High-throughput mediation analysis of human proteome and metabolome identifies mediators of post-bariatric surgical diabetes control

To improve the power of mediation in high-throughput studies, here we introduce High-throughput mediation analysis (Hitman), which accounts for direction of mediation and applies empirical Bayesian linear modeling. We apply Hitman in a retrospective, exploratory analysis of the SLIMM-T2D clinical trial in which participants with type 2 diabetes were randomized to Roux-en-Y gastric bypass (RYGB) or nonsurgical diabetes/weight management, and fasting plasma proteome and metabolome were assayed up to 3 years. RYGB caused greater improvement in HbA1c, which was mediated by growth hormone receptor (GHR). GHR’s mediation is more significant than clinical mediators, including BMI. GHR decreases at 3 months postoperatively alongside increased insulin-like growth factor binding proteins IGFBP1/BP2; plasma GH increased at 1 year. Experimental validation indicates (1) hepatic GHR expression decreases in post-bariatric rats; (2) GHR knockdown in primary hepatocytes decreases gluconeogenic gene expression and glucose production. Thus, RYGB may induce resistance to diabetogenic effects of GH signaling.Trial Registration: Clinicaltrials.gov NCT01073020.

Diet and Exercise Modulate GH-IGFs Axis, Proteolytic Markers and Myogenic Regulatory Factors in Juveniles of Gilthead Sea Bream (Sparus aurata)

The physiological and endocrine benefits of sustained exercise in fish were largely demonstrated, and this work examines how the swimming activity can modify the effects of two diets (high-protein, HP: 54% proteins, 15% lipids; high-energy, HE: 50% proteins, 20% lipids) on different growth performance markers in gilthead sea bream juveniles. After 6 weeks of experimentation, fish under voluntary swimming and fed with HP showed significantly higher circulating growth hormone (GH) levels and plasma GH/insulin-like growth-1 (IGF-1) ratio than fish fed with HE, but under exercise, differences disappeared. The transcriptional profile of the GH-IGFs axis molecules and myogenic regulatory factors in liver and muscle was barely affected by diet and swimming conditions. Under voluntary swimming, fish fed with HE showed significantly increased mRNA levels of capn1, capn2, capn3, capns1a, n3, and ub, decreased gene and protein expression of Ctsl and Mafbx and lower muscle texture than fish fed with HP. When fish were exposed to sustained exercise, diet-induced differences in proteases’ expression and muscle texture almost disappeared. Overall, these results suggest that exercise might be a useful tool to minimize nutrient imbalances and that proteolytic genes could be good markers of the culture conditions and dietary treatments in fish.

Ghrelin-Induced Food Intake, But not GH Secretion, Requires the Expression of the GH Receptor in the Brain of Male Mice

Ghrelin stimulates both growth hormone (GH) secretion and food intake. The orexigenic action of ghrelin is mainly mediated by neurons that co-express agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus (ARH). GH also stimulates food intake and, importantly, ARH AgRP/NPY neurons express GH receptor (GHR). Thus, ghrelin-induced GH secretion may contribute to the orexigenic effect of ghrelin. Here, we investigated the response to ghrelin in male mice carrying GHR ablation specifically in neurons (Brain GHR KO mice) or exclusively in ARH AgRP/NPY neurons (AgRP GHR KO mice). Although Brain GHR KO mice showed normal ghrelin-induced increase in plasma GH levels, these mutants lacked the expected orexigenic response to ghrelin. Additionally, Brain GHR KO mice displayed reduced hypothalamic levels of Npy and Ghsr mRNA and did not elicit ghrelin-induced c-Fos expression in the ARH. Furthermore, Brain GHR KO mice exhibited a prominent reduction in AgRP fiber density in the ARH and paraventricular nucleus of the hypothalamus (PVH). In contrast, AgRP GHR KO mice showed no changes in the hypothalamic Npy and Ghsr mRNAs and conserved ghrelin-induced food intake and c-Fos expression in the ARH. AgRP GHR KO mice displayed a reduced AgRP fiber density (~16%) in the PVH, but this reduction was less than that observed in Brain GHR KO mice (~61%). Our findings indicate that GHR signaling in the brain is required for the orexigenic effect of ghrelin, independently of GH action on ARH AgRP/NPY neurons.

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.

Somatostatin, as a Bridge Between the GH-Axis and the Gth-Axis

Somatostatin (SST) is a 14-amino acid peptide produced in the hypothalamus of vertebrates, including fish. It regulates many physiological processes such as growth development and metabolic processes in the animal’s body. Negative control of growth hormone in vivo and in vitro was characterized in several fish species such as salmon, goldfish, rainbow trout and tilapia. Although very important, the SST/SST-R system in Nile tilapia (Oreochromis niloticus) was not deeply characterized. The somatostatin system in tilapia possess two ligands (Somatostatin1b and Somatostatin 2), and five receptors (SST-R 1-5). Unlike mammals, in fish, FSH and LH are secreted from different cell populations in the pituitary. By performing cell specific transcriptome analysis of double-labelled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, we identified genes specifically enriched in each cell type. Analysis of the RNA-seq discovered 4 types of SST-Rs: sstr2, sstr3, sstr5 and sstr5x3. The specific localization of each SST-R was identified by In Situ hybridization with specific probes for each of the SST-Rs. SST-R2 and SST-R5x3 were expressed on LH and FSH cells, while SST-R5 was exclusively expressed on LH cells. Interestingly, SST-R3, which was expressed on GH secreting cells, was also expressed on both gonadotropin-secreting cells. Transactivation assays, using COS7 cell line transfected with tilapia SST-Rs together with the reporter plasmid CRE-luc, demonstrated an effect through the cAMP/PKA pathway. Signal transduction analysis demonstrated that SST agonist (Octreotide; IC50 = 0.8-60nM) decreased the cAMP/PKA pathway, while an opposite effect was found when SST antagonist (Cyclosomatostatin; EC50 = 0.1 - 188 nM) was used. To understand the physiological effects of somatostatin on gonadotropins and GH release, we examined the effect of ip injection (100 μg/kg BW) of somatostatin agonist and antagonist on plasma FSH, LH and GH levels. SST agonist decreased plasma GH and FSH levels, as fast as two hours post injection and their levels remained low until the end of the experiment. On the other hand, SST antagonist increased LH and FSH levels two hours post injection, but while FSH levels remained high during the entire experiment, LH levels went back to basal levels afterwards. Our results show - for the first time in fish - a direct effect of SST on gonadotropin release, that could serve as a bridge between the GH-axis and the GTH-axis. The research was funded by the Israel Science Foundation (ISF) no. 1540/17.

Circulating levels of growth hormone in postural orthostatic tachycardia syndrome

Postural orthostatic tachycardia syndrome (POTS) is a cardiovascular autonomic disorder with poorly understood etiology and underlying pathophysiology. Since cardiovascular morbidity has been linked to growth hormone (GH), we studied GH levels in patients with POTS. We conducted an age-sex-matched case–control study in patients with POTS (age 31 ± 9 years; n = 42) and healthy controls (32 ± 9 years; n = 46). Plasma GH levels were measured using high-sensitivity chemiluminescence sandwich immunoassay. The burden of orthostatic intolerance symptoms was assessed by the Orthostatic Hypotension Questionnaire (OHQ), consisting of a symptom assessment scale (OHSA) and a daily activity scale (OHDAS). POTS patients had significantly higher composite OHQ score than controls, more symptoms and less activity. Supine heart rate and diastolic blood pressure (BP), but not systolic BP, were significantly higher in POTS. Median plasma GH levels were significantly lower in POTS (0.53 ng/mL) than controls (2.33 ng/mL, p = 0.04). GH levels were inversely related to OHDAS in POTS and supine systolic BP in POTS and controls, but not heart rate neither group. POTS is associated with lower GH levels. Impairment of daily life activities is inversely related with GH in POTS. A higher supine diastolic BP is inversely associated with GH levels in POTS and healthy individuals.

The Therapeutic Effect of Endoscopic Tumor Resection on Acromegalic Cardiomegaly in Patients With Pituitary Adenoma

Background: Acromegaly defines as chronic elevations of insulin-like growth factor-1 (IGF-1) and growth hormone (GH), which results in enlargement of organs and soft tissues. Cardiovascular complications of acromegaly such as cardiomegaly, hypertension contributing to a high risk of cardiovascular events. This study aimed to identify the determinants of the prevalence of cardiomegaly as cardiovascular comorbidity of acromegaly and the potential effect of the curative intervention. Methods: A total of 160 patients with acromegaly due to pituitary adenoma participated in this study. Acromegaly diagnosed was based on clinical manifestations, age-adjusted plasma IGF-I, and elevated plasma GH levels, not suppressible during an oral glucose tolerance test (75 g). Electrocardiogram and chest X-ray obtained in all patients. Treatment approaches included transsphenoidal surgery and hormonal evaluation performed before and six months after surgery. Results: The GH serum level was elevated in all patients before surgery, with a mean of 33.7 ng/ mL that reached 5.7 ng/mL after surgery. Mean IGF-1 was 794.1 ng/mL that reached 395.6 ng/mL postoperatively. The prevalence of cardiomegaly was 15% that improved in 5% of patients after trans sphenoidal surgery. Conclusion: Appropriate surgical intervention in acromegaly that complicated by cardiomegaly may result in significant improvement of the cardiac structure.

295 Awardee Talk: Novel regulation of growth hormone by kisspeptin

Kisspeptin (KP) is critical regulator of reproductive function through it’s potent stimulation of gonadotropin releasing hormone (GnRH). In addition there has been inconclusive evidence to suggest intravenous KP can stimulate growth hormone (GH). Studies in 24 h fasted (but not fed) sheep determined that ICV injection of Kp-10 can increase plasma GH concentrations. This led to questions about the mechanism linking KP and GH. Since fasting is a critical requirement for the Kp effect on GH, studies were focused on factors that are linked to fasting within the hypothalamus and are known regulators of GH. Fasting causes a major upregulation of neuropeptide Y (NPY), a potent appetite regulator and in ruminants, a stimulator of GH. Pretreatment of BIBO 3304, an NPY receptor antagonist, blocked the effect of KP to increase GH and implicated NPY as a mediator of the KP effect. Indeed, KP injected ICV upregulated cFOS in NPY and GH Releasing Hormone (GHRH) cells in the arcuate nucleus and reduced cFOS in Somatostatin (SS) cells in the periventricular nucleus. Another factor, altered by fasting and capable of regulating GH, is Ghrelin. Both blockage of Ghrelin release and central blockade of Ghrelin receptors reduced or blocked release of GH after KP treatment. These experiments suggest an hypothesis that fasting upregulates central Ghrelin and NPY expression permitting the activation of NPY by KP. NPY in turn activates GHRH and inhibits SS to release GH.

Parental Obesity Differentially Associates with Newborn Adiposity. Is Programming of the GH-IGF Axis Involved? (OR09-05-19)

Objectives Maternal obesity is strongly associated with offspring obesity risk. However, studies show that paternal obesity also affects offspring phenotype, especially in males. Programming of the growth hormone (GH) insulin-like growth factor (IGF) axis seems to be an important mechanism in this finding, but evidence regarding this relationship in humans is scarce. The primary purpose of this study was to evaluate associations between maternal and paternal adiposity with that of their offspring. A secondary aim was to investigate associations between paternal adiposity with GH levels in male offspring. Methods Parent-offspring trios (n = 209) from the Glowing study were studied. Percent body fat mass (%FM) was measured using air displacement plethysmography early in pregnancy in parents, and at age 2 weeks in the offspring. Plasma GH levels were measured at birth from umbilical cord blood (n = 31) and at age 2 years (n = 39) in male offspring. Multiple linear regression was used to model infant's %FM (dependent variable) at 2 weeks of age using parental %FM, race, age, IQ, income, delivery method, gestational weight gain, feeding mode, and gestational age (GA) as independent variables. The best fitted models were constructed for girls and boys. Spearman correlations (ρ) between paternal %FM and offspring GH levels were adjusted for maternal %FM. Results Girls (n = 94): Paternal %FM did not associate with female newborn %FM. Instead, maternal %FM (β = 0.12, P = 0.010) and C-section delivery mode (β = 2.56, P = 0.011) had the strongest associations with female newborn %FM. Boys (n = 115): Maternal %FM (β = 0.12, P = 0.006) and GA (β = 1.06, P = 0.003) were positively associated with male newborn %FM, whereas paternal %FM was negatively associated (β = −0.09, P = 0.014) with male offspring adiposity. There was no significant correlation between paternal %FM and GH measured at birth (ρ = 0.19, P = NS) in boys. However, at age 2 years, GH levels in boys decreased with increasing paternal %FM (ρ = −0.40, P = 0.015). Maternal %FM did not correlate with offspring GH level at any age. Conclusions Parental obesity differentially associates with newborn girls’ and boys’ adiposity at age 2 weeks. In line with prior findings in animal models, this preliminary analysis points to programming of the GH/IGF axis in male offspring born to obese fathers. Funding Sources USDA ARS # 6026-51000-010-05S, NIH 1UL1RR029884.