Impaired Expression of Humanin during Adrenocortical Carcinoma.

The discovery of mitochondria-derived peptides (MDPs) has provided a new perspective on mitochondrial function. MDPs encoded by mitochondrial DNA (mtDNA) can act as hormone-like peptides, influencing cell survival and proliferation. Among these peptides, humanin has been identified as a crucial factor for maintaining cell survival and preventing cell death under various conditions. Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy that results from adrenal hormone dysfunction. This study aimed to investigate humanin expression in the adrenal tissue and serum of patients with ACC. For the first time, our study revealed significant reduction in the mRNA expression of humanin in patients with ACC compared to healthy controls. However, no significant changes were observed in the serum humanin levels. Interestingly, we identified a positive correlation between patient age and serum humanin levels and a negative correlation between tumor size and LDL levels. While the impaired expression of humanin in patients with ACC may be attributed to mitochondrial dysfunction, an alternative explanation could be related to diminished mitochondrial copy number. Further investigations are warranted to elucidate the intricate relationship among humanin, mitochondrial function, and ACC pathology.

Impact of Classic Adrenal Secretagogues on mRNA Levels of Urotensin II and Its Receptor in Adrenal Gland of Rats.

Urotensin 2 (Uts2) is a biologically active peptide involved in the regulation of a variety of physiological and pathophysiological processes. In both the human and rat adrenal gland, the expressions of the Uts2 gene and its receptor (Uts2r) have been described. This paper focuses on the description of the hormonal control of the mRNA levels of urotensin II and its receptor in the adrenal gland of the rat, both in vitro and in vivo. The initial in vitro experiments were carried out on freshly isolated rat adrenocortical cells and their primary culture. The obtained results indicated a stimulating PKA-independent effect of ACTH on the Uts2 mRNA level in the tested cells, with no changes in the Uts2r transcript. Subsequent in vivo experiments showed that ACTH-induced adrenal growth was accompanied by an elevated level of the Uts2 mRNA, with unchanged expression of Uts2r. In the other types of in vivo gland growth studied, enucleation-induced adrenal regeneration and compensatory growth of the gland, the mRNA levels of the studied genes showed no significant differences. The only exception was hemiadrenalectomy, which led to a significant increase in Uts2 mRNA expression level 24 h after surgery. In 12-week-old rats of both sexes, gonadectomy led to a significant increase in the level of Uts2 mRNA in the adrenal gland, an effect that was prevented by sex hormones' replacement. No changes in Uts2r transcript levels were observed under these conditions. Thus, this study suggests that the regulation of Uts2 and Uts2r mRNA levels differs significantly in the rat adrenal gland. While Uts2 transcript levels appear to be mainly dependent on ACTH action, Uts2r mRNA levels are not under the control of this hormone.

The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

Zwilch kinetochore protein (ZWILCH) plays a key role in proper cell proliferation. The upregulation of the ZWILCH gene was observed in many types of cancers, but the association of ZWILCH with adrenocortical carcinoma (ACC) was not investigated so far. The main aim of the presented study was to verify if the enhanced level of the ZWILCH gene can be used as a diagnostic marker for ACC development and progression, as well as a predictor of survival time for ACC patients. The performed analyses included investigation of the ZWILCH expression profile in tumors with publicly available TCGA (The Cancer Genome Atlas) datasets and transcriptomic data from the Gene Expression Omnibus (GEO) database, as well as, in human biological samples of normal adrenal, adrenocortical carcinoma and in commercially available tissue microarrays. The findings demonstrate statistically significant higher ZWILCH gene expression in ACC tissue in comparison with normal adrenal glands. Furthermore, there is a strong correlation between ZWILCH upregulation and tumor mitotic rate and the probability of patient survival. The enhanced ZWILCH level is also connected with the activation of genes involved in cell proliferation and the inhibition of genes related to the immune system. This work contributes to a better understanding of the role of ZWILCH as an ACC biomarker and diagnostic tool.

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.

Effects of Resveratrol in Goto-Kakizaki Rat, a Model of Type 2 Diabetes.

Resveratrol exhibits a pleiotropic, favorable action under various pathological conditions, including type 2 diabetes. However, its anti-diabetic effects in animal models and human trials have not been fully elucidated. The aim of the present study was to determine whether resveratrol is capable of inducing beneficial changes in the Goto-Kakizaki rat, a spontaneous model of diabetes, which in several aspects is similar to type 2 diabetes in humans. Goto-Kakizaki (GK) rats and control Sprague-Dawley (SD) rats were treated intragastrically with resveratrol (20 mg/kg b.w./day) for 10 weeks. Then, a glucose tolerance test was performed and levels of some adipokines in blood were measured. Moreover, lipid contents in skeletal muscle and liver tissues, along with the expression and phosphorylation of pivotal enzymes (AMP-activated protein kinase-AMPK, acetyl-CoA carboxylase-ACC, protein kinase B-Akt) in these tissues were determined. Histology of pancreatic islets was also compared. GK rats non-treated with resveratrol displayed a marked glucose intolerance and had increased lipid accumulation in the skeletal muscle. Moreover, upregulation of the expression and phosphorylation of AMPK, ACC and Akt was shown in the muscle tissue of GK rats. Those rats also had an abnormal structure of pancreatic islets compared with control animals. However, treatment with resveratrol improved glucose tolerance and prevented lipid accumulation in the skeletal muscle of GK rats. This effect was associated with a substantial normalization of expression and phosphorylation of ACC and Akt. In GK rats subjected to resveratrol therapy, the structure of pancreatic islets was also clearly improved. Moreover, blood adiponectin and leptin levels were partially normalized by resveratrol in GK rats. It was revealed that resveratrol ameliorates key symptoms of diabetes in GK rats. This compound improved glucose tolerance, which was largely linked to beneficial changes in skeletal muscle. Resveratrol also positively affected pancreatic islets. Our new findings show that resveratrol has therapeutic potential in GK rats.

ZFP91 zinc finger protein expression pattern in normal tissues and cancers.

Zinc finger protein 91 (ZFP91) gene has been recently acknowledged to possess oncogenic properties. To date, its expression has been examined only in a handful of human organs and cancer types. The aim of the present study was to characterize, for the first time, the ZFP91 expression pattern in a range of human tissues and cancer types. ZFP91 mRNA expression was examined using Cancer Survey cDNA sets. Utilized cDNA samples represented 15 human organs and 17 cancer types. ZFP91 mRNA expression was the highest in the testes and lymph nodes. It was downregulated in testis cancer, lymphoma and thyroid cancer, and upregulated in prostate cancer. Among the analyzed cancer types, ZFP91 expression was markedly elevated in sarcomas and melanoma. On a protein level, a large-scale reverse phase protein array was employed providing samples from 11 organ types and from cancers derived from these organs. ZFP91 protein expression was revealed to be generally stable across the tested samples and was only moderately elevated in breast, ovarian and pancreatic cancers. To the best of our knowledge, this is the first study to thoroughly analyze the ZFP91 expression pattern in human tissues and cancers. The obtained results provide the foundation for further work aiming to reveal its full biological significance.

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.

ZFP91: A Noncanonical NF-κB Signaling Pathway Regulator with Oncogenic Properties Is Overexpressed in Prostate Cancer.

Novel molecular targets are being searched to aid in prostate cancer diagnosis and therapy. Recently, ZFP91 zinc finger protein has been found to be upregulated in prostate cancer cell lines. It is a potentially important oncogenic protein; however only limited data regarding its biological function and expression patterns are available. To date, ZFP91 has been shown to be a key factor in activation of noncanonical NF-κB signaling pathway as well as to be involved in HIF-1α signaling in cancer cells. The present study aimed to characterize ZFP91 expression in prostate cancer specimens. Furthermore, since our earlier reports showed discrepancies between ZFP91 mRNA and protein levels, we studied this interrelationship in LNCaP and PC-3 prostate cancer cell lines using siRNA mediated knockdown. QPCR analysis revealed marked upregulation of ZFP91 mRNA in the majority of prostate cancer specimens. Transfection of prostate cancer cells with ZFP91 siRNA resulted in a 10-fold decrease in mRNA levels. On a protein level, however, no inhibitory effect was observed over the time of the cell culture. We conclude that ZFP91 is overexpressed in prostate cancer and that potential accumulation of the ZFP91 protein in studied cells may be of importance in prostate cancer biology.

Lack of expression of preproorexin and orexin receptors genes in human normal and prostate cancer cell lines.

INTRODUCTION: Studies on expression of orexins (OXs) and their receptors in human prostate gland and human prostatic cell lines are scanty and their results contradictory. Regarding this, we carefully reinvestigated this problem on human prostatic cell lines. MATERIAL AND METHODS: Expression of preproorexin (ppOX) (6 primer pairs), and orexin receptors 1 and 2 (OXR1, OXR2) (4 and 2 primer pairs, respectively) was assessed by conventional PCR and QPCR in human normal (PrEC, PrSc, PrSmC) and prostate carcinoma (Du145, LNCaP, and PC3) cell lines. We designed intron spanning primers and also we applied primers from earlier publications and commercially available ones. RESULTS: With the designed primer pairs, in all studied cell lines we failed to demonstrate expression of ppOX, OXR1 and OXR2 genes at the mRNA level, while reaction products were observed in control tissues (human placenta and adrenals). Primers applied in earlier studies did not form amplification products specific for preproorexin or orexin 1 receptor. Some commercially available primers for orexin receptor 1 produced false positive results. CONCLUSIONS: We found no evidence for the presence of preproorexin-orexin receptors system genes' mRNAs in human prostate cell lines. The reported premises for these genes' expression in prostate and prostatic cell lines may have arisen either from the presence of non-prostate cells included in the samples or from faulty PCR settings.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

INTRODUCTION: Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. MATERIAL AND METHODS: Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. RESULTS: By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERß and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERß mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRß were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. CONCLUSIONS: In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRß receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control.

VSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix(®) Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNA levels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11b2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation.

Enucleation-induced rat adrenal gland regeneration: expression profile of selected genes involved in control of adrenocortical cell proliferation.

Enucleation-induced adrenal regeneration is a highly controlled process; however, only some elements involved in this process have been recognized. Therefore, we performed studies on regenerating rat adrenals. Microarray RNA analysis and QPCR revealed that enucleation resulted in a rapid elevation of expression of genes involved in response to wounding, defense response, and in immunological processes. Factors encoded by these genes obscure possible priming effects of various cytokines on initiation of regeneration. In regenerating adrenals we identified over 100 up- or downregulated genes involved in adrenocortical cell proliferation. The changes were most significant at days 2-3 after enucleation and their number decreased during regeneration. For example, expression analysis revealed a notable upregulation of the growth arrest gene, Gadd45, only 24 hours after surgery while expression of cyclin B1 and Cdk1 genes was notably elevated between days 1-8 of regeneration. These changes were accompanied by changes in expression levels of numerous growth factors and immediate-early transcription factors genes. Despite notable differences in mechanisms of adrenal and liver regeneration, in regenerating adrenals we identified genes, the expression of which is well recognized in regenerating liver. Thus, it seems legitimate to suggest that, in the rat, the general model of liver and adrenal regeneration demonstrate some degree of similarity.

Angiogenesis in the course of enucleation-induced adrenal regeneration--expression of selected genes and proteins involved in development of capillaries.

Enucleation-induced rapid proliferation of adrenocortical cells and restoration of adrenals structure requires formation of new blood vessels. The performed studies aimed to select from around 30,000 transcripts, identified by means of Affymetrix(®) Rat Gene 1.1 ST Array, the genes involved in angiogenesis in the course of enucleation-induced adrenal regeneration and to characterize their expression levels in regenerating gland between days 1 and 15 after surgery. At day 1 of regeneration almost 2000 genes showed more than 2-fold up/down-regulation. At days 1-3 after surgery the highest expression demonstrated genes involved in the development of inflammation and blood clot formation. From around 2000 genes we selected genes involved in angiogenesis. During the regeneration 62 genes involved in angiogenesis were identified as up- or down-regulated. Some data were also validated by QPCR. Levels of Vegfa and Kdr (Vegfr-2) mRNAs were very low at day 1 of regeneration and remained unchanged thereafter. The highest expression of Figf gene was found at day 5 while that of Vwf gene at days 1 and 2 after surgery. Levels of Thy1 mRNA increased notably between days 2 and 5 of the experiment. In comparison to control rats, Mc2r (ACTH receptor) expression was lowered at day 1 of the experiment and remained unchanged thereafter. This suggests that enucleation-induced adrenal neoangiogenesis does not require elevated expression of ACTH receptor. Results of our studies strongly suggest that enucleation-induced adrenal regeneration is an angiogenesis-dependent process. Moreover, immunohistochemistry suggests that regenerating adrenal parenchymal cells release numerous angiogenic factors which paracrinally may regulate formation of new vessels.

Ghrelin and obestatin inhibit enucleation-induced adrenocortical proliferation in the rat.

Studies involving the role of ghrelin (GHREL) in regulating the proliferative activity of various cell types have obtained variable results depending primarily on the experimental model applied. It was recently reported that neither GHREL nor obestatin (OBS) affected the proliferative activity of cultured rat adrenocortical cells. In view of the conflicting results, we investigated the effects of GHREL and OBS on the proliferative activity of rat adrenocortical cells in a model of bilateral enucleation-induced adrenocortical regeneration in the rat. Rats were sacrificed 5 or 8 days after surgery. Twenty-four hours before being sacrificed, the appropriate groups were infused with 3 nmol GHREL or OBS/100 g. The mitotic index was assessed using the stachmokinetic method with vincristine. In comparison with intact rats, expression levels of ppGHREL, BAX, JUN-B and JUN-C genes were notably higher in regenerating adrenals, and neither GHREL nor OBS infusion affected these levels. Expression levels of the GHS-R, GPR39v2 and FOS genes were affected neither by adrenal enucleation nor GHREL or OBS infusion. Expression of only two studied genes, GPR39v1 and EGR1, was regulated by OBS. In the regenerating adrenal glands, GPR39v1 and EGR1 mRNA levels were higher than the levels in intact animals. GHREL infusion had no effect while OBS infusion notably stimulated GPR39v1 mRNA levels in the regenerating adrenal gland and evoked an opposite effect on EGR1 mRNA. OBS administration resulted in a potent decrease in the mitotic index of the studied cells, an effect found at both days 5 and 8 of the experiment. GHREL exerted a similar effect only at day 5 of adrenocortical regeneration. Neither GHREL nor OBS had an effect on blood aldosterone concentrations. GHREL infusion lowered plasma corticosterone concentration at day 5 but not 8 of the experiment, while OBS administration was ineffective. Thus, this study is the first to demonstrate that, in vivo, both GHREL and OBS inhibit the growth of the regenerating adrenal cortex. Moreover, the data suggest that the effect of OBS might be, at least in part, mediated by the EGR1 pathway known to be critical in cell proliferation.

Orexin B inhibits proliferation and stimulates specialized function of cultured rat calvarial osteoblast-like cells.

Orexin-A (OXA) and orexin-B (OXB) are polypeptides derived from the same 130 amino acid long precursor (prepro-orexin) that bind and activate two closely related orphan G protein-coupled receptors OX1-R and OX2-R. These hypothalamic neuropeptides stimulate food intake and energy expenditure and play a significant role in sleep-wakefulness regulation. Present studies aimed to investigate the effects of orexins on proliferative activity and osteocalcin secretion by cultured rat calvarial osteoblast-like (ROB) cells. Conventional RT-PCR methods detected expression of the OX1-R gene in freshly isolated ROB cells and cells cultured for 7, 14 and 21 days. In contrast, at all time points tested, expression of prepro-OX or OX2-R genes was not demonstrated. QPCR revealed the highest expression of OX1-R gene in freshly isolated bone cells and a notably lower one in cultured ROB cells. Exposure of cultured cells to both OXA and OXB stimulated expression of the OX1-R gene. However, this effect was seen at the lowest tested concentration (1x10(-10) M). Exposure of cultured ROB cells to OXA for 48 h did not change osteocalcin concentrations in media analyzed at days 7, 14 and 21 of culture. On the contrary, OXB notably stimulated osteocalcin concentrations in media taken at days 14 and 21 of culture. In contrast, OXA exerted a notable inhibitory effect on the proliferative activity of ROB cells at day 7 of culture, while OXB exerted a similar effect at day 14. Thus, the obtained results suggest that: (i)(ROB) cells are provided with functional OX1-R gene; (ii) in ROB cells expression of this gene seems to be up-regulated by low concentrations of both OXA and OXB; (iii) OXB exerts inhibitory effects on proliferative activity and stimulating effects on osteocalcin secretion by cultured ROB cells; (iv) rat calvarial osteoblasts provided with OX receptor may be a target for circulating orexins. Thus, orexins may be included in the expanding group of neuropeptides involved in the physiological regulation of the major bone cell types.