Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Effects of prolonged treatment with adrenocorticotropin on the morphology and function of rat adrenocortical autotransplants.

Regenerated adrenocortical nodules were obtained by implanting in the musculus gracilis of rats fragments of the capsular tissue of their excised adrenal glands. Five months after operation, transplanted rats showed a slightly elevated blood concentration of adrenocorticotropin (ACTH), a moderately reduced plasma level of corticosterone (PBC) and a very low concentration of circulating aldosterone (PAC). Regenerated nodules were well encapsulated, and from the connective capsule some septa dipped into the parenchyma. Subcapsular-outer (OZ) and inner (IZ) cells were similar to those of the zona fasciculata/zona reticularis (ZF/ZR) of the normal gland; juxta-septal (JZ) cells resembled those of the zona glomerulosa (ZG). Prolonged (14 days) ACTH infusion normalized PBC and caused a conspicuous hypertrophy of transplanted tissue, which was coupled with a marked hypertrophy of ZF/ZR-like OZ and IZ cells and a notable rise in the basal in vitro production of corticosterone. Conversely, ACTH infusion strikingly lowered PAC, reduced the number of ZG-like JZ cells, and decreased both basal and stimulated secretion of 18-hydroxylated steroids by transplants in vitro.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on the secretory activity of dispersed rat adrenocortical cells. Evidence for the interaction of VIP with ACTH receptors

VIP dose-dependently increased basal, but not submaximally ACTH (10⁻¹⁰ M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10⁻⁸ M. [4-Cl-D-Phe⁶,Leu¹⁷]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10⁻⁶ M), completely annulled VIP (10⁻⁸M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10⁻¹⁰ M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10⁻⁶ M) and only partially reduced (about −30%) by VIP-A (10⁻⁶ M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.     

Effects of leptin to cultured growth plate chondrocytes

OBJECTIVE: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. METHODS: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. RESULTS: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. CONCLUSION: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.     

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [¹²⁵I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K⁺-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca²⁺-channel activator BAYK-8644, and blocked K⁺- and BAYK-8644-evoked rise in the intracellular Ca²⁺ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca²⁺ channel-mediated Ca²⁺ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.     

Endothelin-1 acutely stimulates the secretory activity of rat zona glomerulosa cells

A bolus IV injection of endothelin-1 (ET-1) (0.5 microgram.kg-1) decreased PRA, without affecting plasma aldosterone (A) concentration. ET-1 exerted a dose-dependent stimulation of basal secretion of A and corticosterone (B) by dispersed zona glomerulosa (ZG) cells, while it did not affect B production by inner adrenocortical cells. ET-1 notably enhanced the secretory response of dispersed ZG cells to a maximal effective concentration of ACTH, but not of either angiotensin II (ANG-II) or potassium. The conclusion is drawn that ET-1 acutely stimulates ZG in rats, by a mechanism probably similar to that underlying the adrenoglomerulotropic actions of ANG-II and potassium.     

Differential role of leptin receptors at the hypothalamic paraventricular nucleus in tonic regulation of food intake and cardiovascular functions

Leptin plays an important role in the central regulation of body weight and arterial pressure via activation of leptin receptors (Ob-Rs) in the hypothalamic area, including the hypothalamic paraventricular nucleus (PVN). The present study was undertaken to investigate whether endogenous leptin in the PVN plays a dual role in the tonic regulation of body weight and arterial pressure. Adult, male normal-weight Sprague-Dawley rats, which were anesthetized and maintained with propofol, were used. A direct bilateral microinjection into the PVN of an antisense oligonucleotide against Ob-R mRNA (ASON1, 50 pmol) significantly increased the daily food intake and body weight gain, effects which lasted for at least 14 days. The same treatment, on the other hand, had no appreciable effect on the basal mean systemic arterial pressure (SAP), heart rate (HR), or power density of the vasomotor components of SAP signals, the experimental index of neurogenic sympathetic vasomotor tone. ASON1 treatment also exerted an insignificant effect on the baroreceptor reflex control of HR. Western blot analysis revealed that a bilateral microinjection into the PVN of ASON1 (50 pmol) significantly decreased the expression of the Ob-R protein in the hypothalamus. The same treatment also attenuated hypertension, tachycardia, and the increase in the power density of the vasomotor components of the SAP signals induced by exogenous bilateral application of leptin (5 or 50 ng) into the PVN. Control application of sense (SON, 50 pmol) or a scrambled antisense Ob-R oligonucleotide (ASON2, 50 pmol) into the bilateral PVN promoted no discernible effect on Ob-R protein expression in the hypothalamus, on daily food intake, or on cardiovascular performance. Our results indicate that whereas the Ob-Rs in the PVN are involved in the tonic regulation of food intake, they might not be actively involved in the tonic regulation of cardiovascular functions.     

Contribution of leptin receptor N-linked glycans to leptin binding

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.     

The adrenal cortex in female rats after estradiol or calcium treatment

Administration of estradiol or calcium, or combined, represents the classical therapeutic approach in the treatment of some menopausal symptoms. We have studied the effects of estradiol dipropionate (EDP) and calcium glucoheptonate (Ca) on morphological and hormonal features of the adrenal gland in 14-month-old female Wistar rats. The animals were treated with EDP (0.625 mg/kg b.w.) or Ca (11.4 mg/kg b.w.) daily for two weeks, with control rats receiving vehicle alone by the same schedule. The cell volumes in the zona glomerulosa (ZG) and zona fasciculata (ZF) were 11.2% and 5.5% greater (P<0.05) and in the zona reticularis (ZR) 13.0% smaller (P<0.05) in the EDP group than in the control group. In the Ca group, cell volume in the ZG was increased by 5.6% (P<0.05), while cell volumes in the ZF and ZR were decreased by 26.0% and 14.7%, respectively (P<0.05), in comparison with control values. Serum aldosterone and corticosterone concentrations were higher in the EDP-treated (by 27.8% and 19.8%, respectively) and Ca-treated (by 80.0% and 24.1%, respectively) groups in comparison with the control group (P<0.05). These data suggest that EDP and Ca treatments have stimulatory effects on the ZG and ZF, but inhibitory effects on the ZR in middle-aged female rats.     

Novel regulatory mechanisms for generation of the soluble leptin receptor: implications for leptin action

Background

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.

Methodology/Principal Findings

To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding.

Conclusions

Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases.     

The effects of chronic SRIH-14 and octreotide administration on the pituitary-adrenal axis in adult male rats

The effects of chronic treatments with SRIH-14 and octreotide on pituitary corticotropes (ACTH cells) and on the adrenal cortex of male Wistar rats were examined. Adult males received two daily s.c. injections of 20 microg/100 g of body weight of either SRIH-14 or octreotide for 28 consecutive days. ACTH cells were studied using a peroxidase-antiperoxidase immunocytochemical procedure. Morpho-metry was used to evaluate the changes in cell and nuclear volumes (microm3) and volume densities (%) of ACTH-immunoreactive cells. The adrenal cortex was analyzed by histological and morphometric methods. A significant (p<0.05) decrease in body weight and in the absolute weights of the pituitary and adrenal glands was observed in both treated groups. Morphometric parameters of ACTH cells in both treated groups were not significantly (p>0.05) different than in control rats. The absolute volumes of the adrenal gland and adrenal cortex were significantly (p<0.05) decreased in both treated groups. The absolute and relative volumes of the zona glomerulosa (ZG), as well as the cellular and nuclear volumes of the ZG were significantly (p<0.05) decreased in the both treated groups. In rats treated with SRIH-14 and octreotide, the absolute and relative volumes of the zona fasciculata (ZF) and zona reticularis (ZR), as well as their stereological parameters, did not change significantly (p>0.05). The aldosterone levels in the SRIH-14 and ocreotide-treated groups were significantly (p<0.05) decreased - by 13% and 19%, respectively. The concentration of ACTH and corticosterone did not change significantly. Together, these findings show that SRIH-14 and octreotide administration affected the morphological characteristics of the adrenal ZG in a similar manner, and brought about a decrease in plasma aldosterone concentration. These treatments did not affect pituitary ACTH cells or adrenal ZF and ZR functioning.     

Detection of Ob-receptor in human adrenal neoplasms and effect of leptin on adrenal cell proliferation.

Leptin, a hormone mainly secreted from adipose tissue, communicates a metabolic signal to the adrenal gland. Ob-Receptor (Ob-R) expression was reported in rat, mice and human adrenal glands. This study intended to investigate possible differences in the Ob-R expression and distribution of Ob-R protein in human adrenal tumors as compared to normal adrenal tissue. Proliferative effects of leptin were analyzed in the human adrenocortical carcinoma cell line (NCI-H295). The full length Ob-R mRNA and the isoforms B219.1 and B219.3 could be demonstrated by RT-PCR in all adrenal tumors (n=8), the tumor cell line (NCI-H295) and normal tissue. In contrast the Ob-R isoform B219.2 was absent in the carcinoma cell line and in most of the adrenal tumors (n=5), whereas it was present in normal adrenals. The Ob-R protein could be demonstrated in benign and malignant adrenocortical tumors. Pheochromocytomas showed only a weak immunostaining with the human Ob-R antibody. Human leptin did not affect the proliferation or variability of adrenal tumor cells as demonstrated by [3H]-thymidine assay and WST-1 test. In conclusion, although functional leptin receptors are expressed in human adrenal tumors, leptin does not regulate tumor cell proliferation.     

Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats

The effect of chronic exposure to light of adult Wistar rats on growth and function of adrenal zona glomerulosa (ZG) and zona fasciculata (ZF) were examined. The females were exposed to continuous light of 600 lux for 95 days, starting on day 30 of age. The controls were kept under a 12:12 h light-dark cycle, at ambient temperature. The rats were sacrificed by decapitation and the left adrenal gland of each animal was dissected out and prepared for morphometric analyses. In animals exposed to chronic lighting, the absolute and relative volume of ZG were insignificantly increased by 5% (p>0.05) compared to controls. The volume of ZG cells and their nuclei were insignificantly changed by 1% (p>0.05) in comparison with corresponding controls. The absolute and relative volume of ZF were significantly increased (by 14 and 9%, respectively; p<0.05), as compared to controls. The volume of ZF cells and their nuclei were significantly increased (by 12 and 9%, respectively; p<0.05). Serum concentration of corticosterone was also significantly (p<0.05) increased by 13% in light-exposed group in comparison with control rats. These findings suggest that continuous exposure of female rats to constant light increased growth and secretory activity of ZF cells.     

Structure-activity studies with ACTH/alpha-MSH fragments on corticosteroid secretion of isolated zona glomerulosa and fasciculata cells

The steroidogenic action of ACTH/alpha-MSH fragments was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(4-7), ACTH-(6-10), ACTH-(4-10) and ACTH-(11-13) stimulated corticosterone production of the zona fasciculata and aldosterone production of the zona glomerulosa cells. ACTH-(7-10) was ineffective. ACTH-(4-7) appeared to be the most potent peptide of the tested fragments. None of the fragments affected the steroidogenic action of ACTH-(1-39). It is suggested that similar to the melanotropic effect of alpha-MSH two 'message' sequences for adrenocortical stimulation exist in the alpha-MSH part of the ACTH molecule.     

Mineralocorticoids in congenital adrenal hyperplasia

While hypertension is observed in only two of the three major subtypes of congenital adrenal hyperplasia (CAH), 11β- and 17-hydroxylase deficiencies, deoxycorticosterone ( (DOC) production is increased in all. The elevated zona fasciculata (ZF) DOC produces mineralocorticoid hypertension with suppressed renin and reduced potassium concentrations. The DOC levels in 21-hydroxylase deficiency are in part produced by renin stimulation of the Zona glomerulosa (ZG) along with aldosterone. Assessment of the mineralocorticoid hormones of the ZF and ZF (17-deoxy steroids) provides additional unique characteristics of each subtype. Dissociation of DOC from cortisol is not unique to CAH. This dissociation is seen in other disorders and contrived conditions. There is a strong suggestion of a non-ACTH regulator of 17-deoxy steroids (DOC) that may contribute significantly to DOC production in general and effect DOC levels in CAH.     

Sex differences in adrenocortical structure and function

Summary Adrenal glands of adult male hamsters are larger and secrete more cortisol than those of females. Stereology was therefore used to study zonal and cellular aspects of development of the adrenal cortex of male and female hamsters. Adrenal glands were studied at weekly intervals from day 21 to day 77 of postnatal ontogenesis. Within this period, body weight did not differ significantly between the sexes. During development, absolute and relative adrenal weights were higher in males; their zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR) become markedly larger than those in females. No marked changes in the volume of individual ZG cells occurred although ZF cells and ZR cells become larger in male than female animals. The total number of adrenocortical cells increased within the period studied, a greater increase being observed in ZG and ZF in males. No distinct sex difference was observed in the number of ZR cells throughout development. From day 56 of postnatal life the adrenal cortex of male hamster contained more parenchymal cells than the female gland. These results thus indicate that sex differences in hamster adrenal cortex depend upon changes in number and size of parenchymal cells.Supported in part by a grant from the Committee of Zoology, Polish Academy of Sciences     

瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达的影响

目的研究瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体(Ob-R)mRNA表达的影响。方法采用六孔板培养皿培养鼠平滑肌细胞,5个孔作为一个浓度组,通过半定量的RT-PCR方法,分别观察了不同浓度的大鼠瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达水平的影响。结果通过半定量RT-PCR,我们检测了短型瘦素受体(Ob-Ra)和长型瘦素受体(Ob-Rb)的mRNA表达水平,瘦素受体(Ob-R)mRNA表达水平随着瘦素及IL-6浓度增加而上升,随着胰岛素的浓度增加而下降。结论瘦素及IL-6可在体外上调平滑肌细胞瘦素受体的表达,胰岛素可下调平滑肌细胞瘦素受体的表达。这三个因子参与了瘦素受体的表达调控,对我们了解瘦素抵抗的发生机制有重要意义。