Anna Hochol

Effects of leptin and leptin fragments on steroid secretion of freshly dispersed rat adrenocortical cells.

The biological actions of leptin on target tissues are mediated via several isoforms of receptors (Ob-Rs), which may differently interact with native leptin and its fragments. Based on the presence in the rat adrenals of at least two Ob-R isoforms and the conflicting findings on the effect of leptin on adrenocortical secretion, we investigated the effects of the native leptin and several leptin fragments (10(-8) and 10(-6)M) on aldosterone and corticosterone secretion from freshly dispersed rat zona glomerulosa (ZG) and zona fasciculata-reticularis (ZF/R) cells. Reverse transcription (RT)-polymerase chain reaction (PCR) showed the expression of Ob-Ra and Ob-Rb mRNAs in both ZG and ZF/R cells. Native murine leptin (1-147) enhanced aldosterone and corticosterone secretion from dispersed ZG and ZF/R cells, and similar effects were elicited by murine leptin fragment 116-130, and human leptin fragments 138-167, 150-167 and [Tyr] 26-39. Human leptin fragment 93-105 was ineffective, while fragment 22-56 decreased corticosterone output without affecting aldosterone secretion. Taken together, our findings indicate that in rat adrenocortical cells leptin and leptin fragments may differently interact with Ob-Rs or interact with different Ob-R isoforms. Moreover, they suggest that (1) the direct adrenocortical secretagogue effect of leptin mainly depends on the C-terminal sequence 116-166; and (2) the N-terminal sequence is not needed for leptin to activate Ob-Rs positively coupled to steroidogenesis, but is possibly responsible for a direct inhibitory effect on glucocorticoid secretion.

Effects of recombinant murine leptin[1-147] and leptin fragment 116-130 on steroid secretion and proliferative activity of the regenerating rat adrenal cortex.

Leptin, the product of the oh gene, is a hormone mainly secreted by the adipose tissue, which acts through specific receptors (Ob-R) widely distributed in the body tissues Ob-Rs are present in the mammalian hypothalamo-pituitary-adrenal axis, and evidence indicates that leptin regulates adrenocortical secretion Moreover, leptin is known to act as a growth promoting factor in some tissues, including the endocrine ovary. We have investigated the effects of three subcutaneous injections of 2 nmol/100 g of native murine leptin[1–147] and of its biologically active fragment 116–130 on the secretory and proliferative activity of the regenerating rat adrenal cortex Leptin[1–147] increased plasma aldosterone concentration at day 8 and plasma corticosterone concentration (PBC) at day 5 of regeneration, without affecting mitotic index. In contrast, leptin[116–130] lowered PBC and mitotic index at both times of adrenal regeneration. In light of the fact that adrenal regeneration is at least in part dependent on the pituitary ACTH, we conclude that (i) native leptin moderately stimulates steroid secretion, acting directly on the adrenal cortex, through signaling mechanisms other than those involved in the ACTH action, (ii) native leptin is unable to enhance the proliferative activity of regenerating adrenals, which conceivably is maximally stimulated by ACTH; (iii) leptin[1–147] and leptin[116–130] differently interact with Ob-Rs or interact with different receptors; and (iv) leptin[116–130] inhibits the signaling pathways mediating both the secretagogue effect of native leptin and the proliferogenic effect of ACTH.

Leptin Prolonged Administration Inhibits the Growth and Glucocorticoid Secretion of Rat Adrenal Cortex

Leptin is an adipose-tissue secreted hormone, that acts to decrease caloric intake and to increase energy expenditure. Some of the leptin effects on the energy balance are known to be mediated by the hypothalamo-pituitary-adrenal (HPA) axis, but the role of this cytokine in the regulation of the growth and steroidogenic capacity of adrenal cortex is still controversial. Therefore, the present study was designed to explore the long-term effects of native leptin[1–147] and its biologically active fragment leptin[116–130] (6 daily subcutaneous injection of 20 nmol/kg) on the rat HPA axis Leptin[1–147] and leptin[116–130] caused a significant adrenal atrophy, which was mainly due to the decrease in the volume of zona fasciculata (ZF) and in the number of its parenchymal cells. Both leptins provoked a marked drop in the plasma concentrations of ACTH and corticosterone, the main hormone produced by ZF cells. The effects of leptin[116–130] were more intense than those of leptin[1–147]. Leptin[1–147], but not its fragment, evoked a clear-cut rise in the plasma concentration of aldosterone. Collectively, these findings indicate that prolonged leptin administration, by inhibiting pituitary ACTH release, exerts a potent suppressive action on the growth and glucocorticoid secretory capacity of the adrenal cortex in the rat. The mechanism(s) underlying the aldosterone secretagogue action of native leptin remain(s) to be investigated.

Sex-related gene expression profiles in the adrenal cortex in the mature rat: microarray analysis with emphasis on genes involved in steroidogenesis.

Notable sex-related differences exist in mammalian adrenal cortex structure and function. In adult rats, the adrenal weight and the average volume of zona fasciculata cells of females are larger and secrete greater amounts of corticosterone than those of males. The molecular bases of these sex-related differences are poorly understood. In this study, to explore the molecular background of these differences, we defined zone- and sex-specific transcripts in adult male and female (estrous cycle phase) rats. Twelve-week-old rats of both genders were used and samples were taken from the zona glomerulosa (ZG) and zona fasciculata/reticularis (ZF/R) zones. Transcriptome identification was carried out using the Affymetrix® Rat Gene 1.1 ST Array. The microarray data were compared by fold change with significance according to moderated t-statistics. Subsequently, we performed functional annotation clustering using the Gene Ontology (GO) and Database for Annotation, Visualization and Integrated Discovery (DAVID). In the first step, we explored differentially expressed transcripts in the adrenal ZG and ZF/R. The number of differentially expressed transcripts was notably higher in the female than in the male rats (702 vs. 571). The differentially expressed genes which were significantly enriched included genes involved in steroid hormone metabolism, and their expression levels in the ZF/R of adult female rats were significantly higher compared with those in the male rats. In the female ZF/R, when compared with that of the males, prevailing numbers of genes linked to cell fraction, oxidation/reduction processes, response to nutrients and to extracellular stimuli or steroid hormone stimuli were downregulated. The microarray data for key genes involved directly in steroidogenesis were confirmed by qPCR. Thus, when compared with that of the males, in the female ZF/R, higher expression levels of genes involved directly in steroid hormone synthesis were accompanied by lower expression levels of genes regulating basal cell functions.

Evidence suggesting that ghrelin O-acyl transferase inhibitor acts at the hypothalamus to inhibit hypothalamo-pituitary-adrenocortical axis function in the rat.

Production of n-octanoyl-modified ghrelin (GHREL), an active form of the peptide requires prohormone processing protease and GHREL O-acyltransferase (GOAT), as well as n-octanoic acid. Recently a selective GOAT antagonist (GO-CoA-Tat) was invented and this tool was used to study the possible role of endogenous GHREL in regulating HPA axis function in the rat. Administration of GOAT inhibitor (GOATi) resulted in a notable decrease in plasma ACTH, aldosterone and corticosterone concentrations at min 60 of experiment. Octanoic acid (OA) administration had no effect on levels of studied hormones. Plasma levels of unacylated and acylated GHREL remained unchanged for 60min after either GOATi or OA administration. Under experimental conditions applied, no significant changes were observed in the levels of GOAT mRNA in hypothalamus, pituitary, adrenal and stomach fundus. After GOATi injection hypothalamic CRH mRNA levels were elevated at 30 min and pituitary POMC mRNA levels at 60 min. Both GOATi and OA lowered basal, but not K(+)-stimulated CRH release by hypothalamic explants and had no effect on basal or CRH-stimulated ACTH release by pituitary slices. Neither GOATi nor OA affected corticosterone secretion by freshly isolated or cultured rat adrenocortical cells. Thus, results of our study suggest that in the rat endogenous GHREL exerts tonic stimulating effect on hypothalamic CRH release. This effect could be demonstrated by administering rats with selected inhibitor of ghrelin O-acyltransferase, the enzyme responsible for GHREL acylation, a process which is absolutely required for both GHSR-1a binding and its central endocrine activities.

Effects of leptin on the response of rat pituitary-adrenocortical axis to ether and cold stresses.

Leptin is a hormone mainly secreted by the adipose tissue, which acts through specific receptors widely distributed in the body tissues, including hypothalamo-pituitary-adrenal axis. We have investigated the effects of a subcutaneous bolus injection of 5 nmol/kg leptin on the pituitary-adrenocortical function in both normal and ether- or cold-stressed rats. Blood concentrations of ACTH, aldosterone and corticosterone were measured by specific RIA 2 or 4 h after the leptin injection. Leptin administration to normal rats resulted in significant rises in the blood levels of ACTH, aldosterone and corticosterone at 2 h, but not at 4 h. Ether and cold stresses markedly increased hormonal blood concentrations at both 2 and 4 h Leptin magnified ACTH response to ether stress at 2 h, but depressed it at 4 h, and enhanced aldosterone response at 2 h, without affecting corticosterone response Leptin increased ACTH response to cold stress at both 2 and 4 h, without altering aldosterone and corticosterone responses. In light of these findings, we conclude that: (i) leptin evokes a middle transient activation of the pituitary-adrenocortical axis of rats under basal conditions; (ii) leptin inhibits the ACTH response to ether stress, but magnifies that to cold stress, and (iii) the leptin-evoked changes in the blood level of ACTH are not paralleled by significant modifications in the secretory activity of the adrenal cortex, which probably undergoes a maximal stimulation under stressful conditions.

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.

PROLONGED CEREBELLIN ADMINISTRATION INHIBITS THE GROWTH, BUT ENHANCES STEROIDOGENIC CAPACITY OF RAT ADRENAL CORTEX

Cerebellin is a 16-amino acid peptide, that has been previously found to acutely stimulate steroid secretion from rat adrenal cortex in vivo and in vitro. We have investigated the effects of a prolonged cerebellin treatment (daily injections of 15 nmoles/kg for 6 consecutive days) on the growth and secretion of rat adrenal cortex. Cerebellin lowered adrenal weight, and morphometry showed that this was due to the decrease in the volume of each adrenocortical zone exclusively ensuing from the reduction in the number of its parenchymal cells. Cerebellin did not alter plasma concentration of ACTH, but it raised the levels of circulating aldosterone and corticosterone. The conclusion is drawn that cerebellin chronic administration evokes a marked hypoplastic atrophy of rat adrenocortical cells, that is coupled with an enhanced ACTH-independent steroidogenic capacity of the remaining parenchymal cells.

Effects of galanin on the secretion and proliferative activity of the immature and regenerating adrenal glands of rats.

The effects of galanin and the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15) on the immature and regenerating rat adrenal glands have been investigated in vivo. Adult female rats with adrenal regeneration and their offpring (20-day-old) were given three subcutaneous injections (28, 16, and 4 h before being killed) of 2 nmol/100 g galanin and/or galanin-A, and 0.1 mg/100 g vincristin 3 h before being killed. Plasma corticosterone concentration was measured by radioimmunoassay, and the mitotic index ( per thousand of metaphase-arrested cells) was evaluated. In immature rats, galanin increased plasma corticosterone concentration, without affecting mitotic index; the secretagogue effect was reversed by galanin-A, which alone was ineffective. In rats with regenerating adrenal, galanin-A increased both blood level of corticosterone and mitotic index; galanin was ineffective, but blocked the effects of galanin-A. These findings allowed us to draw the following conclusions: 1) galanin exerts a moderate glucocorticoid secretagogue action on immature rat adrenals, but endogenous galanin does not play a major physiological role in the functional control of the gland; and 2) endogenous galanin exerts a maximal tonic inhibitory control on both glucocorticoid secretion and proliferative activity of regenerating rat adrenals, whose physiological relevance remains to be investigated.

Arginine–vasopressin and corticotropin-releasing hormone are sequentially involved in the endothelin-1-induced acute stimulation of rat pituitary-adrenocortical axis

The acute effect of endothelin-1 (ET-1) on the hypothalamo-pituitary-adrenal (HPA) axis has been investigated in the rat. The plasma concentrations of arginine-vasopressin (AVP), ACTH, aldosterone and corticosterone have been measured by RIA 30 and 60 min after ET-1 administration. ET-1 (2.0 nmol kg(-1) raised AVP plasma concentration at both 30 and 60 min. ET-1 did not alter the ACTH plasma level at 30 min, but markedly increased it at 60 min. ACTH response was unaffected by the simultaneous administration of AVP-receptor antagonists (AVP-As) Des-Gly-[Phaa1,D-Tyr(Et)2,Lys6,Arg8]-vasopressin or [Deamino-Pen1,Tyr(Me)2,Arg8]-vasopressin (20 nmol kg(-1), but abolished by the corticotropin-releasing hormone (CRH)-receptor antagonist alpha-helical-CRH(9-41) (alpha-CRH, 10 nmol kg(-1). ET-1 evoked significant rises in the blood levels of aldosterone and corticosterone at both 30 and 60 min. AVP-As abrogated the response at 30 min, while alpha-CRH was ineffective. Both AVP-As and alpha-CRH partially reversed adrenocortical secretory response at 60 min. Collectively, these findings confirm that systemically administered ET-1 stimulates rat HPA axis, and provide evidence that the mechanism underlying this effect may involve the sequential activation of AVP and CRH release.