Cholesterol Accumulation in Adrenocortical Mitochondria after ACTH-stimulation

Rat adrenocortical cell suspensions (106 cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min. About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium. Aminoglutethimide at 50 ƒÊM inhibited the steroidogenesis to 16 %. Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined. The mitochondria of the cells incubated without the drugs contained 25.2 fig cholesterol/mg protein. Cholesterol content increased by 10 % in the mitochondria of the ACTHstimulated cells. The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143 % of cholesterol compared to those of the nontreated cells. When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200 % compared to that of the control rats. These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells. The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy. Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter. Mitochondria of steroidogenic tissues contain a cholesterol side chain cleavage enzyme, cytochrome P-450scc, in their inner membrane, and either a reaction catalyzed by this enzyme system or transport of cholesterol to the enzyme catalytic site through the mitochondrial membrane is believed to be a rate-determining step in steroidogenesis (Garren et al., 1971 ; Kimura, 1981 ; Schulster, 1974 ; Simpson, 1979 ; Stone and Hechter, 1954). This poses an interesting question about the status of cholesterol in the mitochondria of these tissues, because the mitochondria of nonsteroidogenic tissues are known not to contain much cholesterol in their membrane. Where cholesterol is localized in the subfraction of adrenocortical mitochondria and how it is transported to the catalytic site of cytochrome P-450scc have been major questions inquired into by a number of biochemists and endocrinologists (Mahaffee et al., 1974 ; Simpson et al., 1978). Received January 10, 1984 178 MUKAI et al. Endocrinol. Japon. April 1984 For instance, Privalle et al. (1983) observed that cholesterol accumulates in the mitochondrial fractions of rat adrenal glands during ether stress when the animal is pretreated with aminoglutethimide. Kido and Kimura (1981) and Ohno et al. (1983) discussed the nature of a labile protein factor which could help cholesterol to move into the mitochondrial inner membrane and to place itself in the catalytic site of the enzyme. Crivello and Jefcoate (1980) reported participation of microtubules and microfilaments in achieving the appropriate transportation of cholesterol, while Hall et al. (1981) discussed a role of calmodulin in the same process. It has been known that a polyene antibiotic, filipin, can specifically bind to membrane cholesterol and form filipin-sterol complexes (De Kruijff and Demel, 1974 ; Kinsky, 1970 ; Norman et al., 1976). These complexes are easily recognized in freezefracture images as protuberances or pits 25-30 nm in diameter (Fujita et al., 1981 ; Fujita et al., 1982 ; Matsuda et al., 1983 ; Montesano, 1979 ; Nakajima and Bridgman, 1981). Although a number of studies have recently been carried out on the distribution of cholesterol in plasma-and cyto-membranes of various organs using freeze-fracture images of filipin-treated tissues, no information is yet available about this subject of adrenocortical mitochondria. In order to confirm the results of previous studies on cholesterol movement in adrenocortical cells and to obtain morphological information about the presence of cholesterol in the mitochondria, we have chemically analyzed the cholesterol content in the adrenocortical mitochondria during the steroidogenic stimulation of the cells in vivo and in vitro, and, at the same time, have microscopically observed the presence of filipincholesterol complexes in the mitochondria of these cells. Materials and Methods Animals and Chemicals Female Sprague-Dawley rats, weighing 160180 g, were used throughout this study. Synthetic ACTH1-24, Cortrosyn, was obtained from Daiichi Seiyaku, Co. Aminoglutethimide was a gift from Ciba-Geigy, Co. Collagenase (Type I) and deoxyribonuclease were purchased from Worthington and Sigma, respectively. Steroids were products of either Makor Chemicals or Steraloids. Filipin was generously donated by the Upjohn Co., Kalamanzoo, Mich. All other chemicals were from local sources and of the highest purity available commercially. Preparation of Adrenocortical Cell Suspensions The cell suspensions were prepared from rat adrenal glands as described by Haning et al. (1970) with some modification. At a time between 8 and 10 am, rats were sacrificed by decapitation. The adrenal glands were quickly excised and placed in Krebs-Ringer bicarbonate buffer (120 mM NaCl, 1.5 mM CaCl2, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 22 mM NaHCO3 and 0.2 % glucose, pH 7.4) containing 1 % bovine serum albumin (KRBGA). Fat tissues around the gland were trimmed off. The glands were cut into small pieces with a razor blade, and the fragments obtained from 30 adrenal glands were suspended in 10 ml of KRBGA containing 2 mg/ml collagenase and 0.05 mg/ml deoxyribonuclease and incubated in an atmosphere of 95 % air and 5 % CO2 for 45 min at 37•Ž. The digested fragments were further dispersed by pipetting. The cell suspensions were centrifuged at 400 •~ g for 7 min. The pellets were washed once by Krebs-Ringer bicarbonate buffer containing 4 % bovine serum albumin and the recovered suspensions were filtered through a Nylon mesh to remove tissue debris. The free cell suspensions thus obtained were preincubated for 60 min at 37•Ž in an atmosphere of 95 (76 air and 5 % CO2, then washed once, and resuspended in KRBGA. Viability of the cells, estimated in 0.04 % Trypan-blue solution, was usually higher than 90 %. According to these procedures, approximately 3 •~ 105 viable cells were obtained from one adrenal gland. Measurement of Steroidogenesis by Adrenocortical Cells and Determination of Cholesterol Vol. 31, No. 2 CHOLESTEROL IN ADRENAL MITOCHONDRIA 179 In a typical experiment, 5 x 105 cells were incubated with, or without, ACTH (40 nM) in 1 ml of KRBGA in an atmosphere of 95 (I) air and 5 % CO2 for 60 min. The incubation was terminated by centrifugation at 1,100 X g for 10 min. The supernatants were measured for steroid content by high performance liquid chromatography (HPLC) as described previously (Kim et al., 1983 ; Momoi et al., 1983 ; Yagi et al., 1983). The pellets were homogenized in 0.25 M sucrose containing 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, using a glass homogenizer with a teflon pestle. The homogenate was centrifuged at 900 ×g for 10 min and the supernatant fraction obtained was again centrifuged at 8,000•~ g for 15 min. The pellets recovered from the second centrifugation were used as the mitochondrial fraction. The mitochondrial pellets were frozen and thawed to disrupt the membrane. To the pellets 5ƒ¿-cholestane was added as an internal standard, and cholesterol was extracted with dichloromethane. Cholesterol was determined by gas chromatography using an OV-1 column (3 mm ×2m) at a column temperature of 250•Ž and an injector temperature of 270•Ž. The content of the ester form of cholesterol was estimated as the difference between the cholesterol content after alkaline digestion (1 M KOH) of the sample and that before the saponification. The experiments were performed at least three times under the same incubation conditions. The average content of the mitochondrial cholesterol in the non-treated cells was 25.2 •} 2.2 fig/mg protein. Preparation of Adrenocortical Mitochondria from Rats Suffering from Ether Stress This experiment was carried out in essentially the same way as described by Privalle et al (1983). Six rats were injected intraperitoneally with 10 mg of aminoglutethimide dissolved in 1 ml of saline. Control groups received saline alone. The animals were immediately transferred into a container saturated with ether gas, and maintained in anesthetic conditions for 20 min. After the ether stress, the animals were killed and the adrenals were excised. The mitochondrial fractions were prepared from the whole adrenal glands in the manner described above. Freeze Fracture Electron Microscopy The mitochondrial pellets were fixed with 2 % glutaraldehyde solution bufferred at pH 7.4 with 0.1 M cacodylate for 5-10 min, and treated with 0.02-0.05 % filipin contained in the same fixative for 12-24 h at room temperature. The samples were soaked in 30 % glycerol solution for 20 min and rapidly frozen in melting nitrogen slush. The specimens were cleaved, shadowed with platinum-palladium, and stabilized with carbon in a JEOL-7000 type universal freeze specimen preparation device. The replicas made were examined in a Hitachi H-500 type electron microscope. Protein Determination Protein concentration was determined in the manner described by Lowry et al. (1951) using bovine serum albumin as the standard.

Mitochondria of steroidogenic tissues contain a cholesterol side chain cleavage enzyme, cytochrome P-450scc, in their inner membrane, and either a reaction catalyzed by this enzyme system or transport of cholesterol to the enzyme catalytic site through the mitochondrial membrane is believed to be a rate-determining step in steroidogenesis (Garren et al., 1971 ;Kimura, 1981 ;Schulster, 1974 ;Simpson, 1979 ;Stone and Hechter, 1954).This poses an interesting question about the status of cholesterol in the mitochondria of these tissues, because the mitochondria of nonsteroidogenic tissues are known not to contain much cholesterol in their membrane.
Where cholesterol is localized in the subfraction of adrenocortical mitochondria and how it is transported to the catalytic site of cytochrome P-450scc have been major questions inquired into by a number of biochemists and endocrinologists (Mahaffee et al., 1974 ;Simpson et al., 1978).
For instance, Privalle et al. (1983) observed that cholesterol accumulates in the mitochondrial fractions of rat adrenal glands during ether stress when the animal is pretreated with aminoglutethimide.Kido and Kimura (1981) and Ohno et al. (1983) discussed the nature of a labile protein factor which could help cholesterol to move into the mitochondrial inner membrane and to place itself in the catalytic site of the enzyme.Crivello and Jefcoate (1980) reported participation of microtubules and microfilaments in achieving the appropriate transportation of cholesterol, while Hall et al. (1981) discussed a role of calmodulin in the same process.
It has been known that a polyene antibiotic, filipin, can specifically bind to membrane cholesterol and form filipin-sterol complexes (De Kruijff and Demel, 1974 ;Kinsky, 1970 ;Norman et al., 1976).These complexes are easily recognized in freezefracture images as protuberances or pits 25-30 nm in diameter (Fujita et al., 1981 ;Fujita et al., 1982 ;Matsuda et al., 1983 ;Montesano, 1979 ;Nakajima and Bridgman, 1981).Although a number of studies have recently been carried out on the distribution of cholesterol in plasma-and cyto-membranes of various organs using freeze-fracture images of filipin-treated tissues, no information is yet available about this subject of adrenocortical mitochondria.
In order to confirm the results of previous studies on cholesterol movement in adrenocortical cells and to obtain morphological information about the presence of cholesterol in the mitochondria, we have chemically analyzed the cholesterol content in the adrenocortical mitochondria during the steroidogenic stimulation of the cells in vivo and in vitro, and, at the same time, have microscopically observed the presence of filipincholesterol complexes in the mitochondria of these cells.In a typical experiment, 5 x 105 cells were incubated with, or without, ACTH (40 nM) in 1 ml of KRBGA in an atmosphere of 95 (I) air and 5 % CO2 for 60 min.The incubation was terminated by centrifugation at 1,100 X g for 10 min.The supernatants were measured for steroid content by high performance liquid chromatography (HPLC) as described previously (Kim et al., 1983 ;Momoi et al., 1983 ;Yagi et al., 1983).The pellets were homogenized in 0.25 M sucrose containing 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, using a glass homogenizer with a teflon pestle.Preparation of Adrenocortical Mitochondria from Rats Suffering from Ether Stress This experiment was carried out in essentially the same way as described by Privalle et al (1983).Six rats were injected intraperitoneally with 10 mg of aminoglutethimide dissolved in 1 ml of saline.Control groups received saline alone.The animals were immediately transferred into a container saturated with ether gas, and maintained in anesthetic conditions for 20 min.After the ether stress, the animals were killed and the adrenals were excised.The mitochondrial fractions were prepared from the whole adrenal glands in the manner described above.

Freeze Fracture Electron Microscopy
The mitochondrial pellets were fixed with 2 % glutaraldehyde solution bufferred at pH 7.4 with 0.1 M cacodylate for 5-10 min, and treated with 0.02-0.05% filipin contained in the same fixative for 12-24 h at room temperature.
The samples were soaked in 30 % glycerol solution for 20 min and rapidly frozen in melting nitrogen slush.The specimens were cleaved, shadowed with platinum-palladium, and stabilized with carbon in a JEOL-7000 type universal freeze specimen preparation device.
The replicas made were examined in a Hitachi H-500 type electron microscope.

Protein Determination
Protein concentration was determined in the manner described by Lowry et al. (1951)   * Six rats were injected with aminoglutethimide , and the animals were subjected to ether stress for 20 min.
Control rats received a saline injection and were exposed to ether.The mitochondrial pellets were prepared from adrenal glands and analyzed for cholesterol as described in Materials and Methods.
The numbers in parentheses represent the percentages of the control.2. The pretreatment with aminoglutethimide caused the accumulation of cholesterol in the mitochondrial fraction during the ether stress.
The increase was almost 100 % compared to the non-treated control group.With regard to these results obtained from the experiments conducted in vitro as well as in vivo on the mitochondrial fraction of rat adrenal glands, the following two points should be noted : (1) the mitochondria of rat adrenal cortex contained an appreciable amount of cholesterol, and (2) the cholesterol Fig.
3 pellets for the purpose of comparison.As shown in Fig. 2, the accumulations of numerous filipin-cholesterol complexes were found mainly in the inner membrane of about one third of the adrenocortical mitochondria as protuberances or pits of 25 to 30 nm in diameter.On the other hand, the rat liver mitochondria did not reveal any filipin-cholesterol complexes at all (Fig. 3).fraction increased to about 180 % in the steroid-producing adrenal glands compared to the non-producing glands.
We also performed the experiments on the animals under the ether tress, and obtained similar results to those of Jefcoate et al. (Table 2).A discrepancy between the degree of accumulation of cholesterol on in vitro (140 %) and in vivo (200 %) stimulation should be explored in the future.A possible explanation may be that the adrenocortical cells in KRBGA were under the unphysiological conditions which could not supply cholesterol from the extracellular source, while the adrenal glands of the animal under the ether stress could obtain cholesterol from the circulation via a low-density lipoprotein pathway.
The second question we asked about the cholesterol in the adrenocortical mitochondria concerned the localization of free cholesterol in the submitochondrial fraction.This question was asked, because it is generally known that mitochondria of the non-steroidogenic tissues do not contain much cholesterol in their membrane.To answer this question, the mitochondrial pellets were treated with filipin-containing fixative, and examined by freeze fracture electron microscopy.
The results clearly indicated the presence of filipin-cholesterol complexes in the inner membrane of mitochondria of the adrenocortical cells, which is the site of the ratedetermining reaction of steroidogenesis.Unfortunately, however, we could not find a significant morphological difference in the amount of the complexes between the mitochondria of the ACTH-stimulated cells and those of the non-stimulated cells.In contrast with these results, the liver mitochondria which had been treated with filipin under the same conditions as those for the adrenocortical mitochondria did not reveal the filipin-cholesterol complexes.These results are consistent with our previous observations obtained on other cells such as adrenal medullary cells, anterior pituitary cells, thyroid follicle cells, pancreatic exocrine cells and ovarian duct cells (Fujita et al., 1981 ;Fujita et al., 1982 ;Matsuda et al., 1983).
Fig. 2. Freeze-fracture image of mitochondria (M) of adrenocortical cells treated with filipin.Note the numerous filipin-cholesterol-complexes (C) in the inner mitochondrial membrane.•~ 37,500 The homogenate was centrifuged at 900 using bovine serum albumin as the standard.