A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles—MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

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.

Triptolide Resistance Was Prevented by Metformin Co-Treatment Under Increased Growth Hormone Signaling Conditions in MDA-MB-231 Cells

Breast cancer is identified as the most common cancer among women and the leading cause of cancer-related deaths. Increased expression of growth hormone (GH) has been found to cause cell growth, drug resistance and invasion-metastasis in breast cancer. Triptolide is a diterpenoid isolated from Tripterygium wilfordii with anti-cancer activity. Our data showed that GH overexpressing MDA-MB-231 cells exhibited a resistance mechanism against tripitolide treatment when compared to wild type MDA-MB-231 cells. To overcome the resistance, we combined triptolide with metformin, an anti-diabetic agent with anti-tumorigenic effect and a potential adjuvant in the management of breast cancer. Our findings indicated that GH+ cells exhibited increased cell viability loss, mitochondrial membrane potential loss and apoptotic cell death with metformin co-treatment. In addition, colony sizes were significantly decreased due to combined treatment. Autophagy is a process of cellular self-degradation during which macromolecules, damaged organelles are delivered to the lysosome in order to control energy level under stressed conditions. Cells can control proliferation or apoptosis by changing the level of autophagy, it has been proposed as a chemoresistance mechanism. Our data showed that there was no significant change in the autophagy induction in MDA-MB-231 wt and GH+ cells treated with triptolide and metformin.

Autocrine Growth Hormone Mediated Curcumin Resistance Overcame by Autophagy Inhibition via Bafilomycin in MDA-MB-231 and T47D Breast Cancer Cells

Curcumin, a plant derived natural compound, has anti-oxidant, anti-proliferative and apoptotic effect on various cancer cells such as prostate, colon and breast cancer. Autocrine growth hormone (GH) expression induced breast cancer invasion-metastasis has been reported in vivo and in vitro cancer models. Autophagy is a vesicule-mediated clearance mechanism and one of the handicap against drug-induced apoptotic cell death. In this study, our aim was to investigate the molecular machinery of curcumin induced apoptotic cell death under autophagy inhibition conditions in autocrine GH expressing MDA-MB-231 and T47D breast cancer cells. Although autocrine GH induced curcumin resistance, this effect was slightly prevented by time-dependent curcumin treatment in MDA-MB-231 and T47D breast cancer cells. In addition, curcumin induced autophagy vacuole formation was determined by acridine orange staining in MDA-MB-231 and T47D wt/GH+ breast cancer cells. Moreover, curcumin triggered autophagy through upregulating Beclin-1, Atg3, Atg12 expressions and LC3 cleavage in each cell line. Concomitantly, BiP, IRE1α and Calreticulin expressions were upregulated following 3 h curcumin exposure in MDA-MB-231 wt and GH+ cells. According to MTT cell viability assay, autocrine GH-mediated curcumin resistance was overcome by bafilomycin and curcumin co-treatment in MDA-MB-231 and T47D GH+ cells. Moreover, curcumin and bafilomycin co-treatment induced cell cycle arrest at G1 phase in MDA-MB-231 GH+ cells, G2/M arrest in T47D GH+ breast cancer cells. In conclusion, autocrine GH-triggered curcumin resistance was overcome by autophagy inhibition condition by bafilomycin treatment in a dose-dependent manner in MDA-MB-231 and T47D GH+ breast cancer cells.

Sex-specific changes in postnatal GH and PRL secretion in somatotrope LEPR-null mice

The developing pituitary is a rapidly changing environment that is constantly meeting the physiological demands of the growing organism. During early postnatal development, the anterior pituitary is refining patterns of anterior hormone secretion in response to numerous genetic factors. Our laboratory previously developed a somatotrope leptin receptor (LEPR) deletion mouse model that had decreased lean body mass, disrupted metabolism, decreased GH stores and was GH deficient as an adult. To understand how deletion of LEPR in somatotropes altered GH, we turned our attention to postnatal development. The current study examines GH, PRL, TSH, ACTH, LH and FSH secretion during postnatal days 4, 5, 8, 10 and 15 and compares age and sex differences. The LEPR mutants have dysregulation of GH (P < 0.03) and a reduced developmental prolactin peak in males (P < 0.04) and females (P < 0.002). There were no differences in weight between groups, and the postnatal leptin surge appeared to be normal. Percentages of immunolabeled GH cells were reduced in mutants compared with controls in all age groups by 35–61% in males and 41–44% in females. In addition, we measured pituitary expression of pituitary transcription factors, POU1F1 and PROP1. POU1F1 was reduced in mutant females at PND 10 (P < 0.009) and PND 15 (P < 0.02) but increased in males at PND 10 (P < 0.01). PROP1 was unchanged in female mutants but showed developmental increases at PND 5 (P < 0.02) and PND 15 (P < 0.01). These studies show that the dysfunction caused by LEPR deletion in somatotropes begins as early as neonatal development and involves developing GH and prolactin cells (somatolactotropes).

Immunohistomorphometric Characteristics Of Pituitary GH Cells In Infant And Peripubertal Female Rats After Treatment With Estradiol Or Human Chorionic Gonadotropin

The effects of estradiol-dipropionate (EDP) or human chorionic gonadotropin (hCG) on immunohistomorphometric characteristics of pituitary GH cells in infant and peripubertal female rats were investigated. The first group of females received five injections of EDP (0.25 mg/kg b.w.) during the neonatal period of life, and was further divided into two subgroups which were sacrificed at the infantile period (17th day) or at the peripubertal period (38th day). The second group received two doses of hCG (50 IU/kg b.w.) on the 15th and 16th day of life in the first subgroup, and on the 36th and 37th days of life in the second subgroup, while they were sacrificed 24 h after the last treatment, respectively. The control females were injected with an equivalent volume of the vehicle and sacrificed according to the appropriate schedules as the hormone treated rats. EDP treatment decreased GH cell volume density in infant and peripubertal females, by 38% and 76% (p<0.05) respectively, in comparison with the controls. The number of GH cells per mm2 in infantile and peripubertal period was decreased in EDP treated animals by 26% and 53% (p<0.05) respectively, compared to the controls. Also, upon EDP treatment in both periods, GH cells were diminished in size and less intensely immunolabelled than in the control groups. The morphometric parameters in animals treated with hCG were insignificantly changed in both analyzed periods, in comparison with the controls. Unlike hCG, EDP manifested clear inhibitory effects on the immunohistomorphometric characteristics of GH cells in examined female rats.

Sex Differences in Somatotrope Dependency on Leptin Receptors in Young Mice: Ablation of LEPR Causes Severe Growth Hormone Deficiency and Abdominal Obesity in Males

Leptin receptor (LEPR) signaling controls appetite and energy expenditure. Somatotrope-specific deletion of the LEPRb signaling isoform causes GH deficiency and obesity. The present study selectively ablated Lepr exon 1 in somatotropes, which removes the signal peptide, causing the loss of all isoforms of LEPR. Excision of Lepr exon 1 was restricted to the pituitary, and mutant somatotropes failed to respond to leptin. Young (2–3 mo) males showed a severe 84% reduction in serum GH levels and more than 60% reduction in immunolabeled GH cells compared with 41%–42% reductions in GH and GH cells in mutant females. Mutant males (35 d) and females (45 d) weighed less than controls and males had lower lean body mass. Image analysis of adipose tissue by magnetic resonance imaging showed that young males had a 2-fold increase in abdominal fat mass and increased adipose tissue density. Young females had only an overall increase in adipose tissue. Both males and females showed lower energy expenditure and higher respiratory quotient, indicating preferential carbohydrate burning. Young mutant males slept less and were more restless during the dark phase, whereas the opposite was true of females. The effects of a Cre-bearing sire on his non-Cre-recombinase bearing progeny are seen by increased respiratory quotient and reduced litter sizes. These studies elucidate clear sex differences in the extent to which somatotropes are dependent on all isoforms of LEPR. These results, which were not seen with the ablation of Lepr exon 17, highlight the severe consequences of ablation of LEPR in male somatotropes.

Adipocyte Versus Pituitary Leptin in the Regulation of Pituitary Hormones: Somatotropes Develop Normally in the Absence of Circulating Leptin

Leptin is a cytokine produced by white fat cells, skeletal muscle, the placenta, and the pituitary gland among other tissues. Best known for its role in regulating appetite and energy expenditure, leptin is produced largely by and in proportion to white fat cells. Leptin is also important to the maintenance and function of the GH cells of the pituitary. This was shown when the deletion of leptin receptors on somatotropes caused decreased numbers of GH cells, decreased circulating GH, and adult-onset obesity. To determine the source of leptin most vital to GH cells and other pituitary cell types, we compared two different leptin knockout models with Cre-lox technology. The global Lep-null model is like the ob/ob mouse, whereby only the entire exon 3 is deleted. The selective adipocyte-Lep-null model lacks adipocyte leptin but retains pituitary leptin, allowing us to investigate the pituitary as a potential source of circulating leptin. Male and female mice lacking adipocyte leptin (Adipocyte-lep-null) did not produce any detectable circulating leptin and were infertile, suggesting that the pituitary does not contribute to serum levels. In the presence of only pituitary leptin, however, these same mutants were able to maintain somatotrope numbers and GH mRNA levels. Serum GH trended low, but values were not significant. However, hypothalamic GHRH mRNA was significantly reduced in these animals. Other serum hormone and pituitary mRNA differences were observed, some of which varied from previous results reported in ob/ob animals. Whereas pituitary leptin is capable of maintaining somatotrope numbers and GH mRNA production, the decreased hypothalamic GHRH mRNA and low (but not significant) serum GH levels indicate an important role for adipocyte leptin in the regulation of GH secretion in the mouse. Thus, normal GH secretion may require the coordinated actions of both adipocyte and pituitary leptin.

Immuno-histomorphometric and -fluorescent characteristics of GH cells after treatment with genistein or daidzein in an animal model of andropause

Somatopause, the complex aspect of andropause, is recognizable by reduced growth hormone - GH/insulin-like growth factor 1 axis function in the ageing male. Soy isoflavones (usually genistein and daidzein), which are known for their beneficial effects in the treatment of ageing symptoms, are active in the pituitary, as well. The immunohistomorphometric and -fluorescent characteristics of pituitary growth hormone secreting cells, in an animal model of andropause, were examined after a treatment with genistein or daidzein. Andropausal Wistar rats were divided into sham operated, orchidectomized and genistein or daidzein treated orchidectomized groups. Genistein or daidzein (30 mg/kg/day) were administered subcutaneously for three weeks, while sham operated and orchidectomized groups received the vehicle alone. Growth hormone secreting cells were identified by the peroxidase-antiperoxidase immuno-histochemical, and immuno-fluorescent procedure. The main characteristic of growth hormone secreting cells in soy isoflavones treated groups is a weaker immuno-histochemical staining and immuno-fluorescent signal compared to sham operated and orchidectomized groups. The growth hormone secreting cell volume in orchidectomized +genistein or +daidzein groups is by 13.8% and 11.9% (p<0.05) smaller respectively, in comparison with the orchidectomized group. In orchidectomized +genistein or +daidzein groups, the growth hormone secreting cells relative volume density is by 62.5% and 61.0% lower (p<0.05) respectively than for the sham operated group, and decreased by 65.4% and 64.0% (p<0.05) respectively, compared to the orchidectomized group. It can be concluded that chronic genistein or daidzein treatment, in an animal model of andropause, attenuates immunohistomorphometric and -fluorescent characteristics of growth hormone secreting cells.

Ghrelin Restoration of Function In Vitro in Somatotropes from Male Mice Lacking the Janus Kinase (JAK)-Binding Site of the Leptin Receptor

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptin's postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P &lt; .002) and the secretion of GH 1.8–3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7–23%). When added with 1–3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.