Plasma GH Levels Research Articles

THU047 Growth Hormone (GH) Signaling In Hepatocytes Controls Hepatic Insulin Sensitivity And Nutrient Disposition

Abstract Disclosure: M. del Rio-Moreno: None. M. Vazquez-Borrego: None. J. Cordoba-Chacon: None. O.P. McGuinness: None. R.D. Kineman: None. It is commonly accepted that GH reduces the actions of insulin. However, we reported plasma GH levels within a physiological range are negatively associated with insulin-mediated glucose uptake, but positively associated with insulin-mediated suppression of hepatic glucose production (HGP) [Cordoba-Chacon J. et al. 2014. AJP Endocrinol Metab]. These findings suggest that GH may promote insulin’s actions on the liver. To determine the direct contribution of GH signaling on hepatic insulin sensitivity, hyperinsulinemic-euglycemic clamps were performed with stable isotope tracers in chow-fed adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd) mice. aHepGHRkd mice show reduced IGF1 and increased GH levels [Cordoba-Chacon J. et al 2015, Diabetes] which indirectly impacts systemic insulin sensitivity and substrate flux to the liver. To normalize plasma IGF1 and GH levels, aHepGHRkd mice were co-treated with adeno-associated virus (AAV) that express IGF1 or a constitutively active form of STAT5b (STAT5bCA). These AAVs have a thyroxin binding globulin promoter, leading to hepatocyte-specific expression. Under both basal and clamped conditions, aHepGHRkd mice exhibited hyperinsulinemia, that was normalized by AAV-mediated IGF1 and STAT5bCA expression. Interestingly, glucose disappearance (Rd), tissue-specific glucose uptake, or HGP (gluconeogenesis and glycogenolysis) did not differ across all groups. However, higher insulin tone in aHepGHRkd mice was required to achieve the same level of Rd and suppression of HGP in the steady-state, which was indicative of systemic and hepatic insulin resistance. These observations are consistent with our previous findings that GH reduces tissue-specific glucose uptake but promotes insulin-mediated control of HGP. In contrast, aHepGHRkd mice exhibited an increased rate of hepatic de novo lipogenesis, and an increase in plasma levels of newly formed triglyceride and triglyceride-bound palmitate, that was normalized by IGF1 and STAT5bCA expression. Taken together, these results indicate loss of hepatocyte GH signaling leads to pathway selective hepatic insulin resistance. This pathway selective hepatic insulin resistance has also been reported in mouse models due to altered substrate delivery to the liver as consequence of excess dietary intake and/or increased adipose tissue lipolysis. However, chow-fed aHepGHRkd mice show hyperinsulinemia that was sufficient to normalize systemic glucose uptake and suppress WAT lipolysis, as noted by normal plasma NEFA levels under both basal and clamped conditions. Therefore, changes in substrate delivery to the liver cannot readily explain the changes observed, suggesting hepatocyte GHR-signaling plays a direct role in partitioning of hepatic nutrients in response to insulin. Presentation: Thursday, June 15, 2023

Cysteamine improves growth and the GH/IGF axis in gilthead sea bream (Sparus aurata): in vivo and in vitro approaches.

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed.

Evaluation of novel ventricular repolarization parameters in patients with acromegaly

Objectives: T wave’s peak and end interval (Tp-e), Tp-e/QT ratio and Tp-e/QTc ratio are novel markers of ventricular repolarization and are associated with ventricular arrhythmias. Increased ventricular arrhythmia incidence is reported in patients with acromegaly. The purpose of this study is to evaluate ventricular repolarization using the Tp-e interval, Tp-e/QT ratio, and Tp-e/QTc ratio in patients with acromegaly. Methods: Thirty-five patients with acromegaly were included in the study. The control group was consisted of forty-one subjects without acromegaly that having similar age, sex ratio and comorbidities. The Tp-e interval, Tp-e/QT ratio, Tp-e/QTc ratio, and other ventricular repolarization parameters of all patients were evaluated using electrocardiography. Results: Tp-e interval, Tp-e/QT ratio and Tp-e/QTc ratio were significantly prolonged in patients with acromegaly compared to the control group. Furthermore, Tp-e interval, Tp-e/QT ratio, and Tp-e/QTc ratio showed a significant correlation with plasma GH levels and LVMI values. Conclusions: Our study revealed that Tp-e interval, Tp-e/QT ratio, and Tp-e/QTc ratio are prolonged in patients with acromegaly. We believe that the Tp-e interval, Tp-e/QT ratio, and Tpe/QTc ratio can be used in the evaluation of increased cardiovascular risk in patients with acromegaly.

The Influence of blood, seminal plasma testosterone, growth hormone and cortisol levels on the sperm quality in merino rams

The research was conducted on 10 Merino rams to determine the effect of testosterone, growth hormone and cortisol levels on the sperm quality. A significant relationship was detected between blood and seminal plasma levels of testosterone (P ˂ 0.01). No GH on a measurable level was detected in the seminal plasma, and it was determined that plasma GH levels, just as testosterone levels began to increase with the approach of the reproductive season, and peaked at the start of the season. Plasma and seminal plasma cortisol levels, on the other hand, while increasing before the season, decreased significantly at the start of the season, and a remarkable correlation between plasma and seminal plasma levels was ascertained (P ˂ 0.01). An important negative relation which was observed between plasma GH levels and seminal plasma cortisol levels in Merino rams. It was found out that the hormones that were examined did not have a significant influence on sperm qualities, but there was an important negative relation only between the plasma GH levels and sperm volumes in the Merino (P ˂ 0.01). In conclusion, it may be stated that in rams, especially plasma testosterone and cortisol levels determine the plasma levels; both plasma and seminal plasma testosterone levels increase to a significant degree in the reproductive season.

Feed Supplementation with the GHRP-6 Peptide, a Ghrelin Analog, Improves Feed Intake, Growth Performance and Aerobic Metabolism in the Gilthead Sea Bream Sparus aurata

The aquaculture sector has experienced rapid and important growth with the subsequent increase of feeding and nutritional issues for sustaining this activity, mainly related to the use of high quality, safe and environmentally friendly feed ingredients. The use of additives in aquafeeds has proven to be a suitable option to improve different productive indicators in farmed fish. In the present study, the effect of adding the GHRP-6 peptide, a ghrelin analog, to a commercial diet of gilthead sea bream (Sparus aurata) was studied at two proportions (100 or 500 μg/kg of feed). Both experimental diets show an increase in growth performance, as well as in feed efficiency after 97 days of experiment. The lower inclusion of GHRP-6 (100 μg/kg) results in a better aerobic metabolism, while the higher inclusion significantly increased plasma GH levels in agreement with the GH secretagogue effects of ghrelin. Similar growth outcome and differences between GHRP-6 levels in aerobic metabolism and GH stimulation suggest that improvements in culture performance by this peptide may occur through different mechanisms. Taken together, this compound can be considered as a viable dietary supplement for increasing production efficiency of sea bream aquaculture, although a better understanding of its dose-specific effects is still required.

LEAP2 deletion in mice enhances ghrelin's actions as an orexigen and growth hormone secretagogue

ObjectiveThe hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. MethodsWe generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. ResultsLEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2× higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10× higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2–119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. ConclusionsLEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.

Ghrelin-induced Food Intake, but not GH Secretion, Requires the Expression of the GH Receptor in the Brain of Male Mice.

Ghrelin stimulates both GH secretion and food intake. The orexigenic action of ghrelin is mainly mediated by neurons that coexpress agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus (ARH). GH also stimulates food intake and, importantly, ARHAgRP/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 knockout [KO] mice) or exclusively in ARHAgRP/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 ARHAgRP/NPY neurons.

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.

The Effect of Two Types of High-Intensity Interval Training on Serum Value of GH and IGF-1 in Overweight Nurses

Background: Growth hormone and insulin-like growth factor 1 are anabolic hormones that play a vital role in the growth of various physical organs. Exercise is one of the stimuli that affect GH and IGF-1 secretion. Objectives: This study aimed to compare the effect of two types of high-intensity interval training (HIIT) on plasma levels of GH and IGF-l in overweight nurses. Methods: In this study, 27 nurses were voluntarily selected and randomly assigned to three groups (9 participants for each group): 1. HIIT (type 1), including eight seconds of spring running and 12 seconds of active recovery, 2. HIIT (type 2), including a 40-meter shuttle run with maximum speed, 3. control. HIIT (type 1) was performed for four weeks, three sessions per week, each session 6 - 9 min with more than 90% HRmax. HIIT (type 2) was applied for four weeks, three sessions per week, with more than 90% HRmax. The control group did not participate in any training protocol. The serum value of GH and IGF-1 were compared in three groups. The data were analyzed by the dependent t-test and ANOVA. One-way analysis of variance (ANOVA) was used to analyze the intergroup data at P ⟨ 0.05. Results: The results showed that HIIT (type 1) and (type 2) significantly increased plasma GH (P = 0.032 in group 2 and P = 0.010 in group 1) and IGF-l (P = 0.004 in group 2 and P = 10.013 in group 1) levels in nurses. The results showed a significant difference in the variables (GH and IGF-l) among HIIT (type 1), HIIT (type 2), and control groups. Conclusions: It can be concluded that four weeks of adverse intermittent exercises are effective in increasing the concentration of GH and IGF-1 serum and decreased percentage body fat in young nurses with overweight, and proportional to the intensity of the exercise protocol response rate is different.

Cortisol regulates insulin-like growth-factor binding protein (igfbp) gene expression in Atlantic salmon parr

The growth hormone (Gh)/insulin-like growth-factor (Igf)/Igf binding protein (Igfbp) system regulates growth and osmoregulation in salmonid fishes, but how this system interacts with other endocrine systems is largely unknown. Given the well-documented consequences of mounting a glucocorticoid stress response on growth, we hypothesized that cortisol inhibits anabolic processes by modulating the expression of hepatic igfbp mRNAs. Atlantic salmon (Salmo salar) parr were implanted intraperitoneally with cortisol implants (0, 10, and 40 μg g−1 body weight) and sampled after 3 or 14 days. Cortisol elicited a dose-dependent reduction in specific growth rate (SGR) after 14 days. While plasma Gh and Igf1 levels were unchanged, hepatic igf1 mRNA was diminished and hepatic igfbp1b1 and -1b2 were stimulated by the high cortisol dose. Plasma Igf1 was positively correlated with SGR at 14 days. Hepatic gh receptor (ghr), igfbp1a, -2a, -2b1, and -2b2 levels were not impacted by cortisol. Muscle igf2, but not igf1 or ghr, levels were stimulated at 3 days by the high cortisol dose. As both cortisol and the Gh/Igf axis promote seawater (SW) tolerance, and particular igfbps respond to SW exposure, we also assessed whether cortisol coordinates the expression of branchial igfbps and genes associated with ion transport. Cortisol stimulated branchial igfbp5b2 levels in parallel with Na+/K+-ATPase (NKA) activity and nka-α1b, Na+/K+/2Cl--cotransporter 1 (nkcc1), and cystic fibrosis transmembrane regulator 1 (cftr1) mRNA levels. The collective results indicate that cortisol modulates the growth of juvenile salmon via the regulation of hepatic igfbp1s whereas no clear links between cortisol and branchial igfbps previously shown to be salinity-responsive could be established.

Establishment of time-resolved fluoroimmunoassays for detection of growth hormone and insulin-like growth factor I in rainbow trout plasma

The GH/IGF-I axis influences many aspects of salmonid life history and is involved in a variety of physiological processes that are related to somatic growth (e.g., reproduction, smoltification, and the response to fasting and stress). As such, fisheries studies utilize GH/IGF-I axis components as indicators of growth and metabolic status. This study established time-resolved fluoroimmunoassays (TR-FIAs) for rainbow trout plasma GH and IGF-I using commercially available reagents. For the GH TR-FIA, the ED80 and ED20 were 0.6 and 28.1 ng/mL, the minimum detection limit was 0.2 ng/mL, and the intra- and inter-assay coefficients of variation (%CV) were 4.1% and 13.4%, respectively. Ethanol remaining from acid-ethanol cryoprecipitation (AEC) of plasma samples to remove IGF binding proteins reduced binding and increased variability in the IGF-I TR-FIA. Drying down and reconstituting extracted samples restored binding and reduced variability. The extraction efficiency of IGF-I standards through AEC, drying down, and reconstitution did not vary over the working range of the assay. For the IGF-I TR-FIA, the ED80 and ED20 were 0.2 and 6.5 ng/mL, the minimum detection limit was 0.03 ng/mL, and the intra- and inter-assay %CV were 3.0% and 6.5%, respectively. Biological validation was provided by GH injection and fasting studies in rainbow trout. Intraperitoneal injection with bovine GH increased plasma IGF-I levels. Four weeks of fasting decreased body weight, increased plasma GH levels, and decreased plasma IGF-I levels. The GH and IGF-I TR-FIAs established herein provide a cost-comparable, non-radioisotopic method for quantifying salmonid plasma GH and IGF-I using commercially available reagents.

The effects of dietary heat-killed probiotics bacteria additives in low-fishmeal feed on growth performance, immune responses, and intestinal morphology in juvenile olive flounder Paralichthys olivaceus

Fishmeal is an important source of protein in the fish diet, but its supply is unstable due to environmental pollution and overfishing of anchovy and herring. In addition, the disease outbreak of fish has been increased, and the abuse of antibiotics and vaccines has been becoming a problem for residual substances. Therefore, studies that address diets with reduced fishmeal content and additives that can increase efficiency and replace antibiotics are needed. Hence, in this study, the probiotics bacteria Bacillus sp. SJ-10 (BSJ-10) and Lactobacillus plantarum (LP) were heat-killed (HK) used as additives in a low-fishmeal diet. The two bacteria with a 45 % fishmeal diet were added at a concentration of 3.34 × 108 CFU/mL, and both of probiotics bacteria were mixed and added to the same concentration in order to produce the diet for HK BSJ-10 (HKBSJ-10), HK LP (HK-LP), and MIX (HKBSJ-10 + HK-LP) groups. We raised four replicates of juvenile olive flounder for 8 weeks. In the HKBSJ-10 group, the plasma insulin, GH, and IGF-I level were higher than the other groups (P < 0.05). Similarly, intramuscular IGF-I activity and signaling pathway were higher in HKBSJ-10 (P < 0.05). The innate immunity lysosome activity was higher in the HKBSJ-10 and MIX groups (P < 0.05), even cholecystokinin manifestation, intestinal wall thickness, and the density of the intestinal folds more evolved in the HKBSJ-10 group. Therefore, the HKBSJ-10 additive could be advanced in growth-related endocrine, innate immunity, and intestinal morphology, positively affecting a low-fishmeal diet for juvenile olive flounder.

PSXIII-42 Plasma levels of growth hormone in laying hens in relation to developmental and hormonal factors

Abstract Growth hormone (GH) is an important positive regulator of ovarian function in vertebrates, including birds, but it can accelerate aging processes. In the present study, GH levels in the blood of laying hens were investigated due to age, reproductive status and ovulatory cycle-related hormones. To this end, the following groups of hens were used: (1) young hens with long clutch (YLC, 29–31 week-old, &amp;gt;10 eggs per clutch), (2) YLC hens treated with LH (YLC-LH), (3) old hens with long clutch (OLC, 62–64 week-old), (4) old hens with short clutch (OSC, 3–6 eggs per clutch). The hens were sacrificed at 1.5 or 14.5 h after assumed ovulation as well as 3.5 h after the LH injection (n = 5–6 per each group). Plasma concentrations of GH were determined by RIA and those of ovarian steroid hormones were measured by ELISA. The data were analyzed by ANOVA. In all compared groups, levels of GH were similar at the beginning and the middle of the ovulatory cycle. The average concentration of GH in OSC hens was higher than in OLC hens (1.28±0.38 vs 0.39±0.09 ng/ml, P &amp;lt; 0.05) but did not differ from that in YLC hens (0.76±0.22 ng/ml). Furthermore, in OSC hens, a positive correlation (r=0.84, P &amp;lt; 0.05) was revealed between GH levels and testosterone levels in the middle of the ovulatory cycle. Injections of LH raised ovarian steroid concentrations (P &amp;lt; 0.001) and did not affect GH concentrations in the plasma of YLC hens. Meanwhile, GH levels correlated with estradiol-17β levels in the YLC-LH group (r=0.81, P &amp;lt; 0.05). The findings suggest that elevated plasma levels of GH are associated with a reproductive impairment in old hens. Besides, a relationship between GH and ovarian steroids depends on the hen age, reproductive status, ovulatory cycle stage, and LH impact. The study was supported partly by RFBR (19-016-00216).

Effect of betaine supplementation on growth performance, nutrient intake and expression of IGF-1 in Karan Fries heifers during thermal stress

Heat stress hampers nutrient utilisation and production of animals, and dietary betaine supplementation can mitigate the adverse effects of heat stress on animals and improve their productivity. The present study was conducted to explore the effects of betaine supplementation on the growth performance of eighteen growing Karan Fries (KF) heifers having similar age and body conditions. The experiment was carried out on three groups (n = 6) of KF heifers viz. control, treatment I (betaine supplemented at 25 g/d/animal), and treatment II (betaine supplemented at 50 g/d/animal). The experiment lasted for eight months covering the three major seasons of Indian tropical conditions viz. hot-dry (temperature humidity index, THI = 83), hot-humid (THI = 85) and thermoneutral season (THI = 73). Blood samples were collected at fortnightly intervals and analysed for plasma growth hormone (GH; competitive ELISA) and total insulin-like growth factor 1 (IGF-1; Sandwich ELISA), as well as expression of IGF-I in peripheral blood mononuclear cells (PBMC) using real-time polymerase chain reaction. Betaine supplementation resulted in significant (p < 0.05) increase in dry matter intake, feed conversion efficiency, body weight gain, plasma GH and IGF-1 levels during all seasons. The concentrations of plasma IGF-1 and the mRNA expression of IGF-1 were higher (p < 0.01) in treatment I as compared to other groups during all seasons. Betaine supplementation at 25 g/d/animal was more cost-effective in improving growth performance of heat-stressed heifers as compared to 50 g/d/animal. The study suggests that the betaine protects intestinal integrity, enhances nutrient utilisation during heat stress and improves growth performance of growing heifers.

Bee Venom Stimulates Hormone Secretion in Rat Somatotroph and Corticotroph Cells: Digital Image Analysis of Secretory Granules.

In this study, we characterized secretory granules in somatotroph (STCs) and corticotroph (CTCs) cells from the anterior pituitary of rats, in conjunction with different experimental treatments with bee venom (BV). In the rats injected for 30 days with daily BV doses equivalent to one sting, we found significant changes in secretory granules' diameter: reduced by 48.15% in STCs and increased by 5.09% in CTCs, and especially a shift to gray into their intensity profile: increased by 237.04% in STCs and by 212.38% in CTCs. In the rats injected with a single high BV dose, the granules' diameter was reduced in both STCs (by 7.14%) and CTCs (by 4.67%-significant) and their gray intensity profile increased by 200% in STCs and by 51.71% in CTCs (both are significant). The changes in the gray profile reflected a reduced content of granules in the cells, consistent with an increase of the plasma levels of GH and ACTH in all cases. We concluded that the reduced hormone cargo of granules in STCs and CTCs resulted from an accelerated cell secretion. The results obtained for the two types of cells correlated, indicating a similar reaction of these secretory cells to the prolonged and acute presence of BV in the organism.

MON-477 Octreotide and ONO-ST-468, a Novel and Potent Somatostatin Receptor Type-2 (SST2) Agonist, Suppress GH Hypersecretion in the Monkey

Purpose: The verification of efficacy of new drug candidates in primate models may address the translational gap between non-clinical and clinical studies and support the prediction of efficacy in humans. Given this, and to support the evaluation of novel SST2 agonists, a GHRH/arginine-induced GH hypersecretion model was established in the monkey. This model was used to evaluate the effect of both octreotide and ONO-ST-468, a novel and potent selective SST2 agonist. Methods: Male cynomolgus monkeys (3-6 years old) were anesthetized with ketamine and maintained with constant infusion of propofol. Octreotide (0.23, 0.47 or 0.70 μg/0.88mL/kg/h), ONO-ST-468 (0.35 or 1.05 μg/1.1mL/kg/h) and saline were continuously administrated by intravenous (i.v.) infusion 2 hours before and after GH hypersecretion (n=7-9 in each group). GH hypersecretion was induced by i.v. bolus administration of GHRH (“time zero”), followed immediately by a 30 minutes i.v. infusion of arginine. Blood samples were collected for 2 hours after GHRH/arginine treatment and the plasma GH level was measured by ELISA. Inhibitory rates of GH hypersecretion were calculated by the area under the curve (AUC0-2h). Results: As a result of examinations for several experimental conditions, excessive GH secretion was induced by i.v. bolus administration of GHRH (30 μg/0.5mL/kg) and the i.v. infusion of arginine (5 g/17.9mL/kg/h). GH AUC0-2h was approximately 72 ng·h/mL in the vehicle group. Octreotide inhibited GHRH/arginine-induced GH hypersecretion in a dose dependent manner. The inhibitory rates (AUC0-2h) by octreotide were 18% (low dose), 59% (medium dose) and 83% (high dose), respectively. The plasma concentration of octreotide in high dose group was 1.76 ng/mL at 2 hours after GHRH/arginine treatment, being similar to effective plasma concentration of octreotide in acromegaly patient (approximately 1 ng/mL). ONO-ST-468 also inhibited GHRH/arginine-induced GH hypersecretion in a dose dependent manner. The inhibitory rates (AUC0-2h) by ONO-ST-468 were 70% (low dose) and 94% (high dose), respectively, demonstrating that the maximum inhibitory effect of ONO-ST-468 was similar to that of octreotide and has the potential to suppress GH hypersecretion in acromegaly patients. Conclusion: Octreotide and ONO-ST-468, a novel small molecule SST2 agonist, suppressed excessive GH secretion in a newly established GHRH/arginine-induced GH hypersecretion model in the monkey.

SUN-321 Rare Case of Hypoglycemia Induced by IGF2 Secreted by an Ovarian Tumor

Introduction: Mesenchymal and epithelial tumors can produced un-cleaved IGF-2 molecules known as “big” IGF-2. These tumors are associated with elevated levels of IGF-2, which can mimic hypoglycemic events of insulin-producing islet-cell tumors by binding and activating insulin receptors. Typically plasma levels of insulin, IGF-1, and GH are low. “Big” IGF-2 molecular testing is not commercially available, however diagnosis of IGF-2 mediated hypoglycemia is based on low insulin and proinsulin in association with a IGF-2 to IGF-1 ratio >3. Clinical Case A 62 year old female with no significant past medical problems presented with 20 lb weight loss and a two month history documented symptomatic hypoglycemia episodes . Her PCP palpated a firm, non-painful mass occupying nearly her entire abdomen on physical exam. CT revealed a pelvic mass measuring 23.4 cm thought to originate from the ovary. Biopsy of the mass showed a well-differentiated neuroendocrine tumor grade 3, Ki 67 of 15%. Lab work by Endocrinology was significant for a serum glucose of 46 mg/dl, insulin <5 uIU/ml, C-peptide <1 ng/ml, cortisol 43 ug/dl, IGF-1 18 ng/ml, IGF-2 163 ng/ml (IGF1:IGF2: 9), pro-insulin 1.7 pmol/L, sulfonyl urea screen was negative, Ca 125: 230 U/ml, Ca 19-9: 7 U/ml, CEA <1 ng/ml, and chromogranin-A 598 ng/ml. Ga DOTATE scan revealed a 21 cm intra-abdominal mass with multiple peritoneal soft tissue implants consistent with a neuroendocrine tumor with peritoneal carcinomatosis. Due to persistent episodes of hypoglycemia, the patient was started on octretotide 200 mg q 8 hrs, Dexamethasone 1 mg q 3 hrs and parenteral nutrition. The patient was evaluated by surgery and deemed to inoperable due to multiple metastasis. She was then treated with cepecidobine and temozolomide with significant improvement in her glycemic control. A week later, the patient was able to be weaned off TPN and dexamethasone and was discharged home on oral diet. She was evaluated a month later as outpatient and had infrequent episodes of hypoglycemia. Conclusion This is the first case report of a patient with IGF-2 induced hypoglycemia treated with a combination of cepecidobine, temozolomide, and octreotide. While octreotide helped to improve her glycemic control by generally inhibiting hormonal secretion, likely of both IGF-2 and insulin production, final symptom relief and clinical stabilization occurred with the combination of capecitabine and temozolomide, which in vitro data has is synergistic for induction of apoptosis in neuroendocrine tumor cell lines .

Somatotropic Axis Regulation Unravels the Differential Effects of Nutritional and Environmental Factors in Growth Performance of Marine Farmed Fishes.

The Gh/Prl/Sl family has evolved differentially through evolution, resulting in varying relationships between the somatotropic axis and growth rates within and across fish species. This is due to a wide range of endogenous and exogenous factors that make this association variable throughout season and life cycle, and the present minireview aims to better define the nutritional and environmental regulation of the endocrine growth cascade over precisely defined groups of fishes, focusing on Mediterranean farmed fishes. As a result, circulating Gh and Igf-i are revitalized as reliable growth markers, with a close association with growth rates of gilthead sea bream juveniles with deficiency signs in both macro- or micro-nutrients. This, together with other regulated responses, promotes the use of Gh and Igf-i as key performance indicators of growth, aerobic scope, and nutritional condition in gilthead sea bream. Moreover, the sirtuin-energy sensors might modulate the growth-promoting action of somatotropic axis. In this scenario, transcripts of igf-i and gh receptors mirror changes in plasma Gh and Igf-i levels, with the ghr-i/ghr-ii expression ratio mostly unaltered over season. However, this ratio is nutritionally regulated, and enriched plant-based diets or diets with specific nutrient deficiencies downregulate hepatic ghr-i, decreasing the ghr-i/ghr-ii ratio. The same trend, due to a ghr-ii increase, is found in skeletal muscle, whereas impaired growth during overwintering is related to increase in the ghr-i/ghr-ii and igf-ii/igf-i ratios in liver and skeletal muscle, respectively. Overall, expression of insulin receptors and igf receptors is less regulated, though the expression quotient is especially high in the liver and muscle of sea bream. Nutritional and environmental regulation of the full Igf binding protein 1–6 repertoire remains to be understood. However, tissue-specific expression profiling highlights an enhanced and nutritionally regulated expression of the igfbp-1/-2/-4 clade in liver, whereas the igfbp-3/-5/-6 clade is overexpressed and regulated in skeletal muscle. The somatotropic axis is, therefore, highly informative of a wide-range of growth-disturbing and stressful stimuli, and multivariate analysis supports its use as a reliable toolset for the assessment of growth potentiality and nutrient deficiencies and requirements, especially in combination with selected panels of other nutritionally regulated metabolic biomarkers.

Ghrelin and Its Receptors in Gilthead Sea Bream: Nutritional Regulation.

Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis.

In vivo effects of 17α-ethinylestradiol, 17β-estradiol and 4-nonylphenol on insulin-like growth-factor binding proteins (igfbps) in Atlantic salmon

Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE2), 17β-estradiol (E2) and 4-nonylphenol (NP). Fry exposed to EE2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr), igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE2 and NP reduced hepatic igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1, and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE2 and E2. EDC exposures did not affect body mass or fork length; however, EE2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE2 and E2 diminished hepatic igfbp1b1, -4 and -6b1, and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals.