 versus 1 mg/kg suggests that 10 mg/kg AZP-3813 may induce near maximal GHR antagonism. To examine the effect of repeated and higher dose treatment of dogs with AZP-3813, young adult, non-fasted, Beagle dogs were injected subcutaneously at rotated dorsal sites with AZP-3813 at doses of 10, 30 or 60 mg/kg (n=2, one male and one female) daily for seven days. Blood samples, collected prior to dosing to establish baseline IGF1 levels and 24 hour after the final, seventh AZP-3813 injection, were assayed for total IGF1 content by radioimmunoassay. Baseline IGF1 levels were lower in the female dogs (136.7 + 36.1 ng/ml) than in the male dogs (197.0 + 11.3 ng/ml); however, the percent IGF1 suppression resulting from AZP-3813 treatment was similar for both sexes. AZP-3813, injected for seven days at 10, 30 and 60 mg/kg, induced highly significant 69.0 + 4.0, 70.5 + 9.5 and 75.0 + 1.0% decreases in IGF1, respectively. The similar magnitude of IGF1 suppression attained with the three doses of AZP-3813 implies that maximal GH antagonism is essentially achieved with the 10 mg/kg dose. The substantial increase in IGF1 suppression achieved by repeated 10 mg/kg injection of AZP-3813, compared to the previously observed suppression after a single 10 mg/kg injection, likely represents compound accumulation (under investigation) as well as a progressive effect of maximal GH antagonism on IGF1 production. These results demonstrate that the potent GHR antagonist activity of AZP-3813 translates to a highly effective suppression of IGF1 in normal dogs and further support the development of AZP-3813 as a potential therapy for acromegaly. Presentation: Thursday, June 15, 2023

OR05-04 Growth Hormone Receptor Antagonism Markedly Improves Therapeutic Response In Mouse Models Of Melanoma, Liver Cancer, Pancreatic Cancer And Cholangiocarcinoma

Abstract Disclosure: R. Basu: None. P. Kulkarni: None. S. Duran Ortiz: None. E. Davis: None. J.J. Kopchick: None. A major hurdle in tackling cancer, the second biggest cause of human mortality, is resistance to therapy. Human growth hormone (GH) mediated activation of the growth hormone receptor (GHR) promotes the initiation and invasive progression of several types of human cancers, known since 1950. Recently, through a series of mechanistic studies using in vitro models and in silico analyses of human cancer patient databases, we have identified a covert role of GH in promoting cancer drug resistance via direct upregulation of: (i) the expression of ATP-binding cassette containing multi-drug transporters (ABC transporters) leading to increased drug efflux, (ii) the epithelial-to-mesenchymal transition (EMT) associated gene expression program, and (iii) the mediators of fibrosis and extracellular matrix remodeling. Additionally, based on the known effects of GH and robust expression of GHR in multiple cell types that constitute the tumor microenvironment, autocrine / paracrine GH putatively exerts potent tumor supportive actions. Therefore, we hypothesized that combining GHR antagonist to chemotherapy can markedly improve therapeutic outcome in human cancers. Melanoma and liver cancer jointly and annually constitute &amp;gt;150,000 patients and &amp;gt;60,000 deaths in the US, much due to resistance to therapy. We used a syngeneic murine model of both cancers using C57BL6 mice transgenic for a GHR antagonist (GHA mice) compared against wild-type (WT) counterparts implanted with either B16F10 melanoma or Hepa-1-6 hepatoma cells. GHA expression drastically sensitized melanoma tumors to cisplatin treatment and hepatoma tumors to sorafenib treatment. Additionally, the five-year survival rates of pancreatic cancer and cholangiocarcinoma are a dismal 10% in 2022, indicating an urgent need for new therapeutic approaches for both of these devastating diseases. For this, we used Nude mice for human pancreatic cancer xenografts treated +/- GHA (Pegvisomant and another in development at our laboratory) combined with gemcitabine. Combination of GHA markedly sensitized pancreatic cancer xenografts to gemcitabine in Nude mice, leading to ∼40% of mice being tumor-free at the end of study. GHA similarly also sensitized human cholangiocarcinoma xenografts in Nude mice to gemcitabine-cisplatin therapy. In all cases, GHA led to a marked reduction in gene expression of multidrug efflux transporters, EMT markers, and fibrosis mediators. Collectively, our data provides strong pre-clinical validation for treatment regimens combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Presentation: Thursday, June 15, 2023

OR01-05 Growth Hormone Action In Somatostatin Neurons Regulates Anxiety And Fear Memory

Abstract Disclosure: W.O. dos Santos: None. V.A. Juliano: None. H.R. Vieira: None. R. Frazao: None. C.D. Munhoz: None. J. Donato: None. Dysfunctions in growth hormone (GH) secretion increase the prevalence of anxiety and depression. Furthermore, GH receptor (GHR) signaling in the amygdala has been associated with fear memory, a key feature of post-traumatic stress disorder. However, it is currently unknown which neuronal population is targeted by GH action to influence neuropsychiatric diseases. Here we showed that approximately 60% of somatostatin (SST)-expressing neurons in the extended amygdala are directly responsive to GH. GHR ablation in SST-expressing cells (SSTΔGHR mice) led to increased anxiety in the light-dark box and elevated plus maze tests, whereas SSTΔGHR females showed no changes in anxiety, compared to control animals. Using auditory Pavlovian fear conditioning, both male and female SSTΔGHR mice exhibited a significant reduction in fear memory. In contrast, GHR ablation in SST neurons did not affect memory in the novel object recognition test. The gene expression was analyzed in a bilateral micropunch comprising the central nucleus of the amygdala and basolateral complex of male mice. Increased mRNA expression of Gabbr2 (GABAB2 receptor), Syp (synaptophysin), and Bdnf were observed in the amygdala of SSTΔGHR mice. In conclusion, GHR expression in SST neurons is necessary to regulate anxiety in males, but not in female mice. GHR ablation in SST neurons also decreases fear memory and affects the gene expression in the amygdala. Our findings identified for the first time a specific neuronal population responsible for mediating the effects of GH on behavioral aspects associated with neuropsychiatric diseases. Presentation: Thursday, June 15, 2023

SAT605 Growth Hormone Action In Somatostatin Neurons Regulates Anxiety And Fear Memory

Abstract Disclosure: W.O. dos Santos: None. V.A. Juliano: None. H.R. Vieira: None. R. Frazao: None. C.D. Munhoz: None. J. Donato: None. Dysfunctions in growth hormone (GH) secretion increase the prevalence of anxiety and depression. Furthermore, GH receptor (GHR) signaling in the amygdala has been associated with fear memory, a key feature of post-traumatic stress disorder. However, it is currently unknown which neuronal population is targeted by GH action to influence neuropsychiatric diseases. Here we showed that approximately 60% of somatostatin (SST)-expressing neurons in the extended amygdala are directly responsive to GH. GHR ablation in SST-expressing cells (SSTΔGHR mice) led to increased anxiety in the light-dark box and elevated plus maze tests, whereas SSTΔGHR females showed no changes in anxiety, compared to control animals. Using auditory Pavlovian fear conditioning, both male and female SSTΔGHR mice exhibited a significant reduction in fear memory. In contrast, GHR ablation in SST neurons did not affect memory in the novel object recognition test. The gene expression was analyzed in a bilateral micropunch comprising the central nucleus of the amygdala and basolateral complex of male mice. Increased mRNA expression of Gabbr2 (GABAB2 receptor), Syp (synaptophysin), and Bdnf were observed in the amygdala of SSTΔGHR mice. In conclusion, GHR expression in SST neurons is necessary to regulate anxiety in males, but not in female mice. GHR ablation in SST neurons also decreases fear memory and affects the gene expression in the amygdala. Our findings identified for the first time a specific neuronal population responsible for mediating the effects of GH on behavioral aspects associated with neuropsychiatric diseases. Presentation: Saturday, June 17, 2023

THU050 The Role Of The GH Receptor Polymorphism As Prognostic Factor Of Vertebral Fractures In Acromegaly Patients Resistant To First Generation SSAs And Treated With GH Receptor Antagonist Or Second Generation Somatostatin Ligand

Abstract Disclosure: F. Costanza: None. S. Chiloiro: None. A. Giampietro: None. P. Mattogno: None. A. Infante: None. F. Angelini: None. L. Lauretti: None. A. Olivi: None. A. Pontecorvi: None. F. Doglietto: None. L. De Marinis: None. A. Bianchi: None. Acromegaly is associated with skeletal fragility and an increased prevalence of vertebral fractures (VF). In recent years several authors have tried to investigate the markers that can predict the risk of bone fragility in this endocrine disorder. Two different isoforms of the GH receptor (GHR) have been described so far, which differ in the presence or absence of a transcript of exon 3 of the GHR gene. Both isoforms produce a functional receptor, but the exon 3-deleted isoforms (d3-GHR) have greater sensitivity to endogenous and recombinant GH than the full-length isoform (fl-GHR). We conducted a longitudinal, retrospective, observational, single-center study of first-generation SSA-resistant acromegaly patients, with the aim of investigating the role of GHR polymorphism as a prognostic factor of incidental VF (I-VF) in first-generation SSA-resistant acromegalic patients and treated with GH receptor antagonist (Pegvisomant) or second-generation somatostatin ligand (Pasireotide Lar).72 patients with acromegaly were included. 28 patients carried d3-GHR isoform (38.9%) and 44 patients carried fl-GHR isoform (61.1%). At baseline, all patients were affected by active acromegaly; 46 patients were treated with Pegvisomant in combination with first generation SSA and 26 were treated with Pasireotide Lar. 18 patients (25%) carried P-VFs. At last follow-up, 58 patients achieved biochemical control of acromegaly (80.6%). 14 patients experienced the occurrence of I-VFs. From the group treated with Pegvisomant in combination with first generation SSA, 32 patients carried fl-GHR polymorphism and 14 carried d3-GHR polymorphism. At baseline, 10 patients (21.7%) were I-VF carriers. At follow-up, 8 patients developed P-VF (17.4%). I-VF occurred more frequently in patients carrying fl-GHR isoform compared to d3-GHR (p=0.04). From the group treated with Pasireotide Lar, 12 patients carried fl-GHR isoform and 14 patients carried d3-GHR isoform. At baseline, 8 patients (30.8%) were I-VF carriers. At follow-up, 6 patients developed P-VF (23.1%). I-VF occurred more frequently in patients carrying d3-GHR isoform than fl-GHR (p=0.01) and in patients with P-VF as compared to patients without P-VF (p=0.05).In conclusion, the GH receptor polymorphism could assume greater relevance as a prognostic factor of VF in acromegaly patients. In the future, the knowledge of the GHR polymorphism may improve the therapeutic approach of acromegaly patients, tailored to the individual patient, in the context of personalized medicine. Presentation: Thursday, June 15, 2023

THU045 WIP1 Phosphatase Is A Novel Specific Target For Epithelial GH Action

Abstract Disclosure: T. Apaydin: None. S. Zonis: None. C. Zhou: None. R. Barrett: None. V. Chesnokova: None. S. Melmed: None. Deficient DNA repair and accumulated DNA damage underlie age-associated pathological changes, tissue fragility, and neoplastic development. ATM kinase, an activator of DNA damage responses (DDR), phosphorylates DDR effectors including CHK2, p53, and γH2AX to ensure DNA repair. We have shown that GH suppresses epithelial DDR activity by suppressing ATM phosphorylation, resulting in DNA damage accumulation due to repressed DNA repair. Wip1 dephosphorylates ATM thereby attenuating DNA damage repair. We found that GH significantly induced Wip1 expression (1.6-fold) in both human normal colon cells (hNCC) and in human 3-dimensional intestinal organoids, resulting in dephosphorylated ATM, CHK2, γH2AX, and p53. Mice bearing GH-secreting tumors with high circulating GH also exhibited induced colon Wip1 expression with lower phospho-ATM, γH2AX, and p53 as well as increased DNA damage. Twofold higher Wip1 expression was observed in blood buffy coats derived from patients with GH-secreting adenoma vs patients with non-functioning pituitary adenoma. Specificity of these observations was validated by showing that in GH-treated cells, blocking GH receptor (GHR) with pegvisomant or blocking STAT5 phosphorylation with the inhibitor CAS 285986-31-4 constrained Wip1 induction and restored ATM phosphorylation, indicating that GH effects on Wip1 are GHR/STAT5-mediated concordant with the observed decreased colon Wip1 expression in GHR-/- mice devoid of GH signaling. Furthermore, Wip1 inhibition with the small molecule inhibitor (GSK2830371) reversed GH-induced DNA damage by restoring phosphorylation of ATM and other key DDR proteins. To determine a mechanism for Wip1 induction, we tested homeodomain-interacting protein kinase 2 (HIPK2) which is regulated by AMPK and potentiates Wip1 proteasomal degradation and its activity. We observed that GH increased phospho-AMPK expression, while blocking AMPK kinase activity with Compound C abrogated GH induction of Wip1. Src upregulates AMPK and triggers HIPK2 cytoplasmic relocation thereby inactivating HIPK2. We found that GH, by activating Src/AMPK phosphorylation, elicits HIPK2 cytoplasmic relocation, thus suppressing Wip1 ubiquitination and increasing its stability. Consistent with these findings, blocking Src phosphorylation with the tyrosine kinase inhibitor dasatinib abolished Wip1 upregulation and induced phosphorylation of its downstream targets. These results indicate that GH suppresses DDR by inducing Wip1 expression, and suppression of Wip1 may prevent epithelial accumulation of unrepaired DNA damage. These findings identify Wip1 as a specific novel target for GH action mediating GH-attenuation of DNA repair. Presentation: Thursday, June 15, 2023

SAT033 Disruption Of GHR In “Middle Aged” Mice Enhances Insulin Sensitivity And Extends Male Lifespan

Abstract Disclosure: S. Duran-Ortiz: None. E.O. List: None. J.A. Young: None. S.C. Mathes: None. R. Basu: None. Y.B. Slama: None. D.E. Berryman: None. Reduced growth hormone (GH) action has been shown to extend healthspan and lifespan in several organisms. In fact, mice and humans with a congenital disruption in the GH receptor (R) gene (GHRKO mice and patients with Laron Syndrome, respectively) have improved insulin sensitivity and are resistant to diabetes, cancer and age-associated cognitive decline. Importantly, the GHRKO mice hold the record for the longest-lived laboratory mice, with a lifespan one week shy of five years of age. GHRKO mice also show reduced markers of aging such as adipose tissue senescence and mTORC1 signaling in liver, kidney, heart and muscle. However, as GH action is required for normal growth and development, we and others have hypothesized that targeted inhibition of GH action at an adult age could be a potent method to extend healthy lifespan. To test this hypothesis and to elucidate how far in development would disruption of GHR extend healthy lifespan, our laboratory recently reported on two mouse lines with post-natal ablation of the GHR at a pubertal age (1.5 months - 1.5mGHRKO mice) and at mature-adult age (6 months - 6mGHRKO mice). We found that ablation of the GHR gene at both 1.5- and 6-months of age extends maximal and mean lifespan preferentially in females. Furthermore, 6mGHRKO mice showed improved oxidative stress resistance while males had improved insulin sensitivity and reduced cancer incidence. With these promising results, we believe that interventions to extend aging should be effective when administered later in life. In the current study, we disrupted the GHR in middle aged mice (12 months - 12mGHRKO mice) using the Cre-Lox system. We found that 12mGHRKO mice exhibit GH insensitivity (high serum GH and low IGF-1 levels), increased adipose tissue mass, reduced lean mass, minimal impact on body weight and length, improved insulin sensitivity and reduced glucose levels in males as well as decreased adipose tissue fibrosis in females compared to males. Moreover, despite the increased fat mass, motor coordination and strength as measured by rotarod and grip strength tests show no deficit in 12mGHRKO mice compared to the controls. Importantly, the male 12mGHRKO mice showed a preferential and significant lifespan extension compared to control mice. Collectively, our results indicate that not only does post-natal GHR disruption does not affect growth and development, but it also confers several crucial healthspan and lifespan advantages. Acknowledgements: This work is supported by the State of Ohio's Eminent Scholar Program, by NIH grant AG059779, by the American Federation for Aging Research (AFAR) and by the Diabetes Institute and by the AMVETS. Presentation: Saturday, June 17, 2023