Increase In Corticosterone Production Research Articles

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis.

Cholesterol is the precursor of all steroids, but how cholesterol flux is controlled in steroidogenic tissues is poorly understood. The cholesterol exporter ABCG1 is an essential component of the reverse cholesterol pathway and its global inactivation results in neutral lipid redistribution to tissue macrophages. The function of ABCG1 in steroidogenic tissues, however, has not been explored. To model this, we inactivated Abcg1 in the mouse adrenal cortex, which led to an adrenal-specific increase in transcripts involved in cholesterol uptake and de novo synthesis. Abcg1 inactivation did not affect adrenal cholesterol content, zonation, or serum lipid profile. Instead, we observed a moderate increase in corticosterone production that was not recapitulated by the inactivation of the functionally similar cholesterol exporter Abca1. Altogether, our data imply that Abcg1 controls cholesterol uptake and biosynthesis and regulates glucocorticoid production in the adrenal cortex, introducing the possibility that ABCG1 variants may account for physiological or subclinical variation in stress response.

Effects of mastication on antibody production under fasting conditions in mice.

Chewing is beneficial not only for digestion and absorption of food, but also for various physiological functions, such as cognition and immunity. In this study, the effect of chewing on hormonal changes and the immune response was investigated under fasting conditions in mice. We investigated leptin and corticosterone levels, which are hormones with well-known associations with immune response and large changes during fasting. To study of effects of chewing under fasting conditions, one group of mice was provided with wooden sticks to stimulate chewing, one group was supplemented with 30% glucose solution, and one group received both treatments. We examined changes in serum leptin and corticosterone levels after 1 and 2 d of fasting. Antibody production was measured 2 weeks after subcutaneous immunization with bovine serum albumin on the last day of fasting. Under fasting conditions, serum leptin levels decreased and serum corticosterone levels increased. Supplementation with 30% glucose solution during fasting increased leptin levels above normal, but had little effect on corticosterone levels. In contrast, chewing stimulation inhibited the increase in corticosterone production, but did not affect the decrease in leptin levels. Antibody production significantly increased under separate and combined treatments. Taken together, our results showed that chewing stimulation during fasting inhibited the increase in corticosterone production and improved antibody production after immunization.

Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue

Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 β-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 β-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum.Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism.Male Sprague–Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 β-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 β-HSD1, and NADPH/NADP+ ratio.Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 β-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions.

Intensive vehicle traffic impacts morphology and endocrine stress response in a threatened amphibian

AbstractAmphibians are considered to be the most threatened group of vertebrates. Among the multiple factors involved in their decline, habitat loss and alteration as a result of human activities is a major threat. At the individual level the effects of habitat alteration are potentially multiple, including a range of morphological and physiological responses. Analysing and understanding these responses is therefore a critical challenge for amphibian conservation. We examined the influence of intensive vehicle traffic (motorbikes and trucks on unpaved pathways) on the body size and condition and on the production of glucocorticoids (i.e. corticosterone) in the yellow-bellied toad Bombina variegata. In particular, we tested the hypothesis that intensive vehicle traffic has a negative influence on body size and body condition, and postulated that it also increases corticosterone production. Using morphometric data and saliva samples collected from four populations in France, we found that intensive vehicle traffic is associated with a decrease in body size and body condition in both males and females. Furthermore, our analysis revealed that corticosterone production was lower in both sexes in populations experiencing intensive vehicle traffic. We suggest that measures should be applied to reduce vehicle traffic intensity on unpaved pathways during toad breeding activity. This is critical for B. variegata, for which man-made ruts and residual puddles could mitigate the loss of natural habitats.

Low-dose Bisphenol A Activates Cyp11a1 Gene Expression and Corticosterone Secretion in Adrenal Gland via the JNK Signaling Pathway.

Certain commonly used compounds that interfere with the functions of the endocrine system are classified as endocrine-disrupting chemicals (EDCs). Bisphenol A (BPA) is an EDC that is widely used in food containers. BPA levels in human sera are commonly observed to be approximately 1-100 nM. Compared with the effects of BPA on the gonads, its effects on the adrenal gland are poorly understood. To investigate the influence of BPA on steroidogenesis, we examined the activity of the steroidogenic gene Cyp11a1 and its regulatory pathways in mouse Y1 adrenal cortex cells. Treatment with BPA at < 100 µM did not cause cell death. However, increased promoter activity and protein expression of Cyp11a1 were induced by low doses of BPA (10-1000 nM). Moreover, BPA induced c-Jun phosphorylation, and a specific inhibitor of c-Jun N-terminal kinase (JNK) significantly suppressed BPA-induced steroidogenesis. Thus, treatment of adrenal cells with low doses of BPA activated Cyp11a1 and increased corticosterone production through the JNK/c-Jun signaling pathway. Identical results were observed in rats after BPA injection. The abnormal induction of hormone synthesis by BPA in the adrenal gland might be linked to human metabolic defects and neuropsychiatric disorders.

Production of corticosteroid hormones in vitro by adrenals in rats with streptozotocin-induced diabetes.

We studied baseline and ACTH-stimulated in vitro production of corticosteroids by rat adrenals. Production of the basic corticosteroids pregnenolone (early precursor in corticosteroid synthesis), progesterone (intermediate precursor in synthesis of gluco- and mineralocorticoid hormones), and corticosterone (major glucocorticoid hormone in rodents) in animals with streptozotocin-induced diabetes was enhanced by 1.8-2.0 times in comparison with the control animals. Addition of ACTH to the incubation medium stimulated pregnenolone production by the adrenals equally in the control and experimental (diabetic) groups, while the increase in corticosterone production was less pronounced in the experimental group. Stimulation of corticosterone production in response to ACTH after saturation of the incubation medium with pregnenolone was also less pronounced in diabetic rats.

Transgenic overexpression of hexose-6-phosphate dehydrogenase in adipose tissue causes local glucocorticoid amplification and lipolysis in male mice

The prereceptor activation of glucocorticoid production in adipose tissue by NADPH-dependent 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has emerged as a potential mechanism in the pathogenesis of visceral obesity and metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) is an endoplasmic reticulum lumen-resident enzyme that generates cofactor NADPH and thus mediates 11β-HSD1 activity. To determine the role of adipose H6PDH in the prereceptor modulation of 11β-HSD1 and metabolic phenotypes, we generated a transgenic (Tg) mouse model overexpressing H6PDH under the control of the enhancer-promoter region of the adipocyte fatty acid-binding protein (aP2) gene (aP2/H6PDH Tg mice). Transgenic aP2/H6PDH mice exhibited relatively high expression of H6PDH and elevated corticosterone production with induction of 11β-HSD1 activity in adipose tissue. This increase in corticosterone production in aP2-H6PDH Tg mice resulted in mild abdominal fat accumulation with induction of C/EBP mRNA expression and slight weight gain. Transgenic aP2/H6PDH mice also exhibited fasting hyperglycemia and glucose intolerance with insulin resistance. In addition, the aP2/H6PDH Tg mice have elevated circulating free fatty acid levels with a concomitant increased adipose lipolytic action associated with elevated HSL mRNA and Ser(660) HSL phosphorylation within abdominal fat. These results suggest that increased H6PDH expression specifically in adipose tissue is sufficient to cause intra-adipose glucocorticoid production and adverse metabolic phenotypes. These findings suggest that the aP2/H6PDH Tg mice may provide a favorable model for studying the potential impact of H6PDH in the pathogenesis of human metabolic syndrome.

Genetic variation of the cutaneous HPA axis: An analysis of UVB-induced differential responses

Mammalian skin incorporates a local equivalent of the hypothalamic–pituitary–adrenal (HPA) axis that is critical in coordinating homeostatic responses against external noxious stimuli. Ultraviolet radiation B (UVB) is a skin-specific stressor that can activate this cutaneous HPA axis. Since C57BL/6 (B6) and DBA/2J (D2) strains of mice have different predispositions to sensorineural pathway activation, we quantified expression of HPA axis components at the gene and protein levels in skin incubated ex vivo after UVB or sham irradiation. Urocortin mRNA was up-regulated after all doses of UVB with a maximum level at 50mJ/cm2 after 12h for D2 and at 200mJ/cm2 after 24h for B6. Proopiomelanocortin mRNA was enhanced after 6h with the peak after 12h and at 200mJ/cm2 for both genotypes of mice. ACTH levels in tissue and media increased after 24h in B6 but not in D2. UVB stimulated β-endorphin expression was higher in D2 than in B6. Melanocortin receptor 2 mRNA was stimulated by UVB in a dose-dependent manner, with a peak at 200mJ/cm2 after 12h for both strains. The expression of Cyp11a1 mRNA — a key mitochondrial P450 enzyme in steroidogenesis, was stimulated at all doses of UVB irradiation, with the most pronounced effect after 12–24h. UVB radiation caused, independently of genotype, a dose-dependent increase in corticosterone production in the skin, mainly after 24h of histoculture. Thus, basal and UVB stimulated expression of the cutaneous HPA axis differs as a function of genotype: D2 responds to UVB earlier and with higher amplitude than B6, while B6 shows prolonged (up to 48h) stress response to a noxious stimulus such as UVB.

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function

Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro. Normotensive subjects (n = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured. Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type. The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.

Adenosine-stimulated adrenal steroidogenesis involves the adenosine A 2A and A 2B receptors and the Janus kinase 2–mitogen-activated protein kinase kinase–extracellular signal-regulated kinase signaling pathway

Adenosine promotes adrenal steroidogenesis in vitro and in vivo. However, the underlying signaling mechanisms of this event and the function of the adenosine receptor subtypes in adrenal cells remain to be elucidated. Expression of A 1, A 2A, A 2B, and A 3 adenosine receptor mRNA in rat adrenal cells was shown by reverse transcription-polymerase chain reaction. Adenosine increased corticosterone production in a time- and dose-dependent manner, and this adenosine effect was mediated by the A 2 adenosine receptors, since the antagonists specific for the A 2A and A 2B adenosine receptors, and specific silencing the A 2A adenosine receptor expression with small interfering RNA significantly blocked the adenosine-induced steroidogenesis. Using pharmacological approaches, we further demonstrated that Janus kinase 2 was the downstream molecule next to the A 2A and A 2B adenosine receptors. Inhibition of Janus kinase 2 prevented the adenosine-induced steroidogenesis and phosphorylation of mitogen-activated protein kinase kinase 1/2 and extracellular signal-regulated kinase 1/2, demonstrating that Janus kinase 2 was the upstream effector of the mitogen-activated protein kinase kinase pathway. Pretreatment with A 2 adenosine receptor, Janus kinase 2, or mitogen-activated protein kinase kinase inhibitors significantly decreased the adenosine-induced phosphorylation of 3′,5′-cyclic adenosine monophosphate responsive element binding protein. In conclusion, these data show that adenosine-stimulated steroidogenesis is mediated via the A 2A and A 2B adenosine receptors, activation of which triggers the Janus kinase 2–mitogen-activated protein kinase kinase–extracellular signal-regulated kinase cascade and 3′,5′-cyclic adenosine monophosphate responsive element binding protein phosphorylation. Based on its stimulatory effect on glucocorticoid production, adenosine is a potential candidate as anti-inflammatory agent.

Anthrax Edema Toxin Sensitizes DBA/2J Mice to Lethal Toxin

Anthrax toxin is made up of three separate protein components: the receptor-binding protective antigen (PA), the adenylyl cyclase edema factor (EF), and the metalloproteinase lethal factor (LF). EF and PA constitute edema toxin (ET), which causes edema when injected subcutaneously. At higher doses, ET causes severe pathologies and death in BALB/cJ mice (A. M. Firoved et al., Am. J. Pathol. 167:1309-1320, 2005). A striking effect of ET at lethal doses is adrenal necrosis. Here we show that low doses of ET (10 microg) that produce no overt signs of illness in mice still cause substantial adrenal lesions. These lesions are not associated with reduced corticosterone production; instead, ET-treated mice have increased corticosterone production. Because the resistance of mice to the other component of anthrax toxin, lethal toxin (LT; LF plus PA), has been shown to be overcome by the perturbation of the endocrine system, we hypothesized that sublethal doses of ET might sensitize LT-resistant DBA/2J mice to LT-mediated lethality. We report that a low dose of ET (5 microg) is sufficient to sensitize DBA/2J mice when given concurrently with LT. Higher doses of ET (e.g., 15 microg) given to male and female DBA/2J mice 18 h prior to LT challenge also sensitize them to LT. This study using highly purified ET and LT demonstrates how the components of anthrax toxin can work together to increase lethality.

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 +/- 2%) and dexamethasone-treated (43 +/- 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.

Early life host–bacteria relations and development: Long-term individual differences in neuroimmune function following neonatal endotoxin challenge

Neonatal animals have a proportionately greater risk, relative to the adult animal, of developing a bacterial infection. Research has revealed that such infections can influence biological processes long after the actual infection has been resolved. Indeed, studies examining the long-term alterations induced by early-life infection, simulated using endotoxin, have indicated that some aspects of the systemic inflammatory response in the adult animal are susceptible to modification. Available evidence suggests that altered inflammatory activities observed in the neonatally endotoxin challenged adult may be the result of potentiated hypothalamic pituitary adrenal (HPA) activity, specifically increased corticosterone production. Few studies, however, have examined whether altered corticosterone production is actually associated with changes in systemic inflammation in the neonatally endotoxin challenged adult animal. The aim, therefore, of the current study was to simultaneously examine the relationship between altered inflammatory activities and corticosterone production in the neonatally endotoxin challenged adult rat. Our findings demonstrate no significant differences exist between adults neonatally treated with saline or endotoxin in terms of their production of corticosterone following endotoxin challenge. While not appearing to influence the production of corticosterone neonatal endotoxin challenge did result in a marked attenuation in the adult's febrile response following endotoxin challenge. Interestingly, circulating levels of IL-1β and TNF-α were found to be equivalent in neonatal treatment groups following endotoxin administration in adulthood, indicating that the reduction in fever was unlikely to be the result of altered pro-inflammatory cytokine production. Further, no differences were found between neonatal treatment groups in net food consumption, water consumption or weight loss following endotoxin challenge in adulthood. Collectively, these findings demonstrate that neonatal endotoxin challenge does not affect a blanket down regulation of inflammatory processes but rather appears to induce highly specific alterations in the adult male Fischer-344 rat.

Effects of beacon on the rat pituitary-adrenocortical axis response to stress

Beacon is a peptide expressed in the rat hypothalamus and adrenal cortex, which is involved in the central regulation of feeding and inhibits basal and agonist-stimulated glucocorticoid secretion from adrenocortical cells. In vivo studies on beacon have not yet been carried out, and therefore we investigated the effects of a subcutaneous (sc) injection of beacon on the response of rat hypothalamo-pituitary-adrenal axis to stress. Handling and sc injection per se elicited a moderate increase in the plasma concentrations of ACTH and corticosterone, which was counteracted by beacon. Similarly, beacon dampened ACTH and corticosterone responses to ether stress. In contrast, beacon did not affect ACTH response to cold stress, although it was able to induce a moderate lowering in the corticosterone response. Taken together, these findings allow us to draw the following conclusions: i) beacon inhibits handling/injection- and ether stress-activated, but not cold stress-activated, neural mechanism(s) responsible for stimulation of ACTH secretion and the ensuing increase in corticosterone production; and ii) the beacon-induced dampening in corticosterone response to stress also involves a direct inhibitory effect on the adrenal-cortex secretory activity. The physiological relevance of beacon as endogenous anti-stress agent remains to be evaluated.

Corticotropin-Releasing Hormone Contributes to the Peripheral Inflammatory Response in Experimental Autoimmune Encephalomyelitis

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh(-/-) mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-gamma production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IkappaBalpha phosphorylation induced by TCR cross-linking was decreased in crh(-/-) T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.

Cecal ligation and perforation is associated with adrenal insufficiency in rats

There is renewed interest in the use of exogenous steroids to treat refractory hypotension in sepsis. Despite the growing body of literature demonstrating the efficacy of steroid therapy in this clinical setting, little is known regarding the mechanism responsible for the observed hypocortisolism. Scant data suggest that TNF inhibits the synthesis and secretion of cortisol by the adrenals. To investigate the pathophysiology of sepsis-induced adrenal insufficiency, we examined the adrenal response to an ACTH stimulation test in a rat model of gram-negative sepsis. Cecal ligation and perforation (CLP) was performed on male Sprague-Dawley rats. Four hours following CLP, adrenal glands were removed, sectioned, and placed in incubation medium. ACTH stimulation tests were performed by the addition of cortrosyn and corticosterone levels measured at 0, 15, 30, and 60 min via radio-immunoassay. The results from CLP animals were compared to those of sham-operated, control rats. Cortrosyn stimulation of adrenal glands collected from control rats resulted in a 4- and 7-fold increase in corticosterone production over baseline at 30 and 60 min, respectively. However, adrenal glands collected from animals following CLP failed to respond to cortrosyn stimulation (Fig. 1) In conclusion, we have shown that cecal ligation and perforation is associated with a significant inhibition of the adrenocortical response to ACTH making this an excellent model to examine the pathophysiology of sepsis-induced adrenal insufficiency.

ACTH and PRL Sensitivity of Highly Differentiated Cell Lines Obtained by Adrenocortical Targeted Oncogenesis

We established cell lines from adrenal tumors of transgenic mice harboring the large T‐antigen of simian virus 40 under the control of the adrenocortical specific promoter of the scavenger aldose reductase‐like akr1b7 gene. Mass spectrometry analyses of serum‐supplemented or serum‐free culture media showed that ATC1 line secreted only corticosterone. These cells, propagated over 25 passages, were characterized with regard to ACTH and PRL responsiveness, as measured by increased corticosterone production, induction of genes involved in the different steps of steroidogenesis (cholesterol delivery, steroid biosynthesis and detoxification of by‐products) and expression of transcriptional regulators (SF‐1 and dax1). Corticosterone secretion (RIA) in serum‐free medium was stimulated over 12‐fold after 6 h treatment with either 10− 9M ACTH or PRL and both hormones seemed equivalent in promoting this secretion (149 ± 14 ng and 145 ± 18 ng/106 cells/6 h, respectively). As expected, Northern blots indicate that ATC1 cells expressed mRNAs for the enzymes of corticosterone metabolism CYP11B1 and CYP21A, as well as those for the proteins SIK, SRB1, StAR, CYP11A1, and AKR1B7. Interestingly, these cells have maintained not only the expression of SF‐1 but also that of dax1. No expression of the zona glomeruloza‐specific cyp11b2 gene was detected. With the exception of cyp21a and mc2r genes which were constitutively expressed, most of the genes above mentioned were induced in a time‐ and dose‐dependent fashion in response to ACTH or PRL while dax1 was repressed. Importantly, hormone‐mediated repression of dax1 gene expression was also observed in vivo in mice adrenals. Altogether these data demonstrate that ATC1 line provided an unique model of well differentiated zona fasciculata immortalized cells suitable for the dissection of molecular events leading to ACTH and PRL regulation of adrenal functions.

An oxidized metabolite of linoleic acid stimulates corticosterone production by rat adrenal cells.

Oxidized derivatives of linoleic acid have the potential to alter steroidogenesis. One such derivative is 12,13-epoxy-9- keto-10-(trans)-octadecenoic acid (EKODE). To evaluate the effect of EKODE on corticosterone production, dispersed rat zona fasciculata/reticularis (subcapsular) cells were incubated for 2 h with EKODE alone or together with rat ACTH (0, 0.2, or 2.0 ng/ml). In the absence of ACTH, EKODE (26 microM) increased corticosterone production from 5.3 +/- 2.3 to 14.7 +/- 5.0 ng. 10(6) cells. h(-1). The stimulatory effect of ACTH was increased threefold in the presence of EKODE (26.0 microM). Cholesterol transport/P-450scc activity was assessed by measuring basal and cAMP-stimulated pregnenolone production in the presence of cyanoketone (1.1 microM). EKODE (13.1 and 26.0 microM) significantly increased basal and cAMP-stimulated (0.1 mM) pregnenolone production. In contrast, EKODE decreased the effect of 1.0 mM cAMP. EKODE had no effect on early or late-pathway activity in isolated mitochondria. We conclude that EKODE stimulates corticosterone biosynthesis and amplifies the effect of ACTH. Increased levels of fatty acid metabolites may be involved in the increased glucocorticoid production observed in obese humans.

Consequences of elevated luteinizing hormone on diverse physiological systems: use of the LHbetaCTP transgenic mouse as a model of ovarian hyperstimulation-induced pathophysiology.

Chronically elevated luteinizing hormone (LH) induces significant pathology in the LHbetaCTP transgenic mouse model, which uses the bovine gonadotropin alpha (alpha)-subunit promoter to direct transgene expression specifically to gonadotropes in the anterior pituitary. Previously, it was shown that female LHbetaCTP mice are infertile due to anovulation, develop granulosa cell tumors, and undergo precocious puberty from elevated LH and steroid hormones that fail to completely repress the alpha-subunit promoter. This chapter will discuss recent studies that further elucidate the impact of chronically elevated LH on diverse physiological systems. Granulosa cell tumors induced by elevated LH are strain dependent and prevented when transgenics are treated with human chorionic gonadotropin (hCG) surges. A granulosa cell tumor-associated transcriptome is generated, revealing several possible gene candidates for ovarian granulosa cell tumorigenesis. Primordial follicles in LHbetaCTP transgenics become depleted and oocytes exhibit increased rates of meiotic segregation defects, although meiotic competency is acquired normally. Anovulation can be rescued in transgenics by superovulation, though pregnancy fails at midgestation due to maternal factors. Uterine receptivity defects prevent implantation of normal embryos following induction of pseuodpregnancy. Transgenics develop Cushing-like adrenocortical hyperfunction with increased corticosterone production following induction of adrenal LH receptor expression. Elevated LH acts as a tumor promoter in the gonads and the adrenal gland, when expressed in conjunction with the inhibin-alpha SV40 transgene. Finally, chronic elevated LH promotes mammary tumorigenesis. The understanding of multiple clinical pathologies--including ovarian cancer, perimenopausal reproductive aging, premature ovarian failure, polycystic ovarian syndrome, Cushing's syndrome, and breast cancer--may be enhanced through further study of this useful transgenic mouse model.