GH Rise Research Articles

KDM1A genotyping and expression in 146 sporadic somatotroph pituitary adenomas.

A paradoxical increase of growth hormone (GH) following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants and somatic loss of heterozygosity of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. We aimed to analyze KDM1A gene sequence and KDM1A and GIPR expressions in somatotroph pituitary adenomas. We conducted a cohort study at university hospitals in France and in Italy. We collected pituitary adenoma specimens from acromegalic patients who had undergone pituitary surgery. We performed targeted exome sequencing (gene panel analysis) and array-comparative genomic hybridization on somatic DNA derived from adenomas and performed droplet digital PCR on adenoma samples to quantify KDM1A and GIPR expressions. One hundred and forty-six patients with sporadic acromegaly were studied; 72.6% presented unsuppressed classical GH response, whereas 27.4% displayed a paradoxical rise in GH after oral glucose load. We did not identify any pathogenic variant in the KDM1A gene in the adenomas of these patients. However, we identified a recurrent 1p deletion encompassing the KDM1A locus in 29 adenomas and observed a higher prevalence of paradoxical GH rise (P = .0166), lower KDM1A expression (4.47 ± 2.49 vs 8.56 ± 5.62, P < .0001), and higher GIPR expression (1.09 ± 0.92 vs 0.43 ± 0.51, P = .0012) in adenomas from patients with KDM1A haploinsufficiency compared with those with 2 KDM1A copies. Unlike in GIP-dependent primary bilateral macronodular adrenal hyperplasia, KDM1A genetic variations are not the cause of GIPR expression in somatotroph pituitary adenomas. Recurrent KDM1A haploinsufficiency, more frequently observed in GIPR-expressing adenomas, could be responsible for decreased KDM1A function resulting in transcriptional derepression on the GIPR locus.

THU064 Clinical, Radiological Characteristics And Response To Somatostatin Analogues Of Acromegaly Patients With Different Patterns Of Growth Hormone Response After Oral Glucose Load

Abstract Disclosure: M. Godlewska: None. A. Boguslawska: None. A. Grochowska: None. A. Hubalewska-Dydejczyk: None. A. Gilis-Januszewska: None. Introduction: Clinical, pathophysiological and prognostic significance of different patterns of growth hormone response in oral glucose tolerance test (OGTT) has been recently a focus of extensive debate. Material and Methods We analyzed 116 consecutive patients with newly diagnosed acromegaly between 2012 and 2022. Finally, 89 patients with an MRI-confirmed pituitary tumor, available results of at least 3 GH values in the OGTT before implementation of any treatment were included. Paradoxical rise of GH in OGTT (PR) was defined as an increase of at least 25% of GH at any time point of OGTT compared to baseline GH concentration. Patients were divided into two groups: with PR (PRpos), without PR (PR neg). Among clinical data, baseline GH, IGF-1 and PRL concentrations (expressed as IGF-1/ULN and PRL/ULN ratios), function of pituitary axes were assessed. Tumor largest diameter, tumor volume, cavernous sinus invasion, compression of the optic chiasm, tumor infarction as well as Signal Intensity Ratio (SIR) of the solid part of the tumor and the grey matter of the temporal lobe were assessed. Response to presurgical somatostatin analogues (SSA)(defined as normalization of IGF-1 and ULN ratio) was assessed in 69 patients. Postsurgical remission was defined as GH suppression &amp;lt;1 ug/ml in OGTT and IGF-1/ULN &amp;lt;1. This study was approved by the local Bioethics Committee (1072.6120.72.2020) and is part of statutory research of the Jagiellonian University Medical College (N41/DBS/000407). Results: PR was observed in 34.8% cases. Female/Male ratio was 32/26 for PRneg and 18/13 for PRpos. Median age upon diagnosis was 40,5 years (33.5-58.0) for PRneg and 51.0 (41.0-61.0) for PRpos (p=0.127). Median diagnostic delay was 4 years (IQR 5) for PRneg and 6.5 years (IQR 7) for PRpos (p=0.05). The frequency of hyperprolactinemia, secondary hypogonadism, secondary hypothyroidism did not differ among groups. Nor any differences in nadir GH concentration in the OGTT, glucose levels in 0, 60, 120 min, PRL and IGF-1 concentrations were found. Median fasting GH concentration was significantly higher in PRneg than in PRpos (10.23 ug/ml, IQR 14.19 vs 7.46 ug/ml, IQR 7.96, respectively; p=0.011). Tumor largest diameter and tumor volume, presence of cavernous sinus invasion, compression of the optic chiasm or radiological signs of tumor infarction and SIR did not differ between groups. The frequency of biochemical control after SSA was 31.6% in PRneg vs 42.9% in PRpos; (p=0.386). After surgery, biochemical cure was confirmed in 59.6% of PRneg and 70.8% of PRpos (p=0.352). Conclusions: In our study, patients in PRneg did not differ from PRpos, especially in terms of tumor intensity and response to preoperative medical treatment, as reported previously. PRpos patients had significantly lower baseline GH concentrations than PRneg. However, further evaluation is required to understand the significance of PR. Presentation: Thursday, June 15, 2023

OR17-05 Lysine Demethylase KDM1A And Ectopic Expression Of GIP-Receptor In Somatotropinomas Of Patients With Paradoxical Response To Oral Glucose

Abstract Disclosure: F. Chasseloup: None. L. Tosca: None. D. Regazzo: None. A. Proust: None. M. Hage: None. E. Kuhn: None. C. Jublanc: None. K. Mokhtari: None. S. Salenave: None. S. Gaillard: None. F. Parker: None. A. Boch: None. G. Tachdjian: None. P. Chanson: None. J. Bouligand: None. G. Occhi: None. P. Kamenicky: None. Introduction: Paradoxical increase of GH following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing’s syndrome. Most patients also displayed a deletion of chromosome 1p, including the KDM1A locus in their adrenal tissues, resulting in complete loss of KDM1A expression. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. The aim of our study was to search for genetic abnormalities of KDM1A in somatotroph pituitary adenomas. Methods: We collected somatotropinoma specimens from acromegalic patients followed in two tertiary endocrine centers in France and one in Italy. Somatic DNA was studied by targeted exome NGS and array-CGH. GIPR and KDM1A expression was quantified in the tumors using digital droplet PCR. Results: We included 186 patients: 108 patients (70.6 %) had a classic pathological GH response after oral glucose load, whereas 45 patients (29.4%) displayed a paradoxical rise of GH concentrations. Patients with a paradoxical response displayed higher IGF-1 levels (360 ± 111.8 % above ULN vs. 309.8 ± 107.3 %, p= 0.0130) and less invasive and smaller tumors (14.5 ± 5.58 mm vs. 18.5 ± 8.73 m, p= 0.0066). Amongst the 146 somatotropinoma specimens analyzed by targeted-NGS, no tumor harbored a KDM1A pathogenic variant, and 35 tumors harbored GNAS mutations (6 with and 29 without paradoxical GH responses). We identified a recurrent 1p deletion encompassing the KDM1A locus in 26 tumors, which were more frequently but not exclusively found in patients with paradoxical GH response compared to those with classic GH response. Somatic deletion of one KMD1A allele was associated with lower KDM1A expression (p=4.5e-5) and higher GIPR expression (p=0.0005). Discussion: Unlike in GIP-dependent PBMAH, we did not identify KDM1A genetic variants in a large cohort of acromegalic patients, independently of their GH response pattern to oral glucose loading. We identified recurrent 1p deletion in some tumors. Pituitary adenomas with a loss of one KDM1A copy due to chromosome 1p deletion harbored higher levels of GIPR transcripts than adenomas diploid for the KDM1A locus. If KDM1A haploinsufficiency leads to partial transcriptional derepression at the GIPR locus and a paradoxical rise of GH after glucose load warrants further investigations. Presentation: Saturday, June 17, 2023

Paradoxical GH increase after oral glucose load in subjects with and without acromegaly

ObjectiveA paradoxical GH rise after the glucose load (GH-Par) is described in about one-third of acromegalic patients. Here, we evaluated the GH profile in subjects with and without acromegaly aiming to refine the definition of GH-Par.DesignObservational case–control study.MethodsOur cohort consisted of 60 acromegalic patients, and two groups of subjects presenting suppressed GH (< 0.4 µg/L) and high (non-acro↑IGF−1, n = 116) or normal IGF-1 levels (non-acro, n = 55). The distribution of GH peaks ≥ 120% from baseline, insulin, and glucose levels were evaluated over a 180-min time interval after glucose intake.ResultsA similar proportion of subjects in all three groups shows a GH ratio of ≥ 120% starting from 120 min. Re-considering the definition of paradoxical increase of GH within 90 min, we observed that the prevalence of GH peaks ≥ 120% was higher in acromegaly than in non-acro↑IGF−1 and non-acro (respectively 42%, 16%, and 7%, both p < 0.001). In patients without GH-Par, a late GH rebound was observed in the second part of the curve. Higher glucose peak (p = 0.038), slower decline after load, 20% higher glucose exposure (p = 0.015), and a higher prevalence of diabetes (p = 0.003) characterized acromegalic patients with GH-Par (with respect to those without).ConclusionsGH-Par response may be defined as a 20% increase in the first 90 min after glucose challenge. GH-Par, common in acromegaly and associated with an increased prevalence of glucose metabolism abnormalities, is found also in a subset of non-acromegalic subjects with high IGF-1 levels, suggesting its possible involvement in the early phase of the disease.

RETRACTED ARTICLE: Polyphenols from Conyza dioscoridis (L.) ameliorate Alzheimer’s disease- like alterations through multi-targeting activities in two animal models

BackgroundRecent investigations suggested that anticancer agents may inhibit the progression of Alzheimer's disease (AD) pathology. Conyza dioscoridis (L.) was demonstrated to have anticancer, antioxidant, anti-inflammatory and antidiabetic effects. This study was carried out to investigate the efficacy of polyphenols from Conyza dioscoridis (L.) extract (PCDE) on AD.MethodsImpacts of 3 doses of PCDE and donepezil, a reference drug, on the features of Alzheimer's disease in two animal models were investigated.ResultsPCDE ameliorated the memory and learning impairment shown in rats following a single dose of scopolamine (scopolamine model) or 17 weeks of high-fat/high-fructose(HF/Hfr) diet coupled with a single dose of streptozotocin, (25 mg/kg) (T2D model). They reduced significantly the high hippocampal cholinesterase activity in the two models of rats. Administration of PCDE for 8 weeks in the T2D model showed a significant reduction in hippocampal GSK-3β, caspase-3 activity and increase in the inhibited glutamate receptor expression (AMPA GluR1 subunit and NMDA receptor subunits NR1, NR2A, NR2B). A significant reduction of HOMA-insulin resistance and serum hypercholesterolemia was observed. The Tau hyperphosphorylation and Aβ 1–42 generation in the hippocampal of T2D rats were significantly decreased by PCDE. Modulation of the oxidative stress markers, (rise in GH and SOD; decrease in MDA levels) and a significant reduction of TNF-α and IL-1β in the hippocampus of T2D rats treated by PCDE extract were important findings in this study. The highest dose tested was 4% of the highest safe dose.ConclusionOur study suggests that PCDE is multi-targeting agent with multiple beneficial activities in combating features of AD. This study may provide a novel therapeutic strategy for AD treatment that warrants clinical studies.

OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Abstract Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Surgical Remission of Acromegaly Resolves Neuroglycopenia and Paradoxical Rise in GH after OGTT

Background: Acromegaly is known to cause insulin resistance through increased gluconeogenesis and reduction in peripheral glucose use; however, hypoglycemia related to acromegaly has not been reported. Clinical Case: A 58-year old man presented for evaluation of several elevated serum IGF1 levels. The patient had reported years of increased body heat but no changes in his hands or feet and no voice deepening. He recently needed 15 dental crowns due to gaps in his teeth. He also had difficult to manage OSA and weight gain. The patient reported neuroglycopenia after a high glycemic meal or drink, although he was never able to objectively measure any low blood glucoses when they occurred; these symptoms improved but did not resolve despite adhering to a low carbohydrate diet. He also had decreased libido and erectile dysfunction. Exam was significant for coarse facial features. Prior testing revealed several elevated IGF1 serum levels, the last one being 227 ng/mL (54-194). One year prior, OGTT resulted in an initial GH level of 0.1 ng/mL with a decrease to <0.1 ng/mL after two hours. Repeat OGTT had an initial GH of 2.98 ng/mL which paradoxically rose to 12 ng/mL. Fasting BG was 90 mg/dL and peaked at 171 mg/dL. Pituitary MRI showed a 5 mm microadenoma, consistent with acromegaly from a GH secreting adenoma. He underwent a TSSC, and his heat intolerance, low libido, and symptom of hypoglycemia resolved completely. Subsequent IGF1 levels and MRI imaging normalized. Postoperatively OGTT showed a peak GH of 0.23 ng/mL with a peak glucose of 134 mg/dL. There was no paradoxical rise in GH. Discussion: Acromegaly is commonly associated with insulin resistance in ~30% of cases; however, there are no reports of associated neuroglycopenia after a carbohydrate-rich meal or OGTT, which in our patient resolved after successful removal of the pituitary microadenoma. His low glucose symptoms could have been a result of reactive hypoglycemia, which is often seen in patients with diabetes or even prediabetes. However this patient had no history of either. He did not have evidence of any tumors causing hypoglycemia and no gastric surgery to suggest a related etiology (e.g, dumping syndrome or nesidioblastosis). Conversely since GH is normally anabolic and stimulates insulin release, the patient’s elevated GH may have caused an abnormal increase in insulin, leading to his hypoglycemia symptoms. Indeed GIP, which stimulates insulin, is thought to be the cause of the paradoxical rise in GH seen in 30% of acromegaly cases. Remarkably, the patient’s hypoglycemia symptoms disappeared after treatment of the acromegaly, which leads us to consider that excess GH was the culprit.

Hepatic GH Receptor Signaling Directly Suppresses Hepatic Steatosis and De Novo Lipogenesis, Independent of Changes in Plasma IGF1 and Insulin

A reduction in GH, as well as IGF1, is associated with non-alcoholic fatty liver disease (NAFLD). However, the relative contribution of changes in circulating GH and IGF1, to hepatic triglyceride accumulation (steatosis), remains to be clearly defined. To study the direct actions of GH on hepatocyte metabolism, we have utilized a mouse model of adult-onset, hepatocyte-specific, GHR knockdown (aHepGHRkd; 10–12 week-old, GHRfl/fl male mice, treated with AAV8-TBGp-Cre). In this and previous reports, we have observed that aHepGHRkd male mice rapidly develop steatosis (after 7 days) associated with enhanced de novo lipogenesis (DNL; measured by deuterated H2O labeling, 10h after 0800h food removal), and low ketone levels, suggestive of reduced hepatic β-oxidation. Of note, aHepGHRkd also reduces plasma IGF1 levels to >80% of GHR-intact controls (GHRfl/fl mice treated with AAV8-TBGp-Null), leading to a rise in GH, due to loss of IGF1 negative feedback to the pituitary/hypothalamus. This reciprocal shift in IGF1/GH is associated with an increase in insulin levels. Therefore, it is possible that the steatosis that develops in aHepGHRkd mice is the consequence of systemic insulin resistance supplying excess substrates (glucose and NEFA) for hepatic lipogenesis. However, inconsistent with this theory is the fact that glucose and NEFA levels are not altered after aHepGHRkd. To tease out the indirect (perhaps driven by high insulin levels) vs. direct effects of GH on hepatocyte lipid accumulation, male aHepGHRkd mice were injected with a vector expressing rat IGF1 (AAV8-TBGp-rIGF1). Reconstitution of hepatocyte IGF1 in aHepGHRkd mice, raised plasma IGF1 and normalized GH, insulin and ketone levels, but hepatic steatosis and DNL remained greater than that of GHR-intact controls, indicating GH directly suppresses hepatic fat accumulation. RNAseq analysis of livers from aHepGHRkd mice showed expression of genes related to carbohydrate metabolism (Gck, Khk) and fatty acid synthesis (Fasn, Srebf1, Usf1), processing (Scd1) and uptake (Cd36) were increased, while genes related to gluconeogenesis (Pck1, Fbp1, G6pc) were reduced. Remarkably, IGF1 reconstitution had no major impact on the hepatic transcriptome of aHepGHRkd mice, with the exception of reducing the expression of Srebf1, consistent with the reduction in circulating insulin levels. Interestingly, carbohydrate-responsive element-binding protein (CHREBP) levels, but not mRNA levels, were greater in aHepGHRkd mice with or without IGF1 reconstitution, consistent with upregulation of CHREBP target genes (Khk and Fasn among others). Taken together, these results suggest GH directly regulates steatosis, at least in part, by suppressing carbohydrate-driven DNL, where additional studies are underway to test this hypothesis.

Role of Growth Hormone in Ghrelin’s Metabolic Actions

Objective: Ghrelin regulates eating, body weight, and blood glucose. Upon binding to its receptor (growth hormone secretagogue receptor; GHSR), administered ghrelin increases food intake, body weight, and blood glucose. In contrast, blocking ghrelin lowers body weight and food intake. Also, mice that lack ghrelin or GHSR develop life-threatening hypoglycemia when submitted to a prolonged caloric restriction protocol providing only 40% of usual daily calories. Although GHSR was first identified in the pituitary, ghrelin was first defined by its ability to stimulate GH secretion via GHSRs, GH replacement prevents hypoglycemia in ghrelin-KO mice undergoing prolonged caloric restriction, and GH is known to modulate body composition, relatively little attention has been devoted to the role of GH-secreting pituitary somatotrophs (“GH cells”) in ghrelin action. The objective here was to determine the requirement for GHSR-expressing GH cells in mediating ghrelin’s metabolic actions. Methods: Mice with GH cell-selective GHSR deletion were generated by crossing novel GH-IRES-Cre mice to novel floxed-GHSR mice. GH cell-selective GHSR knockout mice and three control littermate groups were studied. Plasma GH, food intake, and blood glucose were measured after ip or sc ghrelin administration. Blood glucose and plasma GH were measured over the course of a 15-d calorie restriction protocol providing only 40% of usual daily calories. Results: In mice with GH cell-selective GHSR deletion, ghrelin-induced GH secretion and food intake were attenuated (by 84.1% at 15 min and by 35.3% at 45 min, respectively) as compared to controls; ghrelin-induced blood glucose elevation was unchanged. Mice with GH cell-selective GHSR deletion exhibited an attenuated GH rise (by 76.8%) over the 15-d calorie restriction period, yet they nonetheless resisted life-threatening hypoglycemia which is observed in similarly-treated ghrelin-KO mice, GHSR-null mice, and mice with hepatocyte-selective GH receptor deletion. Conclusions: These results suggest that GH cell-expressed GHSRs are required for ghrelin’s acute orexigenic and GH secretory actions but are dispensable for ghrelin’s glucoregulatory actions, at least in the settings assessed here. Although GH cell-expressed GHSRs are required for the progressive GH elevations associated with prolonged calorie restriction, they are not required for ghrelin’s overall protective effects to block prolonged calorie restriction-associated hypoglycemia.

SUN-LB52 The Protective Effects of Hepatocyte GH Receptor (GHR) Signaling Against Steatosis and Liver Injury Is Sexually Dimorphic and Autonomous of IGF1

GH dysregulation contributes to the development of non-alcoholic fatty liver disease (NAFLD), however debate remains as to the relative contribution of the direct vs indirect effects of GH, via IGF1. Mouse models with congenital, liver-specific knockout of the GHR, JAK2 or STAT5, as adults exhibit steatosis, glucose intolerance, insulin resistance and white adipose tissue (WAT) lipolysis. It is believed that fatty liver is due to the dramatic reduction in circulating IGF1 altering systemic metabolism, due to loss of the insulin-like effects of IGF1 and the loss of IGF1 negative feedback to the pituitary leading to a rise in GH that promotes systemic insulin resistance and WAT lipolysis shifting the flux of fatty acids to the liver. In addition, low IGF1/high GH alters the development of other metabolically relevant tissues, which could indirectly contribute to the liver phenotype observed with congenital loss of hepatic GH signaling. To directly test the actions of GH on adult hepatocyte function, we developed a mouse model of adult-onset, hepatocyte-specific knockdown of the GHR (aHepGHRkd; 12 week-old, GHRfl/fl mice treated with AAV8-TBGp-Cre). aHepGHRkd enhanced hepatic de novo lipogenesis (DNL), rapidly leading to steatosis in males, but not females. In males, enhanced DNL and steatosis was sustained with age and associated with hepatocyte ballooning, inflammation and mild fibrosis. These changes occurred independent of severe systemic insulin resistance and WAT lipolysis, although the aHepGHRkd mice exhibit low IGF1/high GH similar to that of congenital models. To directly test the role of hepatocyte GHR signaling, independent of changes in IGF1, aHepGHRkd mice were treated with a vector expressing rat IGF1 targeted specifically to hepatocytes (AAV8-TBGp-rIGF1). Mice were fed standard chow diet and tissues collected 8m post-AAV. IGF1 replacement elevated plasma IGF1 in aHepGHRkd mice, resulting in a reduction in plasma GH and pituitary expression of Gh, Ghrhr and Ghsr, indicating negative feedback of IGF1 was restored. In male aHepGHRkd mice, IGF1 replacement reduced insulin and whole body lipid utilization and increased WAT, however it did not reduce steatosis or alter hepatic fatty acid composition indicative of DNL and had minimal effects on liver injury markers. RNAseq analysis of liver extracts showed IGF1 replacement also had no major impact on the differentially expressed genes observed after aHepGHRkd. These results demonstrate that steatosis, DNL and liver injury observed in male aHepGHRkd mice are autonomous of IGF1. Despite the fact that hepatic GHR protein levels were not detectable in both female and male aHepGHRkd mice, females maintained moderate levels of IGF1 and were protected from steatosis. The mechanism by which female mice are protected remains to be elucidated, however is consistent with clinical data indicating pre-menopausal women are resistance to NAFLD.

Metabolic insights from a GHSR-A203E mutant mouse model

ObjectiveBinding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. MethodsWe generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. ResultsIn COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. ConclusionsOur data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.

The GH Axis in Relation to Accepting an Early Macronutrient Deficit and Outcome of Critically Ill Patients.

Changes in the GH axis during critical illness resemble fasting in healthy adults and contribute to hypercatabolism, which potentially affects outcome. Accepting macronutrient deficits by withholding parenteral nutrition (PN) during the first week in the intensive care unit (ICU; late PN) reduced complications and accelerated recovery as compared with early use of PN (early PN). To investigate how late PN affects the GH axis in relation to its clinical outcome benefits. Preplanned subanalysis of the Early Parenteral Nutrition Completing Enteral Nutrition in Adult Critically Ill Patients randomized controlled trial. A total of 1128 patients for time-course study, 20 patients investigated for nocturnal GH pulsatility, and 600 patients investigated for muscle weakness, with early PN and late PN patients having comparable baseline characteristics. Withholding PN during the first ICU week (late PN) vs early PN. Changes in serum GH, IGF-I, IGF-binding protein (IGFBP) 3, and IGFBP1 concentrations from ICU admission to day 4 or last ICU day for patients with a shorter ICU stay (d4/LD) and association in multivariable analyses with likelihood of earlier live ICU discharge, risk of new infection, and muscle weakness. Late PN attenuated a rise in serum GH and IGF-I (P < 0.0001), did not affect IGFBP3, and attenuated a decrease in IGFBP1 concentrations from admission to d4/LD (P < 0.0001) as compared with early PN. Late PN decreased nonpulsatile (P = 0.005), but not pulsatile, GH secretion. Adjusting the multivariable models for the observed GH axis alterations increased the independent benefit of late PN for all outcomes. GH axis alterations induced by late PN were independently associated with adverse outcomes (P ≤ 0.03). Accepting macronutrient deficits early during critical illness further suppressed the GH axis, which statistically attenuated its clinical outcome benefits.

The Rise in Growth Hormone during Starvation Does Not Serve to Maintain Glucose Levels or Lean Mass but Is Required for Appropriate Adipose Tissue Response in Female Mice

In mice, GH levels rise in response to short-term fasting or starvation (food restriction to 40% of ad libitum intake), similar to that which occurs in humans in response to fasting or anorexia. Recent studies using acyl-ghrelin knockout mice have suggested that the rise in GH during food restriction is essential to support glucose levels. To directly test this hypothesis, adult-onset isolated GH deficient (AOiGHD) mice and their GH-replete littermate controls were provided 40% of ad libitum food intake for 11 d. As previously shown, food restriction increased GH levels in controls, and this response was not observed in AOiGHD mice. In both controls and AOiGHD, food restriction resulted in an initial decline in glucose, which stabilized to 82-85% of ad libitum-fed values by d 2. In addition, loss of lean mass in response to food restriction was not altered by GH status. However, the loss of fat mass and the associated rise in circulating free fatty acids and ketones was blunted in starved AOiGHD mice compared with controls. Taken together, these results suggest a rise of GH during starvation is not required to support glucose levels and muscle mass but may be important in supporting fat mobilization.

Naloxone decreases the inhibitory effect of somatostatin on GH release induced by cigarette smoking in man

To establish whether somatostatin (SRIH) exerts its inhibitory effect on the nicotine-induced release of GH by interacting with an opioid pathway, normal volunteers were treated with naloxone during (2 no-filter) cigarettes smoking and with SRIH. Nicotine significantly increased serum GH levels about 3.5 fold. Naloxone alone did not change GH rise induced by cigarette smoking. The stimulatory effect of GH by nicotine was completely blocked by SRIH. In the presence of both SRIH and naloxone, GH levels rose 1.5 fold in response to nicotine. Since naloxone only partially reversed the inhibiting action of SRIH, only a partial involvement of opioid peptides in SRIH action might be supposed. Alternatively, SRIH and naloxone-sensitive opiates might produce this inhibiting effect on GH rise in response to cigarette smoking through independent pathways.

Lipid mobilization in subcutaneous adipose tissue during exercise in lean and obese humans. Roles of insulin and natriuretic peptides

The aim of this study was to evaluate the relative contributions of various hormones involved in the regulation of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise and to assess the impact of obesity on this regulation. Eight lean and eight obese men performed a 60-min cycle exercise bout at 50% of their peak oxygen uptake on two occasions: during intravenous infusion of octreotide (a somatostatin analog) or physiological saline (control condition). Lipolysis in SCAT was evaluated using in situ microdialysis. One microdialysis probe was perfused with the adrenergic blockers phentolamine and propranolol while another probe was perfused with the phosphodiesterase and adenosine receptor inhibitor aminophylline. Compared with the control condition, infusion of octreotide reduced plasma insulin levels in lean (from approximately 3.5 to 0.5 microU/ml) and in obese (from approximately 9 to 2 microU/ml), blunted the exercise-induced rise in plasma GH and epinephrine levels in both groups, and enhanced the exercise-induced natriuretic peptide (NP) levels in lean but not in obese subjects. In both groups, octreotide infusion resulted in higher exercise-induced increases in dialysate glycerol concentrations in the phentolamine-containing probe while no difference in lipolytic response was found in the aminophylline-containing probe. The results suggest that insulin antilipolytic action plays a role in the regulation of lipolysis during exercise in lean as well as in obese subjects. The octreotide-induced enhancement of exercise lipolysis in lean subjects was associated with an increased exercise-induced plasma NP response. Adenosine may contribute to the inhibition of basal lipolysis in both subject groups.

Body fat excess and stimulated growth hormone levels in adult patients with Prader–Willi syndrome

It has been suggested that reduced GH secretion in Prader-Willi syndrome (PWS) may simply reflect their excessive fat body mass. However, the GH response to standard provocative tests is significantly lower in PWS adults than obese controls with similar body mass index (BMI). Nevertheless, BMI is an inadequate measure of body composition, because PWS harbor a higher percentage of fat body mass (FM%) than simple obesity, under the same degree of weight excess. This study evaluated either the GH response to combined GHRH + arginine administration and the FM%, by DEXA, in 11 PWS adults (8 females, aged 20.1-41.1 years, BMI 43.6 +/- 1.8, FM% 53.0 +/- 1.5), in comparison to those obtained in 10 patients with essential obesity (8 females, aged 23.5-45.8 years), matched for BMI and FM% (42.9 +/- 0.4 and 51.5 +/- 1.0, respectively). Moreover, IGF-I levels were measured in both groups. The GH response to GHRH + arginine was significantly lower in PWS patients (GH peak 5.4 +/- 1.3 microg/L; area under the curve (AUC) 311.2 +/- 72.5 microg/L/hr) than obese controls (GH peak 21.0 +/- 4.1 microg/L, P < 0.005; AUC 1241.1 +/- 272.8 microg/L/hr; P < 0.01), as well as IGF-I values (110.5 +/- 16.7 microg/L vs. 242.1 +/- 31 microg/L, P < 0.005). The GHRH + arginine induced GH rise in patients with del15q11-q13 was significantly higher than subjects with UPD15 (GH peak 7.7 +/- 1.7 microg/L vs. 2.7 +/- 1.0 microg/L, P < 0.05; AUC 458.5 +/- 91.0 microg/L/hr vs. 134.4 +/- 46.0 microg/L/hr, P < 0.02). These findings suggested that stimulated GH levels are significantly lower in PWS adults when compared to obese controls and that GH response to GHRH + arginine is different in PWS individuals having separate genetic subtypes.

Effects of an Oral Growth Hormone Secretagogue in Older Adults

GH secretion declines with age, possibly contributing to reduced muscle mass, strength, and function. GH secretagogues (GHS) may increase muscle mass and physical performance. We conducted a randomized, double-masked, placebo-controlled, multicenter study to investigate the hormonal, body composition, and physical performance effects and the safety of the orally active GHS capromorelin in older adults with mild functional limitation. INTERVENTION/PARTICIPANTS: A total of 395 men and women aged 65-84 yr were randomized for an intended 2 yr of treatment to four dosing groups (10 mg three times/week, 3 mg twice a day, 10 mg each night, and 10 mg twice a day) or placebo. Although the study was terminated early according to predetermined treatment effect criteria, 315 subjects completed 6 months of treatment, and 284 completed 12 months. A sustained dose-related rise in IGF-I concentrations occurred in all active treatment groups. Each capromorelin dose prompted a rise in peak nocturnal GH, which was greatest with the least frequent dosing. At 6 months, body weight increased 1.4 kg in subjects receiving capromorelin and decreased 0.2 kg in those receiving placebo (P = 0.006). Lean body mass increased 1.4 vs. 0.3 kg (P = 0.001), and tandem walk improved by 0.9 sec (P = 0.02) in the pooled treatment vs. placebo groups. By 12 months, stair climb also improved (P = 0.04). Adverse events included fatigue, insomnia, and small increases in fasting glucose, glycosylated hemoglobin, and indices of insulin resistance. In healthy older adults at risk for functional decline, administration of the oral GHS capromorelin may improve body composition and physical function.

Acute and chronic neuroendocrine effects of interferon‐β 1a in multiple sclerosis

Treatment of multiple sclerosis with interferon-beta (IFN-beta) results in variable responses interindividually. Cytokine-hormone interactions may modulate the therapeutic effects of IFN-beta. Since hyperactivity of the hypothalamo-pituitary-adrenal (HPA) axis and other neuroendorine disturbances occur in multiple sclerosis, we determined the detailed neuroendocrine response of patients with multiple sclerosis to IFN-beta. Longitudinal open-label study. Eight patients with relapsing-remitting multiple sclerosis (four women, age 31.9 +/- 1.5 years, EDSS 1.5-2.5). Plasma ACTH, cortisol, prolactin, GH, TSH, LH and FSH were determined in 30-min intervals during 8 h on four occasions: after intramuscular injection of saline; after the first dose of IFN-beta 1a; after the second IFN-beta dose with oral indomethacin pretreatment; and after 3 months of IFN-beta therapy. Dexamethasone-corticotropin-releasing hormone test was performed before and at 3 months on IFN-beta. Compared to saline, IFN injection resulted in marked rise in plasma ACTH (mean, 370% of baseline), cortisol (214%), prolactin (253%) and GH (756%), between 2 and 6 h after injection. With indomethacin, hormone secretion occurred with reduced peak values. Endocrine response adapted partially after 3 months of treatment. HPA axis activity decreased in most patients, but increased in one patient with frequent relapses. Marked neuroendocrine effects occur in response to IFN-beta in multiple sclerosis. Upon prolonged treatment, these effects partially adapt, and HPA axis hyperactivity is reduced. Prospective studies to determine the relation to individual treatment response can be based on these findings.

Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH and ACTH in rats

To examine the effect of obestatin, a recently identified peptide derived from preproghrelin, on pituitary hormone secretion, obestatin was administered in anesthetized male rats. Intravenous administration of obestatin did not show any effect on plasma GH, PRL, ACTH and TSH levels. Since obestatin has been reported to have opposite effects of ghrelin in regulating food intake, gastric emptying and intestinal contractility, GH suppressive effect, which is opposite effect of ghrelin, was tested. Intravenous administration of GHRH or GHRP-2, a ghrelin receptor ligand, resulted in a marked plasma GH elevation. However obestatin did not show any effect on GHRH- or GHRP-2-induced GH rise. Furthermore intracerebroventricular administration of obestatin also did not influence plasma GH, PRL, ACTH and TSH levels. These findings suggest that obestatin has no effect on pituitary hormone secretions despite the presence of GPR39, a receptor for obestatin, in the pituitary.

The Role of Endocrine Counterregulation for Estimating Insulin Sensitivity from Intravenous Glucose Tolerance Tests

During insulin-modified frequently sampled iv glucose tolerance tests (IM-FSIGT), which allow assessment of insulin action, plasma glucose can markedly decrease. This study aimed to assess the counterregulatory impact of the insulin-induced fall of glucose on minimal model-derived indices of insulin sensitivity (S(I)) and glucose effectiveness. Thirteen nondiabetic volunteers (seven males, six females, aged 26 +/- 1 yr, body mass index 22.1 +/- 0.7 kg/m(2)) were studied. All participants were studied in random order during IM-FSIGT (0.3 g/kg glucose; 0.03 U/kg insulin at 20 min) and during identical conditions but with a variable glucose infusion preventing a decrease of plasma glucose concentration below euglycemia (IM-FSIGT-CLAMP). Five participants additionally underwent euglycemic-hyperinsulinemic (1 mU.kg(-1).min(-1)) clamp tests. Plasma glucose declined during IM-FSIGT to its nadir of 50 +/- 3 mg/dl at 60 min in parallel to a rise (P < 0.05 vs. basal) of plasma glucagon, cortisol, epinephrine, and GH. Glucose infusion rates of 4.6 +/- 0.5 mg.kg(-1).min(-1) between 30 and 180 min during IM-FSIGT-CLAMP prevented the decline of plasma glucose and the hypoglycemia counterregulatory hormone response. S(I) was approximately 68% lower during IM-FSIGT (3.40 +/- 0.36 vs. IM-FSIGT-CLAMP: 10.71 +/- 1.06 10(-4).min(-1) per microU/ml, P < 0.0001), whereas glucose effectiveness did not differ between both protocols (0.024 +/- 0.002 vs. 0.021 +/- 0.003 min(-1), P = NS). Compared with the euglycemic hyperinsulinemic clamp test, S(I) expressed in identical units from IM-FSIGT was approximately 66% (P < 0.001) lower but did not differ between the euglycemic hyperinsulinemic clamp test and the IM-FSIGT-CLAMP (P = NS). The transient fall of plasma glucose during IM-FSIGT results in lower estimates of S(I), which can be explained by hormonal response to hypoglycemia.