The role of cyclic adenosine 3',5'-monophosphate in cholesterol metabolism and steroidogenesis by the human fetal adrenal gland

In the present investigation we studied the role of cAMP as a mediator of ACTH action in human fetal adrenal (HFA) tissue. We have characterized the response to ACTH, dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and cholera toxin (CT) with respect to steroidogenesis, low density lipoprotein (LDL) binding, degradation of LDL, and the rate of de novo synthesis of cholesterol. The rate of dehydroisoandrosterone sulfate secretion was similar in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT. In contrast, cortisol secretion by HFA tissue was more sensitive to dbcAMP and CT than to ACTH. In membrane preparations obtained from HFA tissue maintained in the presence of ACTH, dbcAMP, or CT, there was a 2 to 3-fold increase of specific binding of [125I]iodo-LDL. In HFA tissue maintained in the presence of ACTH or CT, the rate of degradation of LDL was significantly increased compared to tissue maintained in the lipoprotein-poor serum alone. Finally, in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT there was a 6- to 10-fold stimulation of the rate of incorporation of [14C]acetate into cholesterol. We conclude that steroidogenesis, LDL binding, and degradation, as well as de novo synthesis of cholesterol, are probably stimulated in HFA tissue via a cAMP-mediated pathway.     

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.     

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.     

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.     

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.     

Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses

There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH.     

Adenylate cyclase activity in neocortex and fetal zone membrane fractions of the human fetal adrenal gland

Whether peptide hormones other than ACTH may be responsible for the difference in size or rate and pattern of steroidogenesis of the fetal zone (FZ) compared to those of the neocortex (NC) of the human fetal adrenal gland is controversial. In the present investigation, the activity of adenylate cyclase in membrane fractions of separated zones of the human fetal adrenal gland was determined. Basal adenylate cyclase activity was 2- to 3-fold greater in NC than in FZ membrane fractions. The addition of ACTH-(1-24) stimulated adenylate cyclase activity in both zones, but the activity was more sensitive to ACTH (10(-10) M) in NC fractions than in FZ fractions (10(-7) M). In addition to ACTH-(1-24), the effect of other ACTH-related peptides on the activity of adenylate cyclase in the separated zones of the adrenal gland was investigated. 16K fragments 2-36, gamma 3MSH, alpha MSH, beta-endorphin, leu-enkephalin, and met-enkephalin, as well as hCG, FSH, prostaglandin E2, prostaglandin F2 alpha, epinephrine, and norepinephrine did not stimulate adenylate cyclase activity in either zone. It is concluded that basal and ACTH-(1-24)-stimulated adenylate cyclase activities are greater in NC than in FZ membrane fractions. In addition, the results of the present investigation do not support the concept that other ACTH-related peptides or peptide or protein hormones increase steroidogenesis by stimulating adenylate cyclase activity in the human fetal adrenal gland.     

Protein kinase-C in the human fetal adrenal gland

The fetal zone (FZ) of the human fetal adrenal gland undergoes rapid growth and exhibits a high rate of steroidogenesis throughout fetal life. In addition to cAMP-dependent processes regulating steroidogenesis and possibly growth of the FZ, evidence is accumulating that cAMP-independent mechanisms are also involved. The purpose of this study was to determine if the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase-C activity, stimulates steroidogenesis in FZ cells and to characterize protein kinase-C activity in FZ, neocortex zone, and anencephalic adrenal tissues. Adrenal glands were obtained from first and second trimester abortions and two anencephalic fetuses. The FZ was dissected from the neocortex. In some experiments, dispersed FZ cells were incubated in the presence and absence of ACTH and TPA for 3 h. TPA and ACTH stimulated steroidogenesis 2- and 5-fold, respectively. In other experiments, the separated zones and anencephalic adrenal tissues were homogenized, and the homogenates were subjected to DEAE-cellulose column chromatography. A single peak with phospholipid- and calcium-dependent activity was found. Subcellular distribution studies demonstrated greatest activity in the cytosolic fraction. The specific activity of protein kinase-C was significantly greater in FZ than neocortex zone, whether expressed per mg protein or per microgram DNA content. The activity in anencephalic tissue was low. In addition, protein kinase-C (80,000-dalton molecular size protein) was detected in adrenal tissues after electrophoresis and immunoblotting using an antibody directed against protein kinase-C. Greater amounts of protein kinase-C were detected in FZ tissue than in NC or anencephalic adrenal tissue. These results indicate that the lower activities of protein kinase-C in neocortex and anencephalic adrenal tissues were due to low amounts of enzyme rather than inactive enzyme. In summary, TPA-stimulated steroidogenesis in fetal zone cells and fetal zone cells contained greater activity and a greater amount of protein kinase-C than neocortex cells. Minimal activity and enzyme protein were found in anencephalic tissues. These results suggest that cAMP-independent mechanisms may play a role in fetal adrenal steroidogenesis.     

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.     

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.     

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.     

Cholesterol synthesis and accretion within various tissues of the fetal and neonatal rat

The rate of cholesterol synthesis is reported to be higher in fetal relative to adult rats. Along with the observation that maternal diets high in fat and cholesterol are unable to alter the rate of cholesterol synthesis in the fetus, this has been taken as indirect evidence that the fetal rat meets its cholesterol needs through de novo synthesis. This study quantified the rates of cholesterol synthesis and accumulation in the liver, brain, intestine, and carcass of the fetal and neonatal rat and the placenta to determine whether these developing tissues are able to support their own cholesterol needs without the uptake of plasma lipoprotein cholesterol. The rate of cholesterol synthesis was determined in vivo using [3H]water. The rate of cholesterol accumulation was determined by calculating the difference in tissue cholesterol content between 2 subsequent days of development. Total fetal body cholesterol synthesis was sufficient to support the rate of cholesterol accumulation. Fetal and neonatal liver synthesized cholesterol at a rate in excess of cholesterol accumulation, suggesting hepatic secretion of cholesterol into the plasma. Before the onset of suckling, the rates of de novo cholesterol synthesis in the intestine, brain, and carcass were also sufficient but not higher than the need for cholesterol accretion. After the establishment of suckling, the rate of cholesterol accumulation in the intestine and carcass was in excess of synthesis, suggesting that neonatal tissues derive some of their cholesterol from dietary milk or liver. These studies suggest that the perinatal rat does not require exogenous cholesterol to support tissue cholesterol accretion. However, the fetal liver may support cholesterol accretion in other tissues through rates of synthesis in excess of accumulation and secretion into plasma. The placenta may derive some cholesterol from the maternal and/or fetal plasma.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase in outer versus inner cortices of the guinea pig adrenal: effects of adrenocorticotropin and dexamethasone

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis, was examined in the lipid-filled outer cortical cells and smooth endoplasmic reticulum-filled inner cortical cells of guinea pig adrenals. The specific activity of HMG CoA reductase was higher in microsomes obtained from the outer cortex, but was stimulated by ACTH and suppressed by dexamethasone (DEX) in both regions. Immunoblots of the microsomal proteins revealed a higher concentration of immunoreactive HMG CoA reductase (mol wt 94K) in microsomes from outer cortical cells. Changes in the intensity of this band corresponded to changes in activity after treatment with ACTH and DEX. However, quantitative immunoassay revealed that changes in activity in the inner zone after ACTH and in both zones after DEX were greater than could be accounted for by changes in immunodetectable protein, implying that other regulatory factors play a role in these cases. Incubation of outer and inner cortical tissues in vitro showed that outer cortical tissue had a greater capacity to incorporate [14C]acetate into cellular cholesterol (free and esterified) and into secreted steroid than did inner cortical tissue. ACTH enhanced the incorporation of [14C]acetate by outer cortical tissues into secreted steroid, but had little effect on incorporation by inner cortical tissues. Assay of the medium indicated that outer cortical tissues secreted much more steroid and increased secretion in response to ACTH, whereas inner cortical tissues secreted little steroid and were little affected by ACTH. Taken together, these results show that outer cortical cells have a greater capacity for cholesterol synthesis and that enhancement of this capacity after ACTH treatment is correlated with an increase in HMG CoA reductase protein. On the other hand, while the level of HMG CoA reductase immunodetectable protein and activity in inner cortical cell microsomes is considerable and appears to increase after ACTH treatment, it is not translated into an ability to synthesize cholesterol. This suggests that the activity of the immunodetectable HMG CoA reductase in the inner zone is suppressed in vivo and that the prominent smooth endoplasmic reticulum found in inner cortical cells has additional functions. The inability of the inner zone to synthesize cholesterol accounts in part for its low steroid production.     

Sterol sulfate metabolism in the adrenals of the human fetus, anencephalic newborn, and adult

The sulfurylation of pregnenolone (delta 5P), dehydroepiandrosterone (D) and cholesterol in subcellular fractions of the separated zones of normal human fetal adrenals as well as the mitochondrial sterol desmolase activities are investigated. The values obtained are compared to those found in adrenal tissues of anencephalics and adults. The 3 beta-hydroxysteroid sulfotransferase activity is localized principally in the cytosolic fraction of the tissues assayed. The highest rate of sulfurylation of delta 5P and D is found in the cytosol of the fetal zone (5 nmol min-1 mg protein-1). The corresponding activities in fetal neocortex, anencephalic, and adult adrenocortical tissues are one tenth of that in the fetal zone. Cholesterol sulfurylation is not detectable under similar assay conditions. The specific activities of cholesterol and cholesteryl sulfate desmolases are 3- 4-fold greater in the fetal zone than in the neocortex. Cholesterol desmolase activity, but not cholesteryl sulfate desmolase activity, is found in adrenal tissues of the anencephalic newborn and adult adrenal cortex. It is concluded that the high levels of delta 5P sulfate and D sulfate found in human fetal plasma arise not only as a consequence of the high capacity of the human fetal adrenal fetal zone to sulfurylate the unconjugated compounds, but also as a result of the conversion of cholesteryl sulfate to delta 5P sulfate and thence to D sulfate.     

Differential activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the adrenal cortex

Cholesterol metabolism and steroidogenesis in the outer (zona fasciculata/glomerulosa) and inner (zona reticularis) zones of the adrenal cortex were examined in the guinea pig. It is known from previous studies that the content of cholesterol in the inner zone is considerably lower than that in the outer zone, although basal low density lipoprotein (LDL) receptor activity is similar in the two zones. To further explore cholesterol metabolism in the guinea pig adrenal cortex, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis, has been examined for which this paper forms the initial report. It was found that the basal specific activity of HMG-CoA reductase was similar in the outer and inner adrenocortical zones (approximately 230 pmol mevalonate formed/min X mg microsomal protein). The administration of ACTH caused 4- and 5-fold increases in HMG-CoA reductase activity in the outer and inner zones, respectively. In fact, the increase in HMG-CoA reductase activity with ACTH treatment was always greater for the inner zone than for the outer zone. This is in contrast to LDL receptor activity, which does not increase in the inner zone as it does in the outer zone with ACTH treatment. When dexamethasone was administered, HMG-CoA reductase activity decreased in the outer zone by about 50%, while there was no change in reductase activity in the inner zone. The latter finding is similar to what happens with LDL receptor activity during dexamethasone administration. Why suppression of endogenous ACTH had no effect on HMG-CoA reductase activity in the inner zone while exogenous ACTH administration caused a marked increase in enzyme activity is not clear, but may be related to phosphorylation/dephosphorylation mechanisms. Based on the use of sodium fluoride in solutions to block HMG-CoA reductase phosphatase, evidence is presented which indicates that a pharmacological dose of ACTH alters the phosphorylation/dephosphorylation status of HMG-CoA reductase in the inner adrenocortical zone, but not in the outer cortical zone.     

Impaired adrenal reserve in the Watanabe Heritable Hyperlipidemic rabbit: implications for LDL-receptor function in steroidogenesis

The cholesterol required for steroidogenesis may be provided by the novo biosynthesis or through the delivery of cholesterol by the circulating lipoproteins. By studying adrenocortical function, structure and biosynthetic capacity in an animal model devoid of the classical, high-affinity low density lipoprotein (LDL) receptor pathway, the respective roles of de novo cholesterolgenesis and lipoprotein cholesterol delivery were investigated. The Watanabe Heritable Hyperlipemic (WHHL) rabbit lacks the LDL-receptor pathway. The activity of the rate-limiting enzyme in cholesterolgenesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was 4- to 15-fold greater than normal in the WHHL adrenal gland. The basal corticosterone concentrations were normal in the WHHL rabbit; however, the corticosterone concentration increased by less than 50% of normal after an intravenous ACTH injection. Electron-microscopic evaluation of adrenocortical cells from the WHHL rabbits disclosed significantly increased mitochondrial surface area and diminished amounts of cytosolic lipid and lysosomal area. These data indicate that the mammalian adrenal gland utilizes endogenously synthesized cholesterol as well as cholesterol delivered through the LDL-receptor pathway. Moreover, in the absence of the LDL-receptor pathway, endogenously produced cholesterol is sufficient for normal basal glucocorticoid function.     

Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukemia cells

Cholesterol levels are abnormally increased in many acute myeloid leukemia (AML) samples exposed in vitro to chemotherapy. Blocking these acute cholesterol responses selectively sensitizes AML cells to therapeutics. Thus, defining the molecular mechanisms by which AML cells accomplish these protective cholesterol increments might elucidate novel therapeutic targets. We now report that the levels of mRNAs encoding the cholesterol synthesis-regulating enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the cholesterol-importing low-density lipoprotein (LDL) receptor were both increased by daunorubicin (DNR) or cytarabine (ARA-C) treatments in almost three fourths of cultured AML samples. However, less than one third of AML samples significantly increased LDL accumulation during drug treatments, suggesting that de novo synthesis is the primary mechanism by which most AML cells increase cholesterol levels during drug exposures. LDL increments were not correlated with cholesterol increments in ARA-C-treated AML samples. However, LDL and cholesterol increments did correlate in DNR-treated AML samples where they were measured, suggesting that a subset of AMLs may rely on increased LDL accumulation during treatment with particular drugs. Our data suggest that cholesterol synthesis inhibitors may improve the efficacy of standard antileukemia regimens, but that for maximum benefit, therapy may need to be tailored for individual patients with leukemia.     

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 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.     

Steroidogenesis in dispersed cells of human fetal adrenal

Steroidogenesis in dispersed fetal zone cells of midtrimester human fetal adrenal was stimulated acutely by ACTH. Polypeptide hormones such as hCG, alpha MSH, ovine PRL, and LH did not produce a similar stimulation of steroidogenesis. The principal steroid products of ACTH-stimulated fetal adrenal cells were dehydroisoandrosterone sulfate, pregnenolone, pregnenolone sulfate, and 17 alpha-hydroxypregnenolone. Only minimal production of the delta 4-3-ketosteroids, cortisol, corticosterone, and progesterone, was observed. Cyanoketone (2 alpha-cyano-4,4,17 alpha-trimethyl-17 beta-hydroxyandrost-5-en-3-one; an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity) treatment of the cells caused only a minor increase in 3 beta-hydroxysteroid formation, confirming that 3 beta-hydroxysteroid formation is the principal steroidogenic fate of cholesterol in these cells. SU-10603 [7-chloro-3,4-dihydro-2-(3-pyridyl)naphthalen-1-(2H)one; a steroid 17 alpha-hydroxylase inhibitor] treatment of the cells caused a marked accumulation of pregnenolone sulfate, indicating that the C-19 steroids are produced from C-21 steroids in this tissue and possibly that dehydroisoandrosterone sulfate is synthesized directly from pregnenolone sulfate. ACTH-stimulated pregnenolone synthesis was inhibited by AY-9944 [trans-1,4-bis-(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride; an inhibitor of cholesterol biosynthesis]. Thus, cholesterol synthesized de novo was the likely steroidogenic precursor in the acute hormonally stimulated fetal adrenal cells.