Low density lipoprotein binding and de novo synthesis of cholesterol in the neocortex and fetal zones of the human fetal adrenal gland

The binding of low density lipoprotein (LDL) and the de novo synthesis of cholesterol in separated zones of human fetal adrenal (HFA) tissues were investigated. The number of LDL-binding sites was 2-fold greater in membrane fractions prepared from fresh fetal zone tissue than in those from neocortex tissue. The binding capacity for LDL in fetal zone and neocortex membrane preparations of HFA tissues maintained in culture in the presence of ACTH was 2-fold greater than that in membrane fractions of control tissues. The rates of de novo synthesis of cholesterol also were determined in separated zones of HFA tissue by measuring the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal fractions prepared from HFA tissues and by determining the rate of incorporation of tritium from [3H]water into cholesterol in HFA tissue fragments. The rate of de novo synthesis of cholesterol in fresh fetal zone tissue was twice that in neocortex tissue as estimated by these methods. When separated zones of HFA tissue were maintained in culture in the presence or absence of ACTH, the rates of de novo synthesis, as determined by the rate of incorporation of tritium from [3H]water into cholesterol, were stimulated to a similar extent by ACTH in both fetal zone and neocortex tissues. However, the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was increased to a greater extent by ACTH pretreatment in neocortex tissues than in fetal zone tissues. In summary, fetal zone tissues of the HFA gland have a larger number of LDL-binding sites and higher rates of de novo synthesis of cholesterol than do neocortex tissues, and ACTH stimulates LDL binding and de novo synthesis of cholesterol in both zones of the HFA gland.     

Metabolism of high density lipoprotein by human fetal adrenal tissue

The role of lipoproteins as a source of the cholesterol utilized for steroidogenesis by human fetal adrenal (HFA) tissue was investigated previously. It was found that low density lipoprotein (LDL) was the lipoprotein preferred as a source of cholesterol for steroidogenesis by the HFA. [125I]Iodo-LDL was taken up and degraded by HFA tissue in organ culture, and the degradation of [125I]iodo-LDL was stimulated when ACTH (1 microgram X ml-1) was present in the culture medium. Others have shown that high density lipoprotein (HDL) is utilized as a source of cholesterol for steroidogenesis by rat adrenocortical cells in vitro and by the adrenals of the adult rat in vivo. In the present investigation we evaluated the metabolism of [125I]iodo-HDL by HFA tissue. [125I]iodo-HDL uptake by the HFA tissue increased in a linear manner with time and as the concentration of [125I]iodo-HDL in the culture medium was increased. However, there was little degradation of [125I]iodo-HDL by HFA. Moreover, preincubation of HFA tissue in medium containing ACTH (1 microgram X ml-1) or HDL, in various concentrations, did not affect the rate of uptake and degradation of [125I]iodo-HDL. The rate of degradation of [125I]iodo-LDL was found to decrease to low levels as the concentration of nonradiolabeled LDL in the culture medium was increased, whereas nonradiolabeled HDL had little effect on the degradation of [125I]iodo-LDL. HFA tissue fragments were incubated in medium containing ACTH plus lipoprotein-poor serum (LPPS) alone or LPPS plus HDL in various concentrations (50-1000 microgram X ml-1). The medium was changed daily and assayed for dehydroisoandrosterone sulfate and cortisol. In the presence of HDL, steroid secretion rates were no greater than those attained by HFA maintained in medium containing LPPS. It is concluded that the HFA utilizes cholesterol derived from LDL for steroidogenesis and that HDL is not metabolized efficiently by the human fetal adrenal.     

Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland

A model proposed for regulation of steroidogenesis, lipoprotein utilization and cholesterol metabolism in HFA tissue is presented in Fig 17. We envision that the role of ACTH and cAMP in steroidogenesis and cholesterol metabolism is as follows. ACTH binds to specific receptors on the surface of the cells of the HFA gland and as a consequence, adenylate cyclase is activated, leading to increased formation of cAMP. cAMP causes activation of protein kinase that leads, presumably, to phosphorylation of specific proteins. This leads to the initiation of reactions that give rise to increased activity of key enzymes and levels of proteins involved in adrenal cholesterol metabolism. Presumably, the action of ACTH causes an increase in the activity of cholesterol side chain cleavage, the rate-limiting step in the conversion of cholesterol to steroid hormones. We suggest that once the mitochondrial cholesterol side-chain cleavage system is fully activated by ACTH, the supply of cholesterol to the mitochondria becomes rate-limiting for steroidogenesis. To meet this demand for cholesterol, a further action of ACTH results in an increase in the number of LDL receptors. LDL binds to specific receptors on the cell surface that are localized in coated pits. LDL is internalized by a process of adsorptive endocytosis and the internalized vesicles fuse with lysosomes and the protein component of LDL is hydrolyzed by lysosomal proteolytic enzymes to amino acids. The cholesteryl esters of LDL also are hydrolyzed to give rise to fatty acids and cholesterol. The liberated cholesterol is available for utilization in the biosynthesis of steroid hormones and other cellular processes. In addition, ACTH stimulates the activity of HMG CoA reductase and, thus, the rate of de novo cholesterol biosynthesis. In this way sufficient cholesterol is obtained to provide for precursor cholesterol to maintain the high rate of steroid synthesis by the HFA. HDL is not utilized as a source of cholesterol by the HFA. Because of the rapid rate of utilization of LDL by the HFA, fetal plasma levels of LDL are low and the activity of the HFA is a primary determinant of these levels. Thus, in the case of anencephaly, in which the activity of the adrenal is very low, plasma levels of LDL are 2--3 times higher than in normal fetuses, whereas plasma HDL levels are similar. In addition, in the normal neonate plasma LDL levels rise rapidly after birth, and this event is coincident with the involution of the fetal zone of the adrenal. The fetal liver is likely to be the major source ultimately of the LDL-cholesterol utilized by the HFA. Consequently, factors that regulate cholesterol and lipoprotein synthesis in the fetal liver may, in turn, affect the steroidogenic activity of the HFA through regulation of the supply of cholesterol precursor. Thus, if trophic factors for the HFA other than ACTH exist, an important site of their action might be the fetal liver, rather than a direct action to influence the rate of synthesis of steroids by the fetal adrenal.     

The role of calmodulin antagonists on steroidogenesis by fetal zone cells of the human fetal adrenal gland

The adrenal gland of the human fetus (HFA) is relatively large compared to that of the adult and exhibits an extremely high rate of steroidogenesis both in vivo and in vitro. The fetal zone cells make up 80-85% of the volume of the HFA and are the major site of steroid production during fetal development. We have recently demonstrated that calcium is involved in the regulation of steroidogenesis in fetal zone cells of the HFA. There is considerable evidence that many actions of calcium within cells are mediated by the calcium-binding protein calmodulin. The purpose of the present investigation was to determine if calmodulin also plays a role in HFA steroidogenesis. To investigate this possibility, the fetal zone was dissected from fetal adrenals of first and second trimester human abortuses. After collagenase digestion of the tissue, dispersed fetal zone cells were maintained in a Krebs-Ringers medium at 37 C for a 3-h incubation. Cells were incubated with and without ACTH (10(-8) M) in the presence of the calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and calmidazolium (CAL) at concentrations of 5-100 microM. The media were assayed for contents of dehydroepiandrosterone sulfate (DS), cortisol (F), pregnenolone, and cAMP by RIA. The addition of ACTH stimulated F secretion 5- to 10-fold compared to that in control fetal zone cells. DS secretion increased up to 5-fold and pregnenolone about 2-fold in the presence of ACTH compared to values in control cells. ACTH also stimulated cAMP secretion by 10-fold compared to that in control cells. The addition of TFP, CPZ, and CAL significantly inhibited ACTH-stimulated DS, F, and pregnenolone secretion in a dose-related fashion to near-control levels. We observed that TFP, CPZ, and CAL inhibited cAMP accumulation as well as Bu2cAMP-stimulated steroid secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by TFP and CPZ, but not by CAL. These studies suggest that calmodulin plays a role in regulating steroidogenesis in fetal zone cells of the HFA.     

The action of phorbol ester on steroidogenesis in cultured human fetal adrenal cells

The potent mitogen and tumor promoter, phorbol 12-myristate 13-acetate (PMA), has a primary action via activation of calcium-dependent protein kinase C. The treatment of monolayer cultures of human fetal adrenal neocortex (HFA) cells with PMA (50-250 nM) stimulated basal dehydroepiandrosterone sulfate (DS) secretion 2-3 fold. ACTH-treated HFA cells secreted amounts of DS and cortisol (F) 10-50 fold greater than basal secretions. PMA (250 nM) addition with ACTH to HFA cells decreased DS and F secretions at least 75% on days 2 and 3 of treatment. Treatment of HFA cells with 4 alpha-phorbol, which does not activate calcium-dependent protein kinase C, did not inhibit steroidogenesis. The attenuated rates of steroidogenesis after PMA treatment correlated with the decreased amounts of steroid 11 beta, 17 alpha- and 21-hydroxylase cholesterol side-chain cleavage steroid dehydrogenase and sulfotransferase activities. The decrease of steroid 17 alpha-hydroxylase activity correlated with the decreased amount of cytochrome P-450(17) alpha as determined after protein immunoblotting of NaDodSO4 cell lysates. After PMA treatment the ACTH-promoted increases of hydroxysteroid sulfotransferase and dehydrogenase activities of HFA cells were suppressed. PMA (50 nM) inhibited cAMP accumulation in ACTH-treated HFA cells, while 4 alpha-phorbol had no effect. Importantly, dibutyryl cAMP (0.2 mM) treatment of HFA cells did not reverse phorbol ester-promoted attenuation of steroidogenesis. We conclude that, in the presence of ACTH, phorbol ester chronically inhibits both cAMP synthesis and cAMP-dependent protein kinase action with resultant decreased steroidogenic enzyme synthesis and steroid production. This may be a consequence of activation, migration and a slow degradation of protein kinase C activity. These multifaceted actions of phorbol ester and associated protein kinase C activation may have critical effects on the ontogeny of fetal adrenal function.     

The role of serum lipoproteins in steroidogenesis by the human fetal adrenal cortex.

In the present investigation it was found that human fetal adrenal tissue maintained in organ culture secreted appreciable quantities of dehydroisoandrosterone sulfate (DS) and cortisol. Pregnenolone was also secreted in significant amounts, principally as the sulfate ester. The highest rate of secretion of these steroids by fetal adrenal tissue occurred when both ACTH and whole human serum were present in the culture medium. In the absence of ACTH, steroid secretion was low. When whole serum was replaced by lipoprotein-poor serum, the steroidogenic response to ACTH was markedly attenuated but not abolished. On the basis of these findings, it is concluded (1) that the human fetal adrenal can synthesize steroid hormones de novo from cholesterol, (2) that ACTH is an important stimulant of steroidogenesis by the human fetal adrenal, and (3) that plasma lipoproteins are a major source of the cholesterol utilized by the human fetal adrenal for steroidogenesis. Hence, it is likely that factors which regulate the production of fetal plasma lipoproteins are important determinants of fetal adrenal steroidogenic activity.     

Steroidogenesis in the human fetal adrenal: a role for cholesterol synthesized de novo

Primary monolayer cultures of human fetal adrenal cells maintained in either lipoprotein-depleted or lipoprotein-supplemented media responded chronically to ACTH treatment with similarly increased steroid secretion. The principal steroid secreted into each medium was dehydroepiandrosterone sulfate. The presence of human low density lipoprotein (hLDL) in the medium enhanced the secretion of nonsulfoconjugated steroids, especially dehydroepiandrosterone. The secretion rate of 11 beta-hydroxyandrostenedione was similar to that of cortisol. In the absence of hLDL, ACTH increased cholesterologenesis to maintain the high rates of steroid secretion. After ACTH treatment, increased accumulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-determining enzyme of cholesterol biosynthesis, was found. Immunoblot analysis demonstrated that this enzyme was a 97K protein in human fetal adrenal cells. Interestingly, the content of this enzyme in cells treated with ACTH in lipoprotein-depleted medium was similar to that in adrenal fetal zone tissue. This finding suggests that cholesterologenesis de novo in addition to plasma LDL is important as a source of steroid precursor in vivo in the human fetal adrenal gland.     

The role of calcium in steroidogenesis in fetal zone cells of the human fetal adrenal gland

The human fetal adrenal gland is composed primarily of fetal zone (FZ) cells, which have a high rate of steroidogenesis. The purpose of this study was to examine the role of calcium in the regulation of steroidogenesis by FZ cells. Dispersed FZ cells were incubated in Krebs-Ringers medium at 37 C for 3 h in the presence of ACTH, (Bu)2cAMP, or forskolin in addition to various drugs. The medium contents of dehydroepiandrosterone sulfate (DS), cortisol (F), and cAMP were quantified by RIA. After the addition of ACTH (10(-10)-10(-5) M), DS and cAMP secretion increased. The addition of EGTA to the medium inhibited ACTH- and forskolin-stimulated DS, F, and cAMP secretion by 50% as well as (Bu)2cAMP-stimulated steroidogenesis. The addition of calcium (10(-5)-10(-2) M) had only a slight effect on the secretion of DS or F in the absence of ACTH or (Bu)2cAMP. In the presence of ACTH and (Bu)2cAMP, however, increasing amounts of calcium resulted in a 2- to 3-fold increase in the rates of DS and F secretion. The addition of either A23187, a calcium ionophore, or verapamil, a calcium channel blocker, inhibited ACTH-stimulated DS and F secretion by 90%. The rate of cAMP formation was greater after ACTH plus verapamil treatment than after ACTH treatment alone, whereas A23187 inhibited ACTH-stimulated cAMP secretion to basal levels. Both A23187 and verapamil inhibited ACTH- and cAMP-stimulated pregnenolone secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by A23187 and verapamil. In conclusion, our results suggest that extracellular calcium is important for activation of the human adrenal FZ cell adenylate cyclase system, while intracellular calcium plays a multifaceted role in controlling steroid production.     

Estrogen regulates low density lipoprotein metabolism by cultured swine granulosa cells

To test estrogen's possible regulation of lipoprotein metabolism by granulosa cells, swine granulosa cells were cultured under serum-free conditions in the presence or absence of estradiol. Treatment with estradiol significantly enhanced high affinity, saturable, [125I]iodo-low density lipoprotein (LDL) binding with a median 2.85-fold (range 2.3- to 5.6-fold, n = six experiments) increase in the calculated number of LDL receptors and no change in the apparent dissociation constant (Kd) for LDL binding (Kd = 3.4 +/- 0.92 micrograms/ml in control and 4.0 +/- 0.87 micrograms/ml human LDL in estradiol-treated cultures). Estradiol also significantly increased [125I]iodo-LDL internalization by granulosa cells and augmented the maximal rate of LDL degradation by 2.0 to 2.5-fold without altering the apparent Michaelis-Menten constant (Km) for this process. Estrogen's dose-dependent enhancement of [125I]iodo-LDL binding, internalization, and degradation could be observed at minimum estradiol concentrations of approximately 100 ng/ml and was accompanied by increased progesterone secretion by granulosa cells. Further studies indicated that estrogen's stimulation of LDL internalization and degradation was not simply attributable to increased rates of nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrrolidone) or increased steroidogenesis per se (tested by blocking cholesterol side-chain cleavage with aminoglutethimide). We conclude that estradiol amplifies LDL binding by swine granulosa cells by increasing the number of high affinity, saturable LDL receptors with no alteration in their apparent affinity. Moreover, estrogen action is accompanied by enhanced rates of progesterone production in the presence of LDL, and increased rates of LDL internalization and degradation, which could not be accounted for simply by accelerated nonspecific bulk fluid-phase pinocytosis. We suggest that the significant facilitative actions of estradiol on lipoprotein binding and metabolism are likely to assist in preparing granulosa cells for the increased rates of progesterone biosynthesis ultimately required in functional corpora lutea.     

Mechanism of ACTH action

ACTH regulates both the differentiated function (steroidogenesis) and the growth and replicative potential of the adrenal cortex. Cyclic AMP (cAMP) serves as the mediator of ACTH action within the cell. The binding of cAMP to its specific receptor activates a protein phosphokinase enzyme with resultant phosphorylation and altered function of several important substrates. The regulation of steroidogenesis requires protein synthesis and affects the conversion of cholesterol to pregnenolone, the rate-limiting step under hormonal control. In normal adrenal cortical tissue, ACTH stimulates DNA synthesis and replication, and in a functional adrenal tumor in tissue culture, ACTH inhibits DNA synthesis and replication. ACTH and cAMP thus appear to direct the adrenal cell toward the differentiated, maximally functional state.     

Low density lipoprotein receptors in bovine adrenal cortex. I. Receptor-mediated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured adrenocortical cells.

Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free cholesterol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue.     

Adrenal steroidogenic actions of cyclic nucleotide derivatives in the rat.

Fifteen 3',5'-cyclic nucleotides and related compounds were studied for ability to mimic the steroidogenic action of ACTH in rats in which secretion of ACTH and corticosterone were suppressed by treatment with betamethasone, or by hypophysectomy. Subcutaneous administration of 8-chloro-cAMP, at doses of 40 mg/kg or greater, elicited the secretion of corticosterone to normal plasma levels in both betamethasone-treated and hypophysectomized animals. Cyclic AMP, dbcAMP, 8-methylthio-cAMP, 8-hydroxy-cAMP and the 6-chloro-8-aminopurine cyclic ribotide analog of cAMP also displayed steroidogenic activity in the betamethasone-treated rat; cGMP, 8-bromo-cGMP and 8-benzylthio-cGMP were inactive. Each of the steroidogenic derivatives of cAMP also displayed ability to activate steroidogenesis in isolated rat adrenal cells. These experiments demonstrate that various derivatives of cAMP mimic the adrenal steroidogenic action of ACTH, in vivo. Structure-activity comparisons support a steroidogenic mechanism involving direct activation by the nucleotides of cAMP-dependent protein kinase of the adrenal cortex.     

The inhibition of low density lipoprotein metabolism by transforming growth factor-beta mediates its effects on steroidogenesis in bovine adrenocortical cells in vitro

Transforming growth factor-beta (TGF beta) has a differential effect on the growth and function of bovine adrenocortical cells in vitro. TGF beta inhibits basal as well as ACTH- or angiotensin II-stimulated steroid formation, with no evidence of change in cell growth. The major inhibitory effect of TGF beta occurs at a step before cholesterol formation, since treatment of adrenocortical cells with TGF beta decreased not only delta 4-steroid levels but also delta 5-steroid levels. The addition of cholesterol reverses the suppression of steroidogenesis induced by TGF beta. To determine the mechanism of this inhibition, the effect of TGF beta on low density lipoprotein (LDL) metabolism was investigated. Cells treated with TGF beta showed a significant suppression of [125I]iodohuman LDL ([125I]LDL) binding to the cell surface, followed by decreases in internalization and proteolytic degradation of [125I]LDL. Maximal inhibition of LDL metabolism was observed at a concentration of 1 ng/ml (4 X 10(-11) M) TGF beta. The stimulation of LDL metabolism by ACTH was also inhibited by TGF beta, and the inhibition observed correlated well with the inhibition of steroidogenesis. The inhibitory effect of TGF beta on [125I]LDL binding results from the decrease in the maximal LDL-binding capacity. The stimulation of LDL uptake induced by Bu2cAMP, cholera toxin, forskolin, and Ang II was also decreased by treatment with 1 ng/ml TGF beta. The specificity of this effect is quite high, since the inhibitory effects of TGF beta on LDL metabolism were not observed with either inhibin A or activin, two molecules that have considerable structural homology to TGF beta. We conclude that TGF beta specifically suppresses LDL metabolism in bovine adrenocortical cell cultures and that this step may mediate, at least in part, its role as a potent inhibitor of steroidogenesis.     

Mechanism of action of prolactin on adrenocortical steroid secretion in hypophysectomized female rats.

Studies were carried out to determine the actions of PRL on adrenocortical function in hypophysectomized female rats in the presence and absence of ACTH. PRL administration alone decreased 5 alpha-reductase activity but did not significantly affect the rates of corticosterone secretion or peripheral plasma corticosterone concentrations. The activities of several steroidogenic enzymes (cholesterol desmolase, 11 beta-hydroxylase, and 21-hydroxylase) were also unaffected by PRL. Adrenal steroidogenesis was increased by ACTH treatment, as expected, resulting in an increase in corticosterone secretion. However, since adrenal 5 alpha-reductase activity was higher in ACTH-treated hypophysectomized rats than in normal animals with intact pituitary glands, large amounts of 5 alpha-dihydrocorticosterone (DHB) and 3 beta, 5 alpha-tetrahydrocorticosterone (THB) were also secreted. PRL, when administered in combination with ACTH, potentiated the effecte levels. PRL did not affect cholesterol side chain cleavage, 11 beta-hydroxylation, or 21-hydroxylation in ACTH-treated rats. However, administration of PRL to ACTH-treated rats lowered adrenal 5 alpha-reductase activity, decreasing DHB and THB secretion. The decrease in DHB and THB secretion approximated the increase in corticosterone output. The results indicate that, in the presence of ACTH, PRL increases corticosterone secretion by decreasing intraadrenal degradation of corticosterone and not by enhancing steroidogenesis.     

Stimulation of progesterone synthesis in luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-adenosine 3',5'-monophosphate: the effect of low density lipoprotein

Primary cultures of luteinized human granulosa cells reduced progestin secretion when taken from serum- and gonadotropin-containing medium into serum- and hormone-free medium. When added individually hCG, 8-bromo-cAMP and low density lipoprotein (LDL) stimulated progestin secretion by the cells after they spent 48 h in serum- and hormone-free medium. However, combinations of hCG or 8-bromo-cAMP and LDL were most effective in increasing steroidogenesis. The effects of hCG in enhancing steroidogenesis in the presence of LDL were first detectable after 3 h, but were most marked after 20 h of culture. hCG and 8-bromo-cAMP increased the conversion of [3H]cholesteryl linoleate, which had been incorporated into the core of LDL, into [3H]progesterone. hCG also stimulated cellular accumulation of LDL cholesterol, as assessed by incorporation of [1-14C]oleic acid into sterol esters or by measurement of total cellular cholesterol in the presence of amino-glutethimide to block steroidogenesis. In contrast to progesterone secretion, estradiol secretion was not affected by the addition of LDL in the absence or presence of 8-bromo-cAMP. We conclude that LDL cholesterol is required for maximal rates of progestin synthesis by human luteinized granulosa cells. When granulosa cells are stimulated by hCG, uptake of LDL cholesterol is promoted, and there is increased utilization of LDL cholesterol for steroid synthesis.     

Insulin regulates low density lipoprotein metabolism by swine granulosa cells

Insulin synergistically amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells. The mechanisms subserving this facilitative interaction included the following: 1) insulin's synergism with LDL was profoundly attenuated by covalent modification of arginine residues in LDL by 1,2-cyclohexanedione treatment; 2) insulin increased by 2- to 6-fold the number of specific high affinity LDL receptors on granulosa cells, with no change in apparent binding affinity; 3) insulin augmented rates of [125I]iodo-LDL internalization and degradation without enhancing nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrollidone); 4) insulin increased by 2.5- to 3-fold granulosa cell content of free and esterified cholesterol (measured by fluorometry) in response to treatment with unlabeled LDL; 5) insulin stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester, and amplified [3H]progesterone secretion by granulosa cells exposed to [3H]cholesteryl linoleate-labeled LDL; and 6) insulin action was specific in that it was not mimicked by desoctapeptide insulin, epidermal growth factor, fibroblast growth factor, or relaxin. We conclude that insulin and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via specific mechanisms that depend upon 1,2,-cyclohexanedione-sensitive residues within LDL apoprotein. Insulin action results in significantly augmented binding, internalization, and degradation of LDL, which is accompanied by increased effectual delivery of cholesterol substrate into cellular sterol pools that participate in enhanced steroidogenesis.     

Expression of low and high density lipoprotein receptor genes in human adrenals

Corticosteroids are synthesized from cholesterol which may arise from de novo synthesis or from the uptake of low or high density lipoproteins (LDL or HDL). In the present study, we compared the expression and regulation patterns of LDL receptor and CLA-1 (CD36 and LIMPII Analogous-1, an HDL receptor) genes in adult human adrenocortical tissues to shed more light on the relative contribution of LDL and HDL in human adrenal steroidogenesis. By screening 64 normal and pathological adrenal samples by Northern blotting, we found a positive correlation between LDL receptor and CLA-1 mRNA expression in the adrenal tissues (r=0.547; spearman rank correlation test P<0.01). Adrenal tissues adjacent to Cushing's adenomas contained consistently less LDL receptor and CLA-1 mRNA than normal adrenals (Mann-Whitney P<0.05). In primary cultures of normal adrenal cells, accumulation of both LDL receptor and CLA-1 mRNAs was upregulated by ACTH in a dose- and time-dependent manner, with an earlier induction of LDL receptor than CLA-1 mRNA expression. (Bu)(2)cAMP also increased the levels of these two mRNAs. Addition of LDL, but not HDL, into the culture medium increased cortisol production in untreated adrenocortical cells. Both LDL and HDL enhanced ACTH-induced cortisol production, with the effect of LDL much stronger than that of HDL. Our data show that LDL receptor and CLA-1's expression is ACTH-dependent and occurs in parallel in human adrenal tissues. LDL rather than HDL may be used as the preferential source of cholesterol for steroidogenesis in human adult adrenocortical cells.     

Role of plasma lipoproteins in the function of steroidogenic tissues

Steroidogenic cells (adrenal, gonads and placenta) utilize cholesterol as a substrate for hormonal biosynthesis. Cholesterol can be synthetized de novo acetate or can enter the cell as a component of plasma lipoproteins, particularly LDL (low density lipoprotein) and HDL (high density lipoprotein). Steroidogenic cells utilize predominantly lipoproteins. Lipoprotein-cholesterol enters the cell by a specific pathway: binding of lipoproteins, particularly LDL to a membrane specific receptor and internalization of the complex receptor-lipoprotein, followed by dissociation of the complex; the receptor is reutilized, while LDL is hydrolyzed by lysosomal enzymes in amino-acids and free cholesterol. The liberated cholesterol is available for utilization by the cell. In addition, lipoproteins modulate intra-cellular cholesterol metabolism via two enzymatic activities; inhibition of hydroxymethyl-glutaryl coenzyme A reductase activity (HMG CoA reductase) which controls the rate limiting step of cholesterol biosynthesis, and stimulation of acyl CoA cholesterol acyltransferase (ACAT) activity which controls the esterification of cholesterol, before its translocation to lipids droplets. Peptide hormones LH/hCG and ACTH regulate lipoproteins metabolism in their target tissues by increasing LDL receptors number, a way which allow more cholesterol to enter the cell. The role of lipoproteins on steroidogenesis has been studied predominantly in adrenal and to a lesser extent in ovary and placenta and poorly in testis. Using a model of cultured porcine Leydig cells, we have demonstrated that de novo synthesis of cholesterol accounts for only 25% of the maximal steroidogenic capacity of these cells. Moreover the effects of lipoprotein LDL was synergistic with that of gonadotropins.     

Prostaglandin secretion by the neocortex and fetal zones of the human fetal adrenal gland

Previously, we reported that the human fetal adrenal (HFA) gland secretes various prostaglandins (PGs) in vitro and that PG secretion is inhibited by endogenously synthesized glucocorticosteroids. In this investigation, the neocortex (NC) and fetal zone (FZ) of the HFA gland were separated by microdissection and maintained as tissue fragments in organ culture. The rate of PG secretion into the culture medium was determined by measuring various PGs using specific RIAs in media collected at 24-h intervals. During the first 24 h in culture, the secretion rates of PGF2 alpha and PGE2 were 6- and 7-fold greater by NC [14 +/- 5 and 9.9 +/- 3 ng mg protein-1 24 h-1 (mean +/- SE)], respectively, than by FZ tissue (2.5 and 1.4 ng mg protein-1 24 h-1). The secretion rates of PGFM and PGD2 were 2-fold greater in NC tissue than in FZ tissue, but the secretion rates of thromboxane B2 were similar in both zones of HFA tissue. In another study, the patterns of secretion of PGF2 alpha and PGE2 were determined as a function of days in culture. The secretion rates of PGF2 alpha and PGE2 fell rapidly in NC from 19.0 +/- 11 and 38.3 +/- 9.7 ng mg protein-1 24 h-1, respectively, to 1.3 +/- 7.2 and 4.8 +/- 3.3 by day 4. In contrast, the secretion rates of PGF2 alpha and PGE2 rose 8- and 3-fold in FZ tissue (from 0.7 +/- 0.2 and 0.9 +/- 0.6 ng mg protein-1 24 h-1, respectively, to 5.9 +/- 0.5 and 3.1 +/- 1.2 by day 4). The addition of ACTH or dexamethasone inhibited PG secretion in both zones, but to a greater degree in FZ tissue than in NC tissue. In summary, the NC secretes larger quantities of PG than the FZ, and the patterns of secretion are different in the two zones. The secretion of PGs is inhibited more in FZ than in NC tissue by ACTH and glucocorticosteroids.