NO-1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs

It is widely believed that high density lipoprotein-cholesterol (HDL-C) functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport (RCT), a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP binding cassette transporter called ABCA1 mediates the first step of RCT. NO-1886 has been proven to be highly effective at increasing HDL-C and reducing atherosclerosis. However, the mechanism of atherosclerosis inhibition for NO-1886 is not fully understood. In this study, the effects of NO-1886 on ABCA1 were investigated in high-fat/high-sucrose/high-cholesterol-fed Chinese Bama minipigs. Administration of NO-1886 (0.1 g/kg body weight/day) in the diet for 5 months significantly reduced atherosclerosis lesions and significantly increased plasma HDL-C and apolipoprotein A-I levels. The mRNA and protein levels of ABCA1 in the liver, retroperitoneal adipose tissue, and aorta were increased by NO-1886 as well. Multivariate linear regression analysis showed that the levels of LPL in plasma and the levels of ABCA1 in aorta were independently associated with the atherosclerotic lesion area. In addition, NO-1886 upregulated liver X receptor alpha and affected the expression of scavenger receptor class B type I in the liver. These results demonstrate that the mechanism of atherosclerosis inhibition for NO-1886 is associated with its effect on ABCA1.     

NO-1886改善糖尿病小型猪的糖代谢

合成化合物NO-1886是一种脂蛋白脂酶活化剂,已被证明其可降低血浆TG并能升高HDLC的浓度．后又发现其还有降低高脂高蔗糖诱发糖尿病兔血浆葡萄糖浓度的作用．对高脂高蔗糖饲料喂养的小型猪脂肪细胞大小、血浆TNF—α和FFA的水平以及NO-1886对其影响进行了研究,结果发现,脂肪细胞明显肥大．血浆TNF-α和FFA以及空腹血糖水平均增高,且引起胰岛素抵抗．添加了l％NO-1886后．脂肪细胞增大被抑制,血浆TNF—α、FFA和空腹血糖的浓度均显著降低,血浆葡萄糖清除率和胰岛素分泌急性相都有了明显改善．以上结果说明,NO-1886可能通过抑制脂肪蓄积、降低血浆TNF-α和FFA的浓度而改善高脂高蔗糖饲料引起的小型猪的糖代谢紊乱．     

The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells.

The sources of cholesterol for steroid hormone production were examined using bovine adrenocortical (BAC) cells in primary culture. The experiments were designed to determine the effects of lipoproteins on cortisol production and the level of BAC cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human low-density lipoprotein (hHDL); none have used the homologous bovine lipoproteins. BAC cells treated with corticotropin (ACTH) in a medium devoid of lipoproteins increased and maintained cortisol production 7- to 20-fold above basal levels. Under such conditions ACTH also increased the rate of HMG-CoA reductase activity. Inhibition of HMG-CoA reductase with mevinolin inhibited cortisol production by 85%, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40-fold above basal levels if hLDL (100 micrograms/ml) was included in the incubation medium. Human LDL also suppressed the levels of HMG-CoA reductase in a concentration-dependent fashion. Human HDL was without effect on either BAC cell steroidogenesis of HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisol production and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 micrograms/ml) increased the ability of BAC cells to produce cortisol production. Bovine HDL (bHDL) also was able to decrease HMG-CoA reductase, but not to the extent caused by hLDL or bLDL. These data demonstrate that bovine adrenal cells can use bHDL as a source of cholesterol for steroid hormone production. These findings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL.     

Diabetic lipoproteins and adrenal aldosterone synthesis--a possible pathophysiological link?

An increased prevalence of diabetes mellitus (DM) has been reported in patients with primary aldosteronism (PA). DM is associated with abnormal structure and metabolism of circulating lipoproteins, which normally serve as a major source of cholesterol for adrenocortical steroidogenesis. The present study has been designed to investigate the effect of diabetically modified lipoproteins on adrenocortical aldosterone synthesis. Lipoproteins (VLDL, LDL, HDL) isolated from healthy volunteers, were subjected to oxidation or glycoxidation in the presence of sodium hypochlorite (3 mmol/l) or glucose (200 mmol/l), and aldosterone synthesis in human adrenocortical cells (H295R) was examined. Native and glycoxidized VLDL had greatest stimulatory effect on aldosterone production by 15-fold and 14-fold, respectively. At the molecular level, these VLDL produced maximum increases in Cyp11B2 mRNA level up to 17-fold. Experiments with the highly selective scavenger receptor class B type I (SR-BI) inhibitor BLT-1 revealed that cholesterol uptake from native and glycoxidized HDL and VLDL for hormone production is considerably mediated by SR-BI. Western blot analysis of extracellular signal-regulated kinase (ERK 1/2) phosphorylation and experiments with the MEK inhibitor U0126 indicated a specific mechanistic role of the ERK cascade in lipoprotein-mediated steroid hormone release. In summary, diabetic dyslipidemia and modification of circulating lipoproteins may promote adrenocortical aldosterone synthesis.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Genistein administration decreases serum corticosterone and testosterone levels in rats

The phytochemical flavonoid genistein has been shown to act as a potent competitive inhibitor of human adrenocortical 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase activities in vitro [J. Steroid Biochem. Molec. Biol. 2002; 80: 355-363]. In the present study, we evaluated the effects of large amounts of genistein continuously administered to weanling rats, particularly on steroidogenesis at the pubertal stage in vivo. Serum concentrations of free and total genistein were significantly higher in the 40 mg/kg genistein administration group when compared with the control group. In genistein administered rats, adrenal weight was significantly higher. Furthermore, a clear expansion of cells was observed in hematoxylin and eosin stained tissue at the zona fasciculata and zona reticularis of the adrenal cortex. However in the testis, no differences in weights or histologic changes were observed. Serum corticosterone concentration significantly decreased to 50% of control levels by 40 mg/kg genistein administration and testosterone also tended to decrease with this dose of genistein. On the other hand, although serum follicle stimulating hormone was unchanged, adrenocorticotropic hormone and luteinizing hormone levels increased with genistein administration. These results suggest a significant effect of genistein on steroidogenesis in the adrenal gland and testis of rats, and this effect appeared to be more evident on steroid production in adrenals than in testis in vivo.     

Morphologic change and elevation of cortisol secretion in cultured human normal adrenocortical cells caused by mutant p21K-ras protein

In our previous study on the tumorigenesis of human functional adrenal tumors, we observed a high frequency of K-ras point mutations in clinical specimens. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells to express the mutated K-ras gene. The mRNA level of steroidogenic enzymes such as cholesterol sidechain cleavage enzyme (P450SCC), 17alpha-hydroxylase/17,20-lyase (P450c17), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the mutant K-ras stably transfected cells were elevated. Cultured normal adrenocortical cells from donors and patients with adrenocortical tumors were then transfected with mutant K-ras expression plasmids constructed from human adrenal tumors. Stable transfectants grew faster than normal cells. Additionally, morphologic change was observed in the transfected cells. Moreover, when the synthesis of hormones was analyzed, the mRNA of P450SCC, P450C17, and 3betaHSD was found to have increased, and the level of cortisol was 18 to 25 times that in control cells. The increased steroid hormone production in mutant K-ras-transfected cells was reversed by lovastatin, a pharmacologic inhibitor of p21ras function. These results, combined with previous reports of steroidogenic K-ras in bovine adrenocortical cells, suggest that the K-ras oncogene is involved in steroidogenesis in human adrenocortical cells.     

Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 2. Reversibility of taxol's inhibition of basal and ACTH-induced steroidogenesis is unaccompanied by reversibility of taxol-induced changes in cell ultrastructure

Taxol inhibits the basal and ACTH-stimulated steroidogenesis of cultured mouse adrenocortical tumor cells, presumably by preventing the arrival of cholesterol in mitochondria. In these cells, taxol polymerizes and rearranges microtubules, disperses SER masses, disrupts the Golgi, and impedes the formation of cholesterol-containing lysosomes. However, taxol's alterations in ultrastructure appear likely to permit both a microtubule-based organelle transport proposed to bring mitochondria of unstimulated cells close to alternate sources of cholesterol--the SER and lipid droplets--and postulated ACTH-caused increases in these encounters. Conceivably, taxol may prevent the transfer of cholesterol from the SER and lipid droplets to mitochondria, once the meetings are achieved. To investigate this possibility, we determined the reversibility of taxol's ultrastructural effects and inhibition of steroidogenesis. Primary cultured adrenal tumor cells were incubated for 4 hr with and without ACTH (10 mU/ml). with taxol (50 micrograms/ml), and with ACTH and taxol 50 simultaneously. Some cultures from each set were washed with fresh medium and re-incubated for 1.5 hr. with and without ACTH. Media taken from cultures at the ends of pre- and post-washout incubations were analyzed for the presence of secreted steroids. Sample cultures were fixed for electron microscopy at the ends of both incubations. Data derived from pre-washout incubations confirmed previous reports of taxol's ultrastructural changes and inhibition of steroidogenesis. When cells recovered from taxol in the absence of ACTH, the inhibition of steroidogenesis was completely reversed. In the presence of ACTH, ex-taxol-treated cells demonstrated a "rounding up' and an increased steroid production that are characteristic responses to the hormone. However, in all cases, there was a persistence of taxol's alterations in organelle numbers and arrangements. Our findings establish that the ultrastructural effects of taxol which we recorded cannot prevent mitochondria of unstimulated and ACTH-stimulated adrenal tumor cells from gaining cholesterol. They strengthened the possibility that in pre-washout incubations, taxol allowed organelle motility to bring mitochondria adjacent to cholesterol-containing SER tubules and lipid droplets, but inhibited steroidogenesis by preventing the cholesterol transfer. Taxol might limit the availability of a protein required for the transfer, an effect not visible in our electron micrographs.     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Regulation of the cholesterol ester cycle of cultured Leydig tumor cells

The MA-10 Leydig tumor cells take up low-density lipoprotein (LDL) from the medium and store the LDL-derived cholesterol as cholesterol esters that can be subsequently mobilized and used for steroid hormone synthesis. The present studies investigate the mechanisms by which cAMP acutely regulates the cellular content of cholesterol esters. In the absence of cholesterol utilization for steroidogenesis, cAMP stimulates cholesterol ester hydrolysis and ester resynthesis proportionally. The augmentation of ester hydrolysis by cAMP is completely matched by increased activity of the acyl-coenzyme-A:cholesterol acyltransferase and thus does not regulate cellular cholesterol ester concentration per se. The more important action of cAMP is to interrupt the cycle of hydrolysis and ester resynthesis by decreasing cholesterol re-esterification. In cells actively synthesizing steroid hormones, cholesterol reesterification is decreased by 82%. The decrease in cholesterol re-esterification occurs because cAMP directs cholesterol normally destined for re-esterification into steroid synthesis; simply blocking the utilization of cholesterol for steroidogenesis completely prevents net cholesterol ester hydrolysis and increases the cellular rate of cholesterol esterification.     

Studies on lipoprotein and adrenal steroidogenesis: II. Utilization of low density lipoprotein- and high density lipoprotein-cholesterol for steroid production in functioning human adrenocortical adenoma cells in culture

We examined the utilization of human low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol for steroid production in primary monolayer culture cells from adenomas of primary aldosteronism and Cushing's syndrome and an adrenal of nodular hyperplasia of Cushing's syndrome. We compared the data obtained with findings in the case of cultured normal human adrenocortical cells. In the presence of 10(-7) M adrenocorticotropin (ACTH), the addition of either LDL or HDL to the culture medium at a cholesterol concentration of 100 micrograms/ml led to a significant increase in the daily secretion rates of cortisol, dehydroepiandrosterone sulfate (DHEA-S) and aldosterone in the adenoma and nodular hyperplasia cells, as in the normal cells. Although LDL greatly increased the secretion of steroid hormones, no significant difference in steroid secretion following the treatments with LDL and HDL were observed in these cultured cells. The contribution of endogenous cholesterol to steroid production was also high, thereby indicating that the neoplastic transformation did not have untoward effects. Cells from adenomas of primary aldosteronism secreted not only aldosterone, but also cortisol and DHEA-S. The daily secretion rates of these steroids were markedly increased when ACTH was added to the medium. With prolonged exposure to ACTH, however, the rate of aldosterone secretion showed a gradual decrease with the incubation time. This decrease might be due to the impaired conversion of corticosterone to 18-hydroxycorticosterone. In case of adenomas in patients with Cushing's syndrome, the secretion of steroid hormones varied in quantity and quality, depending on the type of plasma cortisol response to the rapid ACTH test in vivo, thereby suggesting that the adrenocortical adenoma of Cushing's syndrome might be divided into two subtypes. These results indicate that human functioning adrenocortical adenoma cells utilize plasma lipoproteins as a source of cholesterol for steroidogenesis during the prolonged stimulation of steroid secretion.     

The effect of hypocholesterolemic drug treatment on adrenocortical cell function

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.     

The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis

Addition of rat or human high density lipoproteins (HDL) or human low density lipoproteins (LDL) to rat adrenocortical cells in vitro was found to enhance steroid production and increase cell cholesterol content. These effects of HDL were not observed in cultured mouse Y-1 adrenal cells, suggesting that rat adrenal cells possess a specific mechanism for uptake of HDL cholesterol not found in Y-1 cells. The effects of HDL were most marked on cells previously stimulated with adrenocorticotropin (ACTH) and depleted of their endogenous cholesterol stores. Such cells were prepared either by treatment in vivo with 4-aminopyrazolopyrimidine or in vitro with ACTH (10(-7) M) in lipoprotein-poor media. Steroid production by treated cells exhibited a saturable dependence on media HDL concentration. In addition to enhancing ACTH stimulated steroid production, addition of HDL also resulted in a saturable concentration-dependent increase in cell cholesterol content. Both aminoglutethimide and cycloheximide were found to inhibit HDL-enhanced steroid production. Finally, addition of HDL to short term incubations (5 1/2 h) of ACTH-treated cells caused no change in the rate of incorporation of 14C-acetate into cholesterol or corticosterone. These results indicate that rat adrenocortical cells possess a specific, saturable, ACTH-dependent mechanism for uptake of HDL cholesterol. Moreover, cellular uptake of HDL cholesterol exceeded by at least 4-fold the amount of cholesterol associated with HDL apoprotein degraded by the cells, suggesting that utilization of HDL cholesterol does not require endocytosis and lysosomal degradation of the entire HDL particle.     

Effects of mevinolin,an inhibitor of cholesterol synthesis,on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture

Summary Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL₃) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosalike into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.     

The effects of taxol, a microtubule-stabilizing drug, on steroidogenic cells

The effects of taxol on steroid production and microtubule polymerization were examined using Y-1 adrenocortical tumor cells, MLTC-1 Leydig tumor cells, and primary cultures of bovine adrenocortical cells. Taxol inhibited the following steroidogenic processes within the Y-1 and MLTC-1 cells: (1) hormonal increase of steroid production, (2) dibutyryl cyclic AMP-increased steroid production, and (3) hormone-stimulated pregnenolone production. The inhibitory action of taxol was concentration dependent and also resulted in an increase in cytoplasmic microtubules. In addition, the inhibitory action of taxol on hormone-stimulated steroid production was reversible. Taxol appeared to inhibit cholesterol movement to the mitochondrial site of cholesterol side-chain cleavage enzyme but did not affect overall protein synthesis. Interestingly, taxol did not affect hormone-stimulated steroid production in bovine adrenocortical cells. This lack of inhibition may correspond to the ultrastructural observation that microtubule bundling after taxol treatment was observed in the tumor cells but not in similarly treated bovine adrenal cells. With this conflicting information between cell types, a direct relationship between taxol treatment and inhibition of steroid production has not been established. However, these results suggest that taxol alters the rate of transport of cholesterol to the cholesterol side-chain cleavage enzyme within the steroidogenic tumor cells.     

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.     

Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells

In previous studies we demonstrated that peripheral-type benzodiazepine receptors (PBR) were coupled to steroidogenesis in several adrenocortical and Leydig cell systems (Mukhin, A.G., Papadopoulos, V., Costa, E., and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9813-9816; Papadopoulos, V., Mukhin, A.G., Costa, E., and Krueger, K.E. (1990) J. Biol. Chem. 265, 3772-3779). The current study elucidates the specific step in the steroid biosynthetic pathway by which PBR mediate the stimulation in steroid hormone production. The adrenocorticotropin (ACTH)-responsive Y-1 mouse adrenocortical cell line was used to compare the mechanisms by which ACTH and PK 11195 (a PBR ligand) stimulate steroidogenesis. The effects of these agents were studied at three stages along the steroid biosynthetic pathway: 1) secretion of 20 alpha OH-progesterone by Y-1 cell cultures; 2) pregnenolone production by isolated mitochondrial fractions; 3) quantities of cholesterol resident in outer and inner mitochondrial membrane fractions. Steroid synthesis stimulated by ACTH was blocked by cycloheximide, an effect documented by other laboratories characterized by an accumulation of mitochondrial cholesterol specifically in the outer membrane. In contrast, PK 11195-stimulated steroidogenesis was not inhibited by cycloheximide, and the magnitude of the stimulation was markedly enhanced when the cells were pretreated with cycloheximide and ACTH. When isolated mitochondria were used, stimulation of pregnenolone production by PK 11195 was largely independent of exogenously supplied cholesterol, indicating that PBR act on cholesterol already situated within the mitochondrial membranes. This phenomenon was found to be the result of a translocation of cholesterol from outer to inner mitochondrial membranes induced by the PBR ligand. These studies therefore suggest that mitochondrial intermembrane cholesterol transport in steroidogenic cells is mediated by a mechanism coupled to PBR.