Steroidogenesis in adrenal glands of young and adult female mice with hyperexpression of the Aguoti gene

Dominant mutation Agouti yellow (AY) leads to ectopic overexpression of the Agouti gene and yellow coat color in mice. Furthermore, the mutation Ay increased adrenal response to emotional stress. The study assessed whether pleiotropic effect of the mutation Ay on adrenals function was dependent on sex and age. 3- and 15-week old female C57B1/6J mice of two agouti-genotypes: Ay/a (ectopic Agouti-gene overexpression) and a/a (absence of Agouti-protein), were investigated. Cyclic AMP level (adenylate cyclase activity) and corticosterone production in adrenal isolated cells stimulated by ACTH and dibutyrul cAMP (db-cAMP) were measured. ACTH increased cAMP accumulation to the same extent in Ay/a- and a/a-mouse adrenal cells of both ages. The dibutyrul cAMP-induced corticosterone production was higher in Ay/a than in a/a-mouse adrenal cells of both ages. The ACTH-induced corticosterone production in 3-week- old Ay/a-m/CQ was lower and in 15-week old Ay/a-mice was higher than in a/a-mice of the respective ages. The ACTH- and db-cAMP-induced steroidogenesis was not changed in Ay/a-mice and decreased in a/a-mice with age. Thus, in females as well as in males, the mutation Agouti yellow did not affect adenylate cyclase activity, increased db-cAMP-induced corticosterone production and disturbed development of adrenal cortex.     

Postnatal development of corticosteroid function of the adrenals in C57BL/6J-Ay mice

We studied postnatal development of corticosteroid function of the adrenals in mice during the period of elevated activity of the hypothalamic-pituitary-adrenal system and the influence of mutant gene Ay on this process. Normally, a corticosterone peak in blood and increased basal and stimulated steroidogenesis in vitro are observed in 3-week old mice. In 3-week old Ay/a mice (hyperexpression of protein agouti) a corticosterone peak in blood is lowered and genotypic differences in steroidogenesis in vitro are absent, as compared to a/A mice (absence of agouti), while at the ages of 10 and 15 weeks, there were no genotypic differences in the blood level of corticosterone and steroidogenesis in vitro was elevated. Thus, a high level of corticosterone during the period of elevated activity of the hypothalamic-pituitary-adrenal system in 3-week old mice is determined by enhanced steroidogenic function of the adrenals. Mutant gene Ay in male mice affected the postnatal development of the adrenal function: the peak of corticosterone in blood was lowered during the period of elevated activity of the system.     

Repeating of emotional stress prevents development of melanocortin obesity and type 2 diabetes in the mice with the Agouti yellow mutation

Brain melanocortin system (MC-system) participates in regulation of energy homeostasis. Dominant mutation yellow of the Agouti gene leads to the hyperphagia, obesity and type 2 diabetes. Stress is known to inhibit food intake and body weight. The aim of the work was to study effects of repeating emotional stress on food intake and lipid-carbohydrate metabolism in Ay-mice. Male mice of C57B1/6J strain predisposed to the obesity (Ay/a-genotype) and normal (a/a-genotype) were used. In control group food intake, body weight and blood levels of insulin and leptin were increased in Ay/a-mice as compared to a/a-mice. Repeating emotional stress (30 min restraint 3 times a week for 5 weeks) did not alter food intake and indices of lipid-carbohydrate metabolism in a/a-mice and decreased food intake, body weight and blood levels of insulin and leptin in Ay/a-mice. Insulin and leptin blood levels were the same in Ay/a- and a/a-mice on 5 week of treatment. The stress increased basal and stress-induced concentrations of corticosterone to an equal degree in Ay/a- and a/a-mice. Thus, light repeating emotional stress hampered development of obesity and 2 type diabetes in the mice with the Agouti yellow mutation.     

Function of the hypothalamo-pituitary-adrenal system in mice with ectopic overexpression of agouti protein

Agouti protein (AP) is known to antagonise the effects of melanocortins (ACTH, MSH) on the melanocortin receptors which participate in regulation of central and peripheral HPA links. This study aimed at estimation of effects of dominant mutation Agouti yellow (Ay-ectopic overexpression Agouti protein) on the HPA axis function in mice. Male mice of C57B1/6J strain of Ay/a- and a/a genotypes (control animals, lack of AP), were used. We demonstrated that basal corticosterone level in the Ay mice matched those of control animals. Stress-activated corticosterone level (p < 0.02) and sensitivity of adrenal to low doses ACTH in vitro and in vivo were higher in Ay mice compared with control mice. Dexamethazone-inhibition of stress-reactivity was more intensive in Ay/a than in a/a mice (p < 0.0007).     

Stress-hyperresponsive period in postnatal development of hypothalamic-hypophyseal-adrenal system in mice

OBJECTIVE OF THE STUDY: to compare response to stress, activity of adenylatcyclase and enzymes of steroidogenesis in adrenal cells in 3-week and adult (15-week) mice. In 3-week mice, basal and stressor level of blood corticosterone was increased. Their response to stress was faster developing as compared with adult mice. In vitro, 3-week mice had enhanced adrenal sensitivity to ACTH, corticosterone release in response to ACTH, exogenous cAMP and progesterone; and the cAMP level in the cells stimulated with ACTH and forscoline. Thus, in 3-week mice, a stress-hyperresponsive period occurs due to increased release of corticosterone and adrenal sensitivity to ACTH. The reason of this involves enhanced activity of adenylatcyclase and intracellular enzymes of steroidogenesis.     

Postnatal development of corticosteroid function of the adrenals in C57BL/6J-<Emphasis Type="Italic">A</Emphasis><Superscript>y</Superscript> mice

We studied postnatal development of corticosteroid function of the adrenals in mice during the period of elevated activity of the hypothalamic-pituitary-adrenal axis and the influence of mutant gene A ^y on this process. Normally, a corticosterone peak in blood and increased basal and stimulated steroidogenesis in vitro are observed in 3-week old mice. In 3-week old A ^y/a mice (hyperexpression of agouti protein) a corticosterone peak in blood is lowered and genotypic differences in steroidogenesis in vitro are absent, as compared to a/a mice (absence of agouti), while at the ages of 10 and 15 weeks, there were no genotypic differences in the blood level of corticosterone and steroidogenesis in vitro was elevated. Thus, a high level of corticosterone during the period of elevated activity of the hypothalamic-pituitary-adrenal axis in 3-week old mice is determined by enhanced steroidogenic function of the adrenals. Mutant gene A ^y in male mice affected the postnatal development of the adrenal function: the peak of corticosterone in blood was lowered during the period of elevated activity of the system.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Hormone-sensitive lipase is required for high-density lipoprotein cholesteryl ester-supported adrenal steroidogenesis

Steroid hormones are synthesized using cholesterol as precursor, with a substantial portion supplied by the selective uptake of lipoprotein-derived cholesteryl esters. Adrenals express a high level of neutral cholesteryl ester hydrolase activity, and recently hormone-sensitive lipase (HSL) was shown to be responsible for most adrenal neutral cholesteryl ester hydrolase activity. To determine the functional importance of HSL in adrenal steroidogenesis, adrenal cells were isolated from control and HSL-/- mice, and the in vitro production of corticosterone was quantified. Results show that, even though adrenal cholesteryl ester content was substantially elevated in both male and female HSL-/- mice, basal corticosterone production was reduced approximately 50%. The maximum corticosterone production induced by dibutyryl cAMP, and lipoproteins was approximately 75-85% lower in adrenal cells from HSL-/- mice compared with control. There is no intrinsic defect in the conversion of cholesterol into steroids in HSL-/- mice. Dibutyryl cAMP-stimulated conversion of high-density lipoprotein cholesteryl esters into corticosterone was reduced 97% in HSL-/- mice. An increase in low-density lipoprotein receptor expression appears to be one of the compensatory mechanisms for cholesterol delivery in HSL-/- mice. These findings suggest that HSL is functionally linked to the selective pathway and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis.     

Pituitary-adrenal function in female mice with mutation Agouti yellow (A(y))

Agouti protein is a paracrine signaling factor modulating action of ACTH and alpha-MSH. Dominant mutation Ay causes ectopic, ubiquitous expression of Agouti protein in mice. It was shown that Ay mutation increased stress-induced hypothalamo-adrenal activity in male mice. There is a sex difference in the hypothalamo-pituitary-adrenal axis in rodents. The aim of this study was to test effects of ectopic overexpression of Agouti protein on pituitary-adrenal function in female mice. Female mice of C57Bl/6J strain with Ay mutation (Ay/alpha) and with mutation nonagouti (alpha/alpha; lack of Agouti protein) were used. Ay/alpha-females had an increased blood level of corticosterone and ACTH after 10-minute restriction as compared with alpha/alpha-females. The adrenal threshold sensitivity and reaction to exogenous ACTH in vivo suggests that increased corticosterone reaction to emotional stress is caused by increased pituitary stimulation.     

Selenium deficiency impairs corticosterone and leptin responses to adrenocorticotropin in the rat

Selenium deficiency causes oxidative stress and impairs steroidogenesis in vitro. Leptin is closely related to the hypothalamo-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis at the central level while corticosteroids have been shown to stimulate leptin secretion in most studies. We hypothesized that oxidative stress impairs adrenal steroidogenesis and decreases leptin production in vivo. The goal of this study was to investigate in rats the effects of selenium deficiency and oxidative stress on adrenal function and on leptin concentrations. Weanling rats were fed a selenium-deficient (Se-) or selenium-sufficient (Se+) diet for 4-10 weeks. Selenium deficiency caused a marked decrease in liver (> or = 99%) and adrenal (> or = 81%) glutathione peroxidase (GPx) activities. Selenium deficiency did not affect basal and short-term adrenocorticotropin (ACTH) stimulated corticosterone or leptin concentrations. In contrast, after long-term ACTH stimulation, selenium deficiency caused a doubling in adrenal isoprostane content and blunted the increase in corticosterone and leptin concentrations observed in Se+ animals. Plasma leptin concentrations were 50% lower in Se- compared to Se+ animals following long-term ACTH. Our results suggest that oxidative stress causes a decrease in circulating corticosterone in response to ACTH, and, as a consequence, a decrease in plasma leptin concentrations.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to 3 x 10(-8) M--3 x 10(-4) M of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostacyclin (PGI2), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE2, PGF2 alpha, PGI2, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10(-5) M) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Effect of adrenalectomy on weight gain and body composition of yellow obese mice (Ay/a).

It has been reported that the adrenal gland is essential to the development of obesity if Ay/a Yellow obese mice (Hausberger and Hausberger 1960). Since the actual body composition data to support this report has not been published, we attempted to duplicate this observation by adrenalectomy of the Yellow mice before the onset of obesity. Two groups of Yellow mice (Ay/a) and normal mice (a/a) were either sham operated or adrenalectomized at two months of age and at four months of age. Body weight was monitored until body gain had stopped. At that time the animals were sacrified and checked for completeness of adrenal removal. Body composition of dry matter, fat, protein, and ash was determined. Adrenalectomy caused a reduction of body fat of 33% and 30% in both the a/a Control and the Ay/a Yellow mice, respectively. The adrenalectomized Yellow mice were still fatter than adrenalectomized Controls (25.3% fat vs. 10.6% fat). Yellow and control adrenalectomized mice showed similar depression in growth rate. These data suggest that while the adrenal gland is essential for complete expression of the genetic potential for fat deposition, it may not be necessary for partial expression indicating a secondary role in the development of obesity in the Ay/a Yellow mouse.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

The LDL receptor is not necessary for acute adrenal steroidogenesis in mouse adrenocortical cells

Steroid hormones are synthesized using cholesterol as precursor. To determine the functional importance of the low density lipoprotein (LDL) receptor and hormone-sensitive lipase (HSL) in adrenal steroidogenesis, adrenal cells were isolated from control, HSL(-/-), LDLR(-/-), and double LDLR/HSL(-/-) mice. The endocytic and selective uptake of apolipoprotein E-free human high density lipoprotein (HDL)-derived cholesteryl esters did not differ among the mice, with selective uptake accounting for >97% of uptake. In contrast, endocytic uptake of either human LDL- or rat HDL-derived cholesteryl esters was reduced 80-85% in LDLR(-/-) and double-LDLR/HSL(-/-) mice. There were no differences in the selective uptake of either human LDL- or rat HDL-derived cholesteryl esters among the mice. Maximum corticosterone production induced by ACTH or dibutyryl cyclic AMP and lipoproteins was not altered in LDLR(-/-) mice but was reduced 80-90% in HSL(-/-) mice. Maximum corticosterone production was identical in HSL(-/-) and double-LDLR/HSL(-/-) mice. These findings suggest that, although the LDL receptor is responsible for endocytic delivery of cholesteryl esters from LDL and rat HDL to mouse adrenal cells, it appears to play a negligible role in the delivery of cholesterol for acute adrenal steroidogenesis in the mouse. In contrast, HSL occupies a vital role in adrenal steroidogenesis because of its link to utilization of selectively delivered cholesteryl esters from lipoproteins.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to

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of prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), prostacyclin (PGI₂), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE₂, PGF_2α, PGI₂, and AA. Although preincubation of dispersed adrenal cells with indomethacin ( ) markedly inhibited PGE₂ synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Interrenal activity in the female lizard Lacerta vivipara J.: in vitro response to ACTH 1-39 and to [Sar1, Val5] angiotensin II (ANG II)

A perifusion system technique was developed in order to determine in vitro the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1-39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar1, Val5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.     

Changes of lipid-carbohydrate metabolism during development of melanocortin obesity in the mice with the Agouti yellow mutation

Melanocortin obesity develops after puberty in mice with Agouti yellow mutation. The aim of the work was to study dynamics of the main characteristics oflipidcarbohydrate metabolism during the obesity development. We used female mice of C57BI/6J strain homozygous for recessive mutation nonagouti (a/a-mice) who were predisposed to the obesity, and heterozygous for dominant mutation Agouti yellow (Ay-mice) who were not predisposed to the obesity. Food consumption and body weights were measured from week 4 to week 22. Some animals were decapitated on 8, 11, 13, 15, 22 weeks. Hyperphagia (6 week) proceeds to body weight increasing (7 week) in the Ay-mice. In the Ay-mice compared to a/a-mice, adiposyte size and blood level of leptin were increased on the 11th week, blood levels of fatty acids and glucose--on the 13th week, blood level of insulin--on the 15th week. The hyperphagia seems to promote development ofmelanocortin obesity, stable disturbances of lipid metabolism appearing before those in glucose metabolism. Complete metabolic obesity syndrome was formed after the 15th week of life.     

HDL is redundant for adrenal steroidogenesis in LDLR knockout mice with a human-like lipoprotein profile

The contribution of HDL to adrenal steroidogenesis appears to be different between mice and humans. In the current study, we tested the hypothesis that a difference in lipoprotein profile may be the underlying cause. Hereto, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice with a human-like lipoprotein profile. Genetic deletion of APOA1 in LDL receptor (LDLR) knockout mice was associated with HDL deficiency and a parallel increase in the level of cholesterol associated with nonHDL fractions. Despite a compensatory increase in the adrenal relative mRNA expression levels of the cholesterol synthesis gene, HMG-CoA reductase, adrenals from APOA1/LDLR double knockout mice were severely depleted of neutral lipids, as compared with those of control LDLR knockout mice. However, basal corticosterone levels and the adrenal glucocorticoid response to stress were not different between the two types of mice. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function when mice are provided with a human-like lipoprotein profile. Our findings provide the first experimental evidence that APOB-containing lipoproteins may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo in mice.