Extracellular Nampt (eNampt/visfatin/PBEF) directly and indirectly stimulates ACTH and CCL2 protein secretion from isolated rat corticotropes.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) acts both as an enzyme in the nicotinamide adenine dinucleotide (NAD) synthesis pathway as well as an extracellular hormone (eNampt). Among its effects, eNampt exerts potent pro-inflammatory effects. We have recently shown that, in rats, eNampt stimulates corticosterone secretion by acting through the pituitary rather than the hypothalamus. OBJECTIVES: To investigate the mechanism of action of eNampt on the secretion of adrenocorticotropic hormone (ACTH) and chemokine (C-C motif) ligand 2 (CCL2), which are cytokines secreted by pituitary neuroendocrine tumors. MATERIAL AND METHODS: The research was carried out on the AtT-20 murine cell line, primary rat pituitary cell culture, isolated pituitary corticotropes, and in vivo. The effects of the performed experiments were examined using the following methods: gene expression profiling using microarrays, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results suggest that eNampt stimulates ACTH secretion from rat corticotropes both directly and indirectly. Indirect action most likely occurs through interleukin (IL)-6 secreted by folliculostellate cells of the pituitary gland. In isolated ACTH cells of the rat pituitary gland, eNampt stimulates the expression of genes involved in the immune response. Among them, the protein encoded by the CCL2 gene seems to also be involved in the regulation of corticotropin-releasing hormone (CRH)-dependent metabolism. Unlike rat corticotropes, murine AtT-20 corticotropic cells do not react to either eNampt or Fk866 (the inhibitor of Nampt enzymatic action). CONCLUSIONS: The eNampt stimulates the secretion of ACTH from rat corticotropes indirectly and directly, likely by stimulating IL-6 secretion from folliculostellate cells of the pituitary gland. This effect was not observed in the AtT-20 corticotropic cell cancer cell line.

Adropin Stimulates Proliferation and Inhibits Adrenocortical Steroidogenesis in the Human Adrenal Carcinoma (HAC15) Cell Line.

Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-ß type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-ß signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Analysis of Transcriptome, Selected Intracellular Signaling Pathways, Proliferation and Apoptosis of LNCaP Cells Exposed to High Leptin Concentrations.

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.

Expression profile of Galp, alarin and their receptors in rat adrenal gland.

BACKGROUND: Galanin-like peptide (Galp) and alarin (Ala) are 2 new members of the galanin peptide family. Galanin (Gal), the "parental" peptide of the entire family, is known to regulate numerous physiological processes, including energy and osmotic homeostasis, reproduction, food intake, and secretion of adrenocortical hormones. Galp and Ala are known to regulate food intake. In the rat, Galp mRNA has been found in the brain, exclusively in the hypothalamic arcuate nucleus (ARC) and median eminence, which are involved in the regulation of energy homeostasis. Alarin-like immunoreactivity is present in the locus coeruleus (LC) and the ARC of rats and mice. OBJECTIVES: The aim of the study was to investigate the expression of Ala, Galp and their receptors in the organs of the hypothalamo-pituitary-adrenal (HPA) axis of the rat. MATERIAL AND METHODS: The expression of the examined genes was measured in different models of adrenal growth of the rat in vivo (postnatal ontogenesis, compensatory adrenal growth, adrenocortical regeneration, adrenocorticotropic hormone (ACTH) administration). The expression was evaluated using the Affymetrix® microarray system or quantitative polymerase chain reaction (qPCR). RESULTS: The expression of Ala gene was observed in each organ of the HPA axis (the hypothalamus, hypophysis and adrenal gland). The elevated level of expression of this gene was observed in the pituitary of 2-day rats, while very low levels of Ala mRNA were observed in the adrenals. Galp mRNA expression was observed only in the hypothalamus and the hypophysis during postnatal ontogenesis. The expression of Gal receptors was demonstrated in the hypothalamus, the hypophysis and the adrenal gland. In different compartments of the adrenal glands of adult, intact male and female rats, the expression of Ala, Galp and galanin receptor 1 (Galr1) genes was negligible, but the expression of galanin receptor 2 (Galr2), galanin receptor 3 (Galr3) and neurotrophic receptor tyrosine kinase 2 (Ntrk2) genes was noticeable. CONCLUSIONS: The examined genes showed different expression levels within the studied HPA axis; some of them were neither expressed in the hypothalamus or the pituitary gland, nor in the adrenal gland.

Effect of ACTH and hCG on the Expression of Gonadotropin-Inducible Ovarian Transcription Factor 1 (Giot1) Gene in the Rat Adrenal Gland.

Gonadotropin-inducible ovarian transcription factor-1 (Giot1) belongs to a family of fast-responsive genes, and gonadotropins rapidly induce its expression in steroidogenic cells of ovaries and testes of rats. Gonadal Giot1 gene expression is regulated by cyclic adenosine monophosphate (cAMP) -dependent protein kinase A pathway, with essential role of orphan nuclear receptor NR4A1 transcription factor (nuclear receptor subfamily 4, group A, member 1). A recent study reports that Giot1 is also expressed in adrenals, however, the mechanism of its regulation in adrenal gland is yet to be identified. Therefore, the aim of this study was to characterise the changes in Giot1 gene expression in male and female rat adrenals using wide range of in vivo and in vitro experimental models. Special emphasis was directed at the Giot1 gene regulation by ACTH and gonadotropin. In our study, we found that ACTH rapidly stimulates Giot1 expression both in vivo and in vitro. However, gonadotropin does not affect the adrenal Giot1 gene expression, presumably due to the low expression of gonadotropin receptor in adrenals. Both testosterone and estradiol administered in vivo had inhibitory effect on Giot1 gene expression in the adrenals of post-gonadectomized adult rats. Further, our studies revealed that the intracellular mechanism of Giot1 gene regulation in rat adrenals is similar to that of gonads. As in the case of gonads, the expression of Giot1 in adrenal gland is regulated by cAMP-dependent signaling pathway with essential role of the NR4A1 transcription factor. The results of our studies suggest that Giot1 may be involved in the regulation of rat adrenocortical steroidogenesis.

Ghrelin as a potential molecular marker of adrenal carcinogenesis: In vivo and in vitro evidence.

CONTEXT: Adrenal tumours belong to one of the most prevalent neoplasms. It is a heterogeneous group with different aetiology, clinical manifestation and prognosis. Its histopathologic diagnosis is difficult and identification of differentiation markers for tumorigenesis is extremely valuable for diagnosis. DESIGN: To assess ghrelin expression and the relationship among ghrelin, IGF2 and the clinicopathological characteristics of adrenal tumours. To investigate the influence of ghrelin on ACC cell line proliferation. MATERIALS AND METHODS: Expression of ghrelin and IGF2 in a total of 84 adrenal tissue samples (30 adenoma, 12 hyperplasia, 8 myelolipoma, 20 pheochromocytoma, 7 carcinoma and 7 unchanged adrenal glands) were estimated. Every operated patient from whom samples were obtained underwent clinicopathological analysis. All the parameters were compared among the groups examined and correlations between these were estimated. H295R cell line was incubated with ghrelin to assess its effect on proliferation and migration rate. RESULTS: The highest ghrelin expression was observed in carcinoma samples and the lowest in the control group. Ghrelin expression was 21 times higher in carcinoma (P = .017) and 2.4 times higher in adenoma (P = .029) compared with controls. There were no statistically significant differences between myelolipoma (P = .093) and pheochromocytoma (P = .204) relative to the control. Ghrelin level was significantly higher in carcinoma compared to adenoma (P = .049) samples. A positive correlation between ghrelin and IGF2 expression was observed only in myelolipoma (P = .001). Ghrelin at concentrations of 1 × 10-6  mol/L and 1 × 10-8  mol/L significantly stimulated proliferation and migration rate in the H295R cell line. CONCLUSION: Ghrelin appears to be an essential factor in driving adrenal tumours development.

Nicotinamide phosphoribosyltransferase and the hypothalamic-pituitary-adrenal axis of the rat.

Nicotinamide phosphoribosyltransferase (Nampt), also termed visfatin, catalyses the rate­limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. In addition to its intracellular function (iNampt), extracellular Nampt (eNampt) also affects numerous intracellular signalling pathways. The current study investigated the role of Nampt in the regulation of the hypothalamic­pituitary­adrenal (HPA) axis in rats. At 1 h after intraperitoneal administration of eNampt (4 µg/100 g) in adult male rats, serum adrenocorticotropic hormone(ACTH) and aldosterone levels remained unchanged, while corticosterone levels were notably elevated compared with the control group, as determined by ELISA. The results of reverse transcription­quantitative polymerase chain reaction (RT­qPCR) demonstrated that, in the hypothalami of eNampt­treated rats, the mRNA expression levels of Fos proto­oncogene, which is also termed c­Fos, were not significantly different compared with the control group; however, the mRNA expression levels of proopiomelanocortin (POMC) were markedly increased in the pituitary gland of eNampt­treated rats compared with the control group. Furthermore, in hypothalamic explants, ELISA results demonstrated that the addition of the eNampt protein exhibited no effect on corticotropin­releasing hormone (CRH) release into the incubation medium and prevented potassium ion­induced CRH release. Additionally, the eNampt­induced increase in ACTH output by pituitary gland explants was not statistically significant, compared with the control group. However, RT­qPCR indicated that exposure of pituitary gland explants to eNampt and CRH increased the levels of POMC mRNA expression; the effect of eNampt, but not CRH, was inhibited by FK866, which is a specific Nampt inhibitor. In primary rat adrenocortical cell cultures, eNampt exhibited no effect on basal aldosterone or corticosterone secretion, while increases in aldosterone and corticosterone levels in response to ACTH were retained. To assess the potential role of iNampt in the regulation of adrenal steroidogenesis, experiments involving a specific Nampt inhibitor, FK866, were performed. Exposure of cultured cells to FK866 notably lowered basal aldosterone and corticosterone output compared with the control group, and completely eliminated the response of cultured cells to ACTH. The results of the present study indicated that the injected eNampt may have increased the corticosterone serum levels by acting at the pituitary level. In addition, iNampt may exert a tonic stimulating effect on the secretion of aldosterone and corticosterone from rat adrenocortical cells, as normal iNampt levels were required to retain the response of cultured rat adrenocortical cells to ACTH. Thus, these data suggest an important physiological role of both iNampt and eNampt in the regulation of the HPA axis activity in the rat.

Effects of leptin on leptin receptor isoform expression and proliferative activity in human normal prostate and prostate cancer cell lines.

Results of studies on the expression of leptin and its receptors in the human prostate gland and human prostate cell lines are contradictory. Regarding this, we carefully reinvestigated this issue using human normal prostate (PrEC, PrSC, PrSMC) and prostate cancer (DU145, LNCaP, PC3) cell lines. Expression of leptin receptor isoforms was assessed by qPCR while the effects of leptin on cell proliferative activity was determined by real-time cell analyzer (RTCA). Expression of the leptin receptor variant 1 was not detected in LNCaP and PrSMC cell lines, but it was found in the remaining cell lines. In contrast, in all examined cell lines, isoforms 1-3 and 2 and 4 of the leptin receptor were found. The expression of isoforms 3 and 6 of the leptin receptor was observed in PC3, PrEC, PrSMC and PrSC cell lines, but not in LNCaP and DU145 cells. Expression of the leptin receptor isoforms 4-6 and 5 was not demonstrated in any of the tested cell lines. We also studied the effects of leptin on the expression of its receptor isoforms in all tested cell lines. At a wide range of concentrations, leptin did not change the expression of leptin receptor variant 1 in the DU145, PrEC and PC3 cell lines. In contrast, in the PrSC cell line, leptin significantly increased the expression of this gene. In all prostate cell lines tested, leptin did not alter the expression levels of variants 1-3 of the leptin receptor isoforms. Leptin did not alter the expression of isoforms 2 and 4 of the leptin receptor in the PC3 and LNCaP cell lines. In the DU145 and PrEC cell lines, leptin inhibited expression of these receptor isoforms while an opposite effect was noted in the PrSC cells. Leptin did not affect the expression level of variants 3 and 6 of its receptor in the PrEC and PC3 cell lines. However, in PrSMC cells, leptin inhibited the expression of variants 3 and 6 of its receptor, while in the PrSC cell line this cytokine significantly increased their expression levels. As assessed by RTCA, leptin stimulated the proliferative activity of DU145 cells, but inhibited this activity in LNCaP cells. At all concentrations tested, leptin did not change the proliferation rate of the PC3, PrEC and PrSMC cells. In contrast, leptin notably stimulated the proliferative activity of the PrSC (prostate stromal cell) cell line. Thus, our study demonstrated that in all tested human normal prostate and prostate cancer cell lines, transcription variants 4, 5 and 6 of the leptin receptor were not expressed. Leptin receptor transcription variants 1, 2 and 3 showed differential expression, which was present in the PC3, PrEC and PrSC cell lines. Our data also suggest that the stimulatory effects of leptin on proliferative activity of the studied cell lines require the expression of leptin receptor variant 1 in the affected cells.

Transcriptome Profile of Rat Adrenal Evoked by Gonadectomy and Testosterone or Estradiol Replacement.

Sex differences in adrenal cortex structure and function are well known in different species. In the rat, they are manifested as larger adrenal cortex and higher corticosterone secretion by females compared with males. These sex differences depend, among others, on functioning of the hypothalamic-pituitary-adrenal axis (HPA). In this aspect, it is widely accepted that testosterone exerts an inhibitory and estradiol stimulatory effect on the said axis. The molecular bases of these sex-related differences are poorly understood. Therefore, we performed studies aimed to demonstrate the effect of testosterone and estradiol on the expression of differentially regulated genes in rat adrenal gland. The classical method applied in the study-gonadectomy and gonadal hormone replacement-allows obtaining results suggesting a physiological role of the tested hormone (testosterone or estradiol) in the regulation of the specific genes. Adult male and female rats were either gonadectomized or sham operated. Half of orchiectomized rats were replaced with testosterone while ovariectomized ones with estradiol. Transcriptome was identified by means of Affymetrix® Rat Gene 2.1 ST Array. Differentially expressed genes were analyzed by means of DAVID web-based bioinformatic tools and confirmed by means of Gene Set Enrichment Analysis. For selected genes, validation of the results was performed using QPCR. Performed experiments have provided unexpected results. Contrary to expectations, in orchiectomized rats, testosterone replacement stimulates expression of numerous genes, mainly those associated with lipids and cholesterol metabolism. However, in ovariectomized animals, estradiol replacement inhibits the expression of genes, mainly those involved in intracellular signaling pathways. The physiological relevance of these findings awaits further research.