The role of bovine lipoproteins in the
regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells
William E. Rainey, Raymond J. Rodgers,f’ and J. Ian Mason* Department of Obstetrics and Gynecology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, and *Department of Biochemistry, The University Texas Southwestern Flinders University
Medical Center, Dallas, Texas; and tDepartment of South Australia, Bedford Park, South Australia.
of
of Medicine,
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-methylglutatyl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human lowdensity lipoprotein (hLDL) and human high-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 ZO-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 8570, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40,fold above basal levels ifhLDL (100 uglml) 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 or HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisolproduction and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 uglml) 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 jindings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL. (Steroids 571167-173, 1992)
Keywords: sterol metabolism; lipoproteins;
bovine HMG-CoA reductase; bovine adrenal; cholesterol;
Introduction Adrenocortical cells require cholesterol as substrate for the production of steroid hormones.’ The uptake and use of cholesterol for steroid production by adrenal cells from plasma lipoproteins is well established in a number of mammalian species.*-” In addition, although the rate of cholesterol synthesis varies greatly from
Address reprint requests to William E. Rainey, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX 752354032. Received May 31, 1991; accepted October 16, 1991.
8 1992 Butterworth-Heinemann
cortisol
species to species, most adrenocortical cells examined have been shown to incorporate radiolabeled acetate or water into cholesterol and have quantifiable levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 2,3,5-9 Thus, adrenocortical cells have at least two sources of cholesterol for steroid synthesis. In vivo, 4-aminopyrazolopyrimidine treatment of rats blocks lipoprotein secretion from the liver, thus depriving the adrenal of an external source of cholesterol.‘OAdministration of this drug elevates cholesterol synthesis within the adrenal, suggesting at least a partial compensation for the depleted serum sources of cholesterol. Subsequent infusion of lipoproteins suppresses cholesterol synthesis. Such experiments detail a close association between plasma lipoproteins and Steroids,
1992, vol. 57, April
167
Papers
cholesterol biosynthesis within the adrenal gland. We have demonstrated such an association using in vitro primary cell cultures of bovine adrenocortical (BAC) cells.’ Cholesterol biosynthesis within these cells was elevated in the absence of exogenous lipoprotein after stimulation of steroid production. Addition of human low-density lipoprotein (hLDL) to the medium prevented this elevation.9 Thus the BAC cells in monolayer culture, under appropriate conditions, can apparently mimic the in vivo situation. These studies raised the question as to the requirements of homologous bovine lipoproteins in steroidogenesis and their regulation of cholesterol biosynthesis within the BAC cell. Previous studies have shown specific receptors for both human high density lipoproteins (hHDL) and hLDL on BAC cells.“*r2 Importantly, the level of bHDL in bovine plasma greatly exceeds the level of bovine LDL (bLDL), in contrast to the situation in human plasma. i3,i4 However, the role of the homologous lipoproteins in BAC cell steroidogenesis has not been examined. In the present study we demonstrate that bLDL, like hLDL, was capable of increasing the steroidogenic capacity of BAC cells. In addition, we show that the major bovine plasma lipoprotein, bovine HDL (bHDL), also could increase the rate of steroidogenesis in the presence of ACTH. Finally, bLDL but not bHDL was capable of fully suppressing the ACTH-promoted induction of HMG-CoA reductase activity.
Experimental Cell isolation and culture Dispersed adrenal cells were prepared from minced bovine adrenal cortices after treatment with collagenase (3.3 mglml from Clostridium histolyticus) and DNAse (0.1 mg/ml) in Hanks’ balanced salt solution containing HEPES (10 mM, pH 7.4) and bovine serum albumin (10 mglml). The collagenase and DNAse were obtained from Boehringer Mannheim (Indianapolis, IN, USA). The cells were harvested, washed, and placed in 35mm cell culture dishes (5 x 10’ cells per dish) in Ham’s F12 and Dulbecco’s modified Eagle’s (DME/F12) high-glucose medium (1 : 1, v/v) with fetal calf serum (lo%), gentamicin sulfate (O.Ol%), and HEPES (15 mM, pH 7.4). After the cells reached confluence (after approximately 5 days), the medium was changed to a lipoprotein-depleted medium (LPDM), that is, one containing DME/F12 high-glucose medium with insulin (5 pg/ml), transferrin (5 pg/ml), selenous acid (5 ng/ml), gentamicin sulfate (O.Ol%), HEPES (15 mM, pH 7.4), and lipoprotein-depleted fetal calf serum (1%). Experiments were initiated on cells that were conditioned in this medium for 24 hours. ACTH (Cortrosyn) was obtained from Organon (West Orange, NJ, USA). Mevinolin was obtained from Merck, Sharp & Dohme (West Point, PA, USA).
Lipoprotein
isolation
Blood was collected from bovine heifers after slaughter at the local abattoir and the blood was allowed to clot overnight. Adult bovine serum was also obtained freshly from Research Biogenics (Bastrop, TX, USA). Cellular elements were removed by centrifugation (2,000 rpm) and lipoproteins were isolated by sequential differential flotation in potassium bromide solutions. Ultracentrifugation was performed in Ti60 or Ti70 fixed-angle rotors in a Beckman (Porterville, CA, USA) L8-M centrifuge (60,000 rpm,
166
Steroids,
1992, vol. 57, April
4 C). Dialysis was performed against 0.15 M NaCl solution containing EDTA (O.Ol%, pH 7.4) using Spectraphor number 2 dialysis tubing (Spectrum Medical Industries Inc., Los Angeles, CA, USA) with molecular weight cutoff limits of 12,000-14,000. Total lipoproteins were first concentrated from serum by raising the buoyant density to p = 1.215 g/ml followed by ultracentrifugation (48 hours). The lipoprotein solution was then dialyzed and the density adjusted to p = 1.019 g/ml and further centrifuged (24 hours). The upper band of very low density lipoproteins was discarded. The lower band of lipoproteins was collected and adjusted to p = 1.063 g/ml. After centrifugation (24 hours), an upper band was collected, adjusted to p = 1.090 g/ml, and centrifuged (24 hours) to yield a fraction of LDL. The lower fraction was also collected, adjusted to p = 1.125 g/ml, and centrifuged (24 hours) to yield a floating band of HDL. Both LDL (1.019 < p < 1.063 g/ml) and HDL (1.090 < p < 1.215 g/ml) were dialyzed and sterilized by filtration through a Millipore filter. Protein concentrations were determined by the method of Lowry et a1.i5 The total cholesterol (nonesterified plus esterified) content of the various lipoprotein fractions were estimated directly using a coupled enzyme assay with cholesterol oxidase, cholesterol esterase, peroxidase, 2-hydroxy-3,5-dichlorobenzenesulfonic acid, and 4-aminoantipyrine.’ Lipoprotein-depleted serum was made from fetal calf serum (GIBCO Laboratories, Grand Island, NY, USA) after adjusting the density to I .215 g/ml and centrifugation for 48 hours. The lower band, containing lipoprotein-depleted serum, was dialyzed and sterilized by filtration. Human LDL (1.019 < p < 1.063) was prepared as previously described.16 Proteins in the lipoprotein fractions and in lipoprotein-depleted fetal calf serum were analyzed after electrophoresis on polyacrylamide gels (10%) containing sodium dodecyl sulfate. Before electrophoresis, each sample was heated at 100 C in buffer” containing sodium dodecyl sulfate (I%), mercaptoethano1 (0.2 M), and EDTA (1 mM). Subsequently, gels were fixed and stained with Coomassie blue.
Steroid measurement The cortisol contents of the various media were quantified directly by radioimmunoassay with antiserum and procedures obtained from Radioassay Systems Laboratories (Carson City, CA, USA). The relative reactivities of the antiserum that was raised against cortisol-21-hemisuccinate-albumin were cortisol, 100%; corticosterone, 5.6%; and 1I-deoxycortisol, 15%.
Determination
of HMG-CoA
reductase activity
Confluent monolayers of bovine adrenocortical cells were washed once with buffer A (50 mM Tris, 0.14 M NaCl, pH 7.4) after experimental treatment. Cells were removed from the dishes in 1 ml of buffer B (50 mM potassium phosphate, pH 7.4, 0.2 M KCl, 5 mM EDTA, and 5 mM dithiothreitol) and centrifuged at 13,000 x g for 3 min, and the pellet was frozen at - 20 C in buffer B (0.1 ml) for subsequent assay of HMG-CoA reductase activity. To assay the reductase activity, cells were dispersed in an additional 0.1 ml of solution added such that the final assay volume (0.2 ml) contained potassium phosphate (100 mM, pH 7.4), glucose-6-phosphate (20 mM), NADP (2.5 mM), dithiothreito1 (9 mM), and glucose-6-phosphate dehydrogenase (2 IUlml). Cell lysis was accomplished by a freeze-thaw procedure and by mechanical dispersion with an automatic dispensing pipette. The enzyme reaction was initiated by the addition of [3-i4C]HMG-CoA (20 nmol in 0.02 ml) to attain a final assay concentration of 100 PM HMG-CoA. The assay period was 20 minutes at 37 C. In previous experiments, we established the
Bovine lipoproteins
and steroidogenesis:
Rainey et al.
validity of this assay with regard to linearity with time and its saturability with substrate and cofactors.” The reaction was terminated by addition of 5 M HCl (0.02 ml) and a further 20minute incubation to ensure complete conversion to mevalonolactone. [3H]Mevalonolactone was then added as a recovery marker. The reaction mixture was applied directly on the preabsorbant strip of precoated silica gel G thin-layer chromatographic plates. The chromatograms were developed using the solvent system benzene/acetone (1: 1). The region on each plate, corresponding to the location of mevalonolactone, was removed into a scintillation vial and a scintillation cocktail (15 ml), containing Omnifluor and methanol (10%) in toluene, was added. Carbon14 and tritium in each sample were quantified after liquid scintillation spectrometry, and the fractional conversion .of [“C]HMG-CoA to [t4C]mevalonolactone was computed. The results represented the mean of duplicate determinations of activities of duplicate samples.
Results Analysis of lipoprotein fractions Four batches of LDL and HDL, and two of lipoproteindepleted fetal calf serum, used to conduct this study were analyzed; the staining patterns from all batches were qualitatively similar. A representative example is shown in Figure 1 and proteins were identified on the basis of their molecular weight and the results were similar to those reported by Cordle et al.‘* Apo B-like proteins, having apparent molecular weights in excess of 200,000, were present in LDL but not HDL. Apo A-l (28,000) was also a major protein in LDL. In HDL Apo A-l was by far the most dominant protein, and several C-apolipoproteins, smaller than the lowest molecular weight standard used (14,400), were also present as observed previously.‘8-20 No APO-E was detectable in either LDL or HDL, although a protein of molecular weight 40,000 was just detectable. A protein of this molecular size has only been reported in bovine lipoproteins of density between 1.006 and 1.020 g/m1.21Proteins of molecular weight of approximately 50,000 were detectable in some HDL preparations, and proteins presumed to be albumin (the electrophoretic mobility was similar to this major protein of the fetal calf lipoprotein-depleted serum) were detectable in both HDL and LDL fractions. A small amount of Apo A-l was detected in lipoprotein-depleted serum. The total cholesterol contents of the bLDL, bHDL, hLDL, and hHDL fractions used in this investigation were 2.2, 3.0, 3.1, and 1.2 pmol/mg protein, respectively.
Figure 1 Major proteins from bovine LDL (50 rg protein, Lane 1) bovine HDL (50 pg protein, Lane 2) and bovine fetal lipoproteindepleted serum (25 pg protein, Lane 3) resolved after electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate, and stained with Coomassie Blue.
Steroid production
Monolayer cultures of bovine adrenocortical cells were treated experimentally in an LPDM supplemented with various concentrations of lipoproteins (both human and bovine). Experiments lasted 60 hours with medium changed at 1Zhour intervals. As we demonstrated previously,’ ACTH addition to cells in LPDM caused an increase in cortisol release of 7- to 20-fold. This elevated level of release was maintained throughout the 60 hours of incubation (Figure 1). The production of cortisol was dependent on de novo synthesis of cholesterol because the HMG-CoA reductase inhibitor, mevi-
nolin, inhibited both basal and ACTH-stimulated steroidogenesis (Table 1). Exposure of BAC cells to ACTH in medium containing hLDL (O-100 pg protein/ml) increased cortisol production in a dosedependent manner during the O-hour incubation period (Figure 1). hLDL (100 pg protein/ml) increased ACTH-stimulated cortisol production five-fold above levels observed in LPDM alone. The rate of cortisol production with hLDL was seen to be maximal during Steroids,
1992, vol.
57, April
169
Papers Table 1
Effects of mevinolin
Treatment
on BAC cortisol production Cortisol (nmol/mg
Control Mevinolin ACTH ACTH + mevinolin
0.61 0.25 13.57 3.46
-c f + ”
TREATMENT PERIOD (h) _I
protein)
36-46
46-60
0.16 0.08 1.07 0.13
Confluent cultures of BAC cells were placed in medium depleted of lipoprotein for 24 h. Cells were then treated with mevinolin (IO PM), ACTH (50 nM), or the combination for 24 h. The control represents the basal levels of steroid release. Values represent the mean 2 SD for four separate dishes of cells and are representative of three experiments.
the third 12-hour period (24-36 hours) of ACTH stimulation. Addition of bLDL (O-100 pug protein/ml medium) did not produce a typical dose-related stimulation of cortisol release by BAC cells (Figure 2). Significant increases in steroidogenesis were only observed when 100 pg/ml of bLDL was added to the medium. At this concentration of LDL, cortisol release in response to ACTH was increased three-fold. bHDL also caused an increase in ACTH-stimulated cortisol release at a concentration of 100 pg protein/ml (Figure 3). Cortisol release was maximal after 24 hours of ACTH treatment and the rate of release of cortisol was approximately 2.5 times that observed from cells in LPDM alone. HMG-CoA
reductase adrenocortical cells
Steroids,
Figure 2 Cortisol release from BAC cells exposed to ACTH (50 nM) in the presence of hLDL. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with hLDL (1, 10,and.lOOpg protein/ml medium) plus ACTH (50 nM). The media were changed every 12 hours for 60 hours. Cortisol content of the media was quantified after radioimmunoassay. Each value represents the mean 2 SE for triplicate samples. When ACTH was not added to the experimental medium, cortisol production was less than 1 nmol/mg protein/12 h. Statistical comparisons were made with cells treated with ACTH alone. l0.5; **O.Ol; ***O.OOl.
activity of bovine
We previously demonstrated that BAC cells increase and maintain elevated HMG-CoA reductase activity in response to ACTH treatment in LPDM.9 In the present study we examined the effects of human and bovine lipoproteins on the ACTH induction of HMG-CoA reductase. BAC cells were preincubated in LPDM for 24 hours and then treated with ACTH and the various lipoproteins (O-100 pg protein/ml). ACTH treatment lasted 24 hours with the medium changed after 12 hours. In the absence of lipoprotein, HMG-CoA reductase activity increased eight-fold (Figure 4). hLDL decreased HMG-CoA reductase activity in a dose-dependent manner. HMG-CoA reductase activity decreased to that seen in untreated cells after addition of 100 pup/ml of LDL. In contrast, hHDL did not alter the ACTH-stimulation of reductase activity (Figure 4) at concentrations up to 100 pg protein/ml. In order to approach more closely the in vivo situation with respect to lipoprotein homology, we examined the effects of bovine lipoproteins on HMG-CoA reductase activity. bLDL, like hLDL, decreased HMG-CoA reductase activity induction by ACTH in a concentration-dependent manner (Figure 5). Activity decreased from an eight-fold induction to basal levels with bLDL (100 pg protein/ml). Unlike hHDL, bHDL (100 pg protein/ml) reduced ACTH-stimulated reductase activity by approximately 20% (Figure 6). 170
HUMAN LDL CONCENTRATION(pg protein/ml)
1992, vol. 57, April
TREATMENT PERIOD (h) 4Orm
o-12
I
12-24 **
I_
24-36
-
36-46
-
46-60
l*
BOVINE LDL CONCENTRATION (pg protein/ml)
Figure 3 Cortisol release by BAC ceils treated with ACTH in the presence of bLDL. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with bHDL (1, 10, and 100 pg protein/ml medium) and ACTH (50 nM). The media were changed every 12 hours for 80 hours. Cortisol contents of the media were quantified after radioimmunoassay. Each value represents the mean + SE for triplicate samples. In the absence of ACTH, cortisol production was less than 1 nmol/mg protein/l2 h. Statistical comparisonswere made with cells treated with ACTH alone. **O.Ol.
Bovine lipoproteins and steroidogenesis: Rainey et al. TREATMENT PERK%)(h) o-12
40-----
ss-48
24-36
12-24
48-80
r
s $3 9s P%
Em
d-
E; C
K Control
0
0 -1loioo
0
0
IlolcG
0 IiOloo
0 11oloo
0 lloloo
1
10
30
100
Bovine Lipoprotein Concentration (pg protein/ml)
BOVINE KIL CONCENTRATION(Pg prolrln/ml)
Figure 4 Cortisol
release
from
bovine
adrenocortical
cells ex-
to ACTH in the presence of bHDL. Confluent cultures of BAC cells were placed in medium depleted of lipoprotein (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with bovine HDL (1.10, and 1OOwg protein/ml medium) and ACTH (50 nM). The media changed every 12 hours for 50 hours. Cortisol contents of the media were quantified after radioimmunoassay. Each value represents the mean f SE for triplicate samples. In the absence of ACTH, cottisol production was less than 1 nmol/mg protein/l2 h. Statistical comparisons were made with cells treated with ACTH alone. *0.05; **O.Ol. posed
Figure 6 Effect of bovine LDL and HDL on the ACTH induction of HMG-CoA reductase activity in BAC cells. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM supplemented with bLDL (0) or bHDL (0) plus ACTH (50 nM). The control (A)represents cells in LPDM alone. The experiment lasted 24 hours with the media changed every 12 hours. After treatment, cells were washed, harvested, and frozen in buffer B until assay of HMG-CoA reductase. Values represent the mean and range of four determinations on duplicate samples. Statistical comparisons were made with cells treated with ACTH alone. *0.05; l*0.01; l**0.001.
Discussion
150 -
i 0
\
I
Control 1 Human Lipoprotein
10
30
100
COMXntratiOt'I (pgprotein/ml)
Figure 5 Effect of hLDL and hHDL on the ACTH induction of HMG-CoA reductase activity in BAC cells. Confluent cultures of BAC cells were placed in medium depleted of lipoprotein (LPDM) for 24 hours. The medium was then replaced with LPDM supplemented with hLDL (0) or hHDL (0) plus ACTH (50 nM). The control (A) represents cells in LPDM alone. The experiment lasted 24 hours with the media changed after 12 hours. After treatment, cells were washed, harvested, and frozen in buffer B until assay of HMG-CoA reductase. Values represent the mean and range of four determinations on duplicate samples. Statistical comparisons were made with cells treated with ACTH alone. *0.05; **0.01; ***0.001.
This report constitutes the first examination of BAC cell utilization of homologous lipoproteins for steroid hormone production. Our findings demonstrate that bLDL and bHDL can increase the level of cortisol production. This observation may be important when one considers the bovine plasma lipoprotein profile where 8040% of the lipoprotein is HDL.13*14*22 As a measure of steroid production by BAC cells, we examined the release into the medium of cortisol, which represented 60-80% of the total amount of steroid released in response to ACTH by BAC cells when grown under the described conditions.’ We have demonstrated that ACTH increases cortisol release 7- to 20fold in the absence of lipoproteins and that the source of cholesterol for steroidogenesis under such lipoproteindepleted conditions is de novo synthesis of cholesterol.’ Herein we reaffirm this observation showing that ACTH stimulated and maintained cortisol production for the f30-hour period examined. Supplementation of the medium with hLDL increased the ACTH-stimulated production of cortisol by five-fold. These data confirm other reports that addition of hLDL can increase steroid production of BAC cells.2*23The increase in steroid production presumably resulted from the uptake and degradation of the hLDL particle, which released cholesterol for use as additional substrate.2*24 Bovine LDL was not as effective at increasing ACTHstimulated steroid production as hLDL. Kovanen et aL2 previously demonstrated that both hLDL and bLDL bound to the same receptors on BAC cells. Steroids,
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Papers
Whereas bLDL was observed to have a higher affinity for the receptor, hLDL was observed to be degraded at a two-fold higher rate than bLDL. This increase in degradation may explain our observation that hLDL increased cortisol production to higher levels than bLDL. Newton and Shawl’ have shown that the bovine adrenal cortex has, in addition to LDL receptors, specific binding sites for HDL. In the present study the addition of bHDL increased the level of cortisol production after ACTH treatment, compared with cells treated in the absence of lipoprotein. Together, these data suggest that bHDL is binding to BAC cell surface receptors, and can supply cholesterol for steroid production. This observation is similar to that reported by Durand et al. using fetal sheep adrenal cells in culture. These cells were able to use sheep HDL as a source of cholesterol for steroidogenesis.” To better understand the sources of cholesterol for steroid production in BAC cells, we examined the level of the enzyme HMG-CoA reductase. HMG-CoA reductase is considered a key regulatory enzyme in the pathway of cholesterol biosynthesis. The levels of this enzyme are also known to change within the adrenal depending on the availability of lipoproteins for steroid hormone production. 9*‘oWhen BAC cells were treated with ACTH for 24 hours the level of HMG-CoA reductase increased by eight-fold. Maintenance of steroid hormone production was dependent on the activity of HMG-CoA reductase. This fact was supported by the ability of the HMG-CoA reductase inhibitor, mevinolin, to inhibit cortisol production. When cells were treated in LPDM containing hLDL, HMG-CoA reductase activity decreased with increasing concentrations of hLDL. Human and bLDL have previously been shown to decrease de novo synthesis of cholesterol in BAC cells.* However, the BAC cells used in these studies did not increase steroid production or HMG-CoA reductase activity in response to ACTH treatment. Thus, the effects generated by lipoprotein addition to the medium were manifested on basal reductase activity. The BAC cells used in the current study were able to maintain steroidogenesis by an increase in cholesterol biosynthesis in lipoprotein-depleted conditions. We demonstrate that both hLDL and bLDL can donate cholesterol and repress the need for an increase in cholesterol biosynthesis. The addition of hHDL to the experimental medium did not affect the ACTH stimulation of HMG-CoA reductase activity in BAC cells. Human HDL addition to culture medium of other cell types also was not observed to decrease reductase activity.2323 Therefore, it was surprising when bHDL slightly suppressed reductase activity of BAC cells. The suppression coincided with the concentration of bHDL, which increased cortisol production to levels observed in the presence of bLDL. The retention of HMG-CoA reductase activity suggested that the BAC cells were using both exogenous cholesterol and cholesterol synthesized de novo in order to maintain steroidogenesis. Our present observations on the requirements of 172
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lipoproteins in BAC cell steroidogenesis are indicative of roles for both bLDL and bHDL, as well as cholesterol synthesis de novo. Importantly, bHDL, the major lipoprotein of bovine plasma, may donate cholesterol for steroid synthesis while still permitting the BAC cell to increase its own ability to produce cholesterol. Such information reinforces the view of a close association between exogenous and endogenous sources of cholesterol for substrate in adrenal steroidogenesis.
Acknowledgments The authors appreciate the technical assistance of Ruby Lu, Glenna Lynch and Lakshmeswari Ravi, and the editorial assistance of E. Ann Whisenand. This research was supported by Grants AG-08175 from the National Institutes of Health, Department of Health and Human Services, and American Heart Association Grant 916-082. R.J.R. was a recipient of a grant-in-aid from the Chilton Foundation.
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173
regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells
William E. Rainey, Raymond J. Rodgers,f’ and J. Ian Mason* Department of Obstetrics and Gynecology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, and *Department of Biochemistry, The University Texas Southwestern Flinders University
Medical Center, Dallas, Texas; and tDepartment of South Australia, Bedford Park, South Australia.
of
of Medicine,
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-methylglutatyl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human lowdensity lipoprotein (hLDL) and human high-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 ZO-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 8570, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40,fold above basal levels ifhLDL (100 uglml) 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 or HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisolproduction and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 uglml) 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 jindings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL. (Steroids 571167-173, 1992)
Keywords: sterol metabolism; lipoproteins;
bovine HMG-CoA reductase; bovine adrenal; cholesterol;
Introduction Adrenocortical cells require cholesterol as substrate for the production of steroid hormones.’ The uptake and use of cholesterol for steroid production by adrenal cells from plasma lipoproteins is well established in a number of mammalian species.*-” In addition, although the rate of cholesterol synthesis varies greatly from
Address reprint requests to William E. Rainey, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Cecil H. and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, TX 752354032. Received May 31, 1991; accepted October 16, 1991.
8 1992 Butterworth-Heinemann
cortisol
species to species, most adrenocortical cells examined have been shown to incorporate radiolabeled acetate or water into cholesterol and have quantifiable levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 2,3,5-9 Thus, adrenocortical cells have at least two sources of cholesterol for steroid synthesis. In vivo, 4-aminopyrazolopyrimidine treatment of rats blocks lipoprotein secretion from the liver, thus depriving the adrenal of an external source of cholesterol.‘OAdministration of this drug elevates cholesterol synthesis within the adrenal, suggesting at least a partial compensation for the depleted serum sources of cholesterol. Subsequent infusion of lipoproteins suppresses cholesterol synthesis. Such experiments detail a close association between plasma lipoproteins and Steroids,
1992, vol. 57, April
167
Papers
cholesterol biosynthesis within the adrenal gland. We have demonstrated such an association using in vitro primary cell cultures of bovine adrenocortical (BAC) cells.’ Cholesterol biosynthesis within these cells was elevated in the absence of exogenous lipoprotein after stimulation of steroid production. Addition of human low-density lipoprotein (hLDL) to the medium prevented this elevation.9 Thus the BAC cells in monolayer culture, under appropriate conditions, can apparently mimic the in vivo situation. These studies raised the question as to the requirements of homologous bovine lipoproteins in steroidogenesis and their regulation of cholesterol biosynthesis within the BAC cell. Previous studies have shown specific receptors for both human high density lipoproteins (hHDL) and hLDL on BAC cells.“*r2 Importantly, the level of bHDL in bovine plasma greatly exceeds the level of bovine LDL (bLDL), in contrast to the situation in human plasma. i3,i4 However, the role of the homologous lipoproteins in BAC cell steroidogenesis has not been examined. In the present study we demonstrate that bLDL, like hLDL, was capable of increasing the steroidogenic capacity of BAC cells. In addition, we show that the major bovine plasma lipoprotein, bovine HDL (bHDL), also could increase the rate of steroidogenesis in the presence of ACTH. Finally, bLDL but not bHDL was capable of fully suppressing the ACTH-promoted induction of HMG-CoA reductase activity.
Experimental Cell isolation and culture Dispersed adrenal cells were prepared from minced bovine adrenal cortices after treatment with collagenase (3.3 mglml from Clostridium histolyticus) and DNAse (0.1 mg/ml) in Hanks’ balanced salt solution containing HEPES (10 mM, pH 7.4) and bovine serum albumin (10 mglml). The collagenase and DNAse were obtained from Boehringer Mannheim (Indianapolis, IN, USA). The cells were harvested, washed, and placed in 35mm cell culture dishes (5 x 10’ cells per dish) in Ham’s F12 and Dulbecco’s modified Eagle’s (DME/F12) high-glucose medium (1 : 1, v/v) with fetal calf serum (lo%), gentamicin sulfate (O.Ol%), and HEPES (15 mM, pH 7.4). After the cells reached confluence (after approximately 5 days), the medium was changed to a lipoprotein-depleted medium (LPDM), that is, one containing DME/F12 high-glucose medium with insulin (5 pg/ml), transferrin (5 pg/ml), selenous acid (5 ng/ml), gentamicin sulfate (O.Ol%), HEPES (15 mM, pH 7.4), and lipoprotein-depleted fetal calf serum (1%). Experiments were initiated on cells that were conditioned in this medium for 24 hours. ACTH (Cortrosyn) was obtained from Organon (West Orange, NJ, USA). Mevinolin was obtained from Merck, Sharp & Dohme (West Point, PA, USA).
Lipoprotein
isolation
Blood was collected from bovine heifers after slaughter at the local abattoir and the blood was allowed to clot overnight. Adult bovine serum was also obtained freshly from Research Biogenics (Bastrop, TX, USA). Cellular elements were removed by centrifugation (2,000 rpm) and lipoproteins were isolated by sequential differential flotation in potassium bromide solutions. Ultracentrifugation was performed in Ti60 or Ti70 fixed-angle rotors in a Beckman (Porterville, CA, USA) L8-M centrifuge (60,000 rpm,
166
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1992, vol. 57, April
4 C). Dialysis was performed against 0.15 M NaCl solution containing EDTA (O.Ol%, pH 7.4) using Spectraphor number 2 dialysis tubing (Spectrum Medical Industries Inc., Los Angeles, CA, USA) with molecular weight cutoff limits of 12,000-14,000. Total lipoproteins were first concentrated from serum by raising the buoyant density to p = 1.215 g/ml followed by ultracentrifugation (48 hours). The lipoprotein solution was then dialyzed and the density adjusted to p = 1.019 g/ml and further centrifuged (24 hours). The upper band of very low density lipoproteins was discarded. The lower band of lipoproteins was collected and adjusted to p = 1.063 g/ml. After centrifugation (24 hours), an upper band was collected, adjusted to p = 1.090 g/ml, and centrifuged (24 hours) to yield a fraction of LDL. The lower fraction was also collected, adjusted to p = 1.125 g/ml, and centrifuged (24 hours) to yield a floating band of HDL. Both LDL (1.019 < p < 1.063 g/ml) and HDL (1.090 < p < 1.215 g/ml) were dialyzed and sterilized by filtration through a Millipore filter. Protein concentrations were determined by the method of Lowry et a1.i5 The total cholesterol (nonesterified plus esterified) content of the various lipoprotein fractions were estimated directly using a coupled enzyme assay with cholesterol oxidase, cholesterol esterase, peroxidase, 2-hydroxy-3,5-dichlorobenzenesulfonic acid, and 4-aminoantipyrine.’ Lipoprotein-depleted serum was made from fetal calf serum (GIBCO Laboratories, Grand Island, NY, USA) after adjusting the density to I .215 g/ml and centrifugation for 48 hours. The lower band, containing lipoprotein-depleted serum, was dialyzed and sterilized by filtration. Human LDL (1.019 < p < 1.063) was prepared as previously described.16 Proteins in the lipoprotein fractions and in lipoprotein-depleted fetal calf serum were analyzed after electrophoresis on polyacrylamide gels (10%) containing sodium dodecyl sulfate. Before electrophoresis, each sample was heated at 100 C in buffer” containing sodium dodecyl sulfate (I%), mercaptoethano1 (0.2 M), and EDTA (1 mM). Subsequently, gels were fixed and stained with Coomassie blue.
Steroid measurement The cortisol contents of the various media were quantified directly by radioimmunoassay with antiserum and procedures obtained from Radioassay Systems Laboratories (Carson City, CA, USA). The relative reactivities of the antiserum that was raised against cortisol-21-hemisuccinate-albumin were cortisol, 100%; corticosterone, 5.6%; and 1I-deoxycortisol, 15%.
Determination
of HMG-CoA
reductase activity
Confluent monolayers of bovine adrenocortical cells were washed once with buffer A (50 mM Tris, 0.14 M NaCl, pH 7.4) after experimental treatment. Cells were removed from the dishes in 1 ml of buffer B (50 mM potassium phosphate, pH 7.4, 0.2 M KCl, 5 mM EDTA, and 5 mM dithiothreitol) and centrifuged at 13,000 x g for 3 min, and the pellet was frozen at - 20 C in buffer B (0.1 ml) for subsequent assay of HMG-CoA reductase activity. To assay the reductase activity, cells were dispersed in an additional 0.1 ml of solution added such that the final assay volume (0.2 ml) contained potassium phosphate (100 mM, pH 7.4), glucose-6-phosphate (20 mM), NADP (2.5 mM), dithiothreito1 (9 mM), and glucose-6-phosphate dehydrogenase (2 IUlml). Cell lysis was accomplished by a freeze-thaw procedure and by mechanical dispersion with an automatic dispensing pipette. The enzyme reaction was initiated by the addition of [3-i4C]HMG-CoA (20 nmol in 0.02 ml) to attain a final assay concentration of 100 PM HMG-CoA. The assay period was 20 minutes at 37 C. In previous experiments, we established the
Bovine lipoproteins
and steroidogenesis:
Rainey et al.
validity of this assay with regard to linearity with time and its saturability with substrate and cofactors.” The reaction was terminated by addition of 5 M HCl (0.02 ml) and a further 20minute incubation to ensure complete conversion to mevalonolactone. [3H]Mevalonolactone was then added as a recovery marker. The reaction mixture was applied directly on the preabsorbant strip of precoated silica gel G thin-layer chromatographic plates. The chromatograms were developed using the solvent system benzene/acetone (1: 1). The region on each plate, corresponding to the location of mevalonolactone, was removed into a scintillation vial and a scintillation cocktail (15 ml), containing Omnifluor and methanol (10%) in toluene, was added. Carbon14 and tritium in each sample were quantified after liquid scintillation spectrometry, and the fractional conversion .of [“C]HMG-CoA to [t4C]mevalonolactone was computed. The results represented the mean of duplicate determinations of activities of duplicate samples.
Results Analysis of lipoprotein fractions Four batches of LDL and HDL, and two of lipoproteindepleted fetal calf serum, used to conduct this study were analyzed; the staining patterns from all batches were qualitatively similar. A representative example is shown in Figure 1 and proteins were identified on the basis of their molecular weight and the results were similar to those reported by Cordle et al.‘* Apo B-like proteins, having apparent molecular weights in excess of 200,000, were present in LDL but not HDL. Apo A-l (28,000) was also a major protein in LDL. In HDL Apo A-l was by far the most dominant protein, and several C-apolipoproteins, smaller than the lowest molecular weight standard used (14,400), were also present as observed previously.‘8-20 No APO-E was detectable in either LDL or HDL, although a protein of molecular weight 40,000 was just detectable. A protein of this molecular size has only been reported in bovine lipoproteins of density between 1.006 and 1.020 g/m1.21Proteins of molecular weight of approximately 50,000 were detectable in some HDL preparations, and proteins presumed to be albumin (the electrophoretic mobility was similar to this major protein of the fetal calf lipoprotein-depleted serum) were detectable in both HDL and LDL fractions. A small amount of Apo A-l was detected in lipoprotein-depleted serum. The total cholesterol contents of the bLDL, bHDL, hLDL, and hHDL fractions used in this investigation were 2.2, 3.0, 3.1, and 1.2 pmol/mg protein, respectively.
Figure 1 Major proteins from bovine LDL (50 rg protein, Lane 1) bovine HDL (50 pg protein, Lane 2) and bovine fetal lipoproteindepleted serum (25 pg protein, Lane 3) resolved after electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate, and stained with Coomassie Blue.
Steroid production
Monolayer cultures of bovine adrenocortical cells were treated experimentally in an LPDM supplemented with various concentrations of lipoproteins (both human and bovine). Experiments lasted 60 hours with medium changed at 1Zhour intervals. As we demonstrated previously,’ ACTH addition to cells in LPDM caused an increase in cortisol release of 7- to 20-fold. This elevated level of release was maintained throughout the 60 hours of incubation (Figure 1). The production of cortisol was dependent on de novo synthesis of cholesterol because the HMG-CoA reductase inhibitor, mevi-
nolin, inhibited both basal and ACTH-stimulated steroidogenesis (Table 1). Exposure of BAC cells to ACTH in medium containing hLDL (O-100 pg protein/ml) increased cortisol production in a dosedependent manner during the O-hour incubation period (Figure 1). hLDL (100 pg protein/ml) increased ACTH-stimulated cortisol production five-fold above levels observed in LPDM alone. The rate of cortisol production with hLDL was seen to be maximal during Steroids,
1992, vol.
57, April
169
Papers Table 1
Effects of mevinolin
Treatment
on BAC cortisol production Cortisol (nmol/mg
Control Mevinolin ACTH ACTH + mevinolin
0.61 0.25 13.57 3.46
-c f + ”
TREATMENT PERIOD (h) _I
protein)
36-46
46-60
0.16 0.08 1.07 0.13
Confluent cultures of BAC cells were placed in medium depleted of lipoprotein for 24 h. Cells were then treated with mevinolin (IO PM), ACTH (50 nM), or the combination for 24 h. The control represents the basal levels of steroid release. Values represent the mean 2 SD for four separate dishes of cells and are representative of three experiments.
the third 12-hour period (24-36 hours) of ACTH stimulation. Addition of bLDL (O-100 pug protein/ml medium) did not produce a typical dose-related stimulation of cortisol release by BAC cells (Figure 2). Significant increases in steroidogenesis were only observed when 100 pg/ml of bLDL was added to the medium. At this concentration of LDL, cortisol release in response to ACTH was increased three-fold. bHDL also caused an increase in ACTH-stimulated cortisol release at a concentration of 100 pg protein/ml (Figure 3). Cortisol release was maximal after 24 hours of ACTH treatment and the rate of release of cortisol was approximately 2.5 times that observed from cells in LPDM alone. HMG-CoA
reductase adrenocortical cells
Steroids,
Figure 2 Cortisol release from BAC cells exposed to ACTH (50 nM) in the presence of hLDL. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with hLDL (1, 10,and.lOOpg protein/ml medium) plus ACTH (50 nM). The media were changed every 12 hours for 60 hours. Cortisol content of the media was quantified after radioimmunoassay. Each value represents the mean 2 SE for triplicate samples. When ACTH was not added to the experimental medium, cortisol production was less than 1 nmol/mg protein/12 h. Statistical comparisons were made with cells treated with ACTH alone. l0.5; **O.Ol; ***O.OOl.
activity of bovine
We previously demonstrated that BAC cells increase and maintain elevated HMG-CoA reductase activity in response to ACTH treatment in LPDM.9 In the present study we examined the effects of human and bovine lipoproteins on the ACTH induction of HMG-CoA reductase. BAC cells were preincubated in LPDM for 24 hours and then treated with ACTH and the various lipoproteins (O-100 pg protein/ml). ACTH treatment lasted 24 hours with the medium changed after 12 hours. In the absence of lipoprotein, HMG-CoA reductase activity increased eight-fold (Figure 4). hLDL decreased HMG-CoA reductase activity in a dose-dependent manner. HMG-CoA reductase activity decreased to that seen in untreated cells after addition of 100 pup/ml of LDL. In contrast, hHDL did not alter the ACTH-stimulation of reductase activity (Figure 4) at concentrations up to 100 pg protein/ml. In order to approach more closely the in vivo situation with respect to lipoprotein homology, we examined the effects of bovine lipoproteins on HMG-CoA reductase activity. bLDL, like hLDL, decreased HMG-CoA reductase activity induction by ACTH in a concentration-dependent manner (Figure 5). Activity decreased from an eight-fold induction to basal levels with bLDL (100 pg protein/ml). Unlike hHDL, bHDL (100 pg protein/ml) reduced ACTH-stimulated reductase activity by approximately 20% (Figure 6). 170
HUMAN LDL CONCENTRATION(pg protein/ml)
1992, vol. 57, April
TREATMENT PERIOD (h) 4Orm
o-12
I
12-24 **
I_
24-36
-
36-46
-
46-60
l*
BOVINE LDL CONCENTRATION (pg protein/ml)
Figure 3 Cortisol release by BAC ceils treated with ACTH in the presence of bLDL. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with bHDL (1, 10, and 100 pg protein/ml medium) and ACTH (50 nM). The media were changed every 12 hours for 80 hours. Cortisol contents of the media were quantified after radioimmunoassay. Each value represents the mean + SE for triplicate samples. In the absence of ACTH, cortisol production was less than 1 nmol/mg protein/l2 h. Statistical comparisonswere made with cells treated with ACTH alone. **O.Ol.
Bovine lipoproteins and steroidogenesis: Rainey et al. TREATMENT PERK%)(h) o-12
40-----
ss-48
24-36
12-24
48-80
r
s $3 9s P%
Em
d-
E; C
K Control
0
0 -1loioo
0
0
IlolcG
0 IiOloo
0 11oloo
0 lloloo
1
10
30
100
Bovine Lipoprotein Concentration (pg protein/ml)
BOVINE KIL CONCENTRATION(Pg prolrln/ml)
Figure 4 Cortisol
release
from
bovine
adrenocortical
cells ex-
to ACTH in the presence of bHDL. Confluent cultures of BAC cells were placed in medium depleted of lipoprotein (LPDM) for 24 hours. The medium was then replaced with LPDM that was supplemented with bovine HDL (1.10, and 1OOwg protein/ml medium) and ACTH (50 nM). The media changed every 12 hours for 50 hours. Cortisol contents of the media were quantified after radioimmunoassay. Each value represents the mean f SE for triplicate samples. In the absence of ACTH, cottisol production was less than 1 nmol/mg protein/l2 h. Statistical comparisons were made with cells treated with ACTH alone. *0.05; **O.Ol. posed
Figure 6 Effect of bovine LDL and HDL on the ACTH induction of HMG-CoA reductase activity in BAC cells. Confluent cultures of BAC cells were placed in medium depleted of lipoproteins (LPDM) for 24 hours. The medium was then replaced with LPDM supplemented with bLDL (0) or bHDL (0) plus ACTH (50 nM). The control (A)represents cells in LPDM alone. The experiment lasted 24 hours with the media changed every 12 hours. After treatment, cells were washed, harvested, and frozen in buffer B until assay of HMG-CoA reductase. Values represent the mean and range of four determinations on duplicate samples. Statistical comparisons were made with cells treated with ACTH alone. *0.05; l*0.01; l**0.001.
Discussion
150 -
i 0
\
I
Control 1 Human Lipoprotein
10
30
100
COMXntratiOt'I (pgprotein/ml)
Figure 5 Effect of hLDL and hHDL on the ACTH induction of HMG-CoA reductase activity in BAC cells. Confluent cultures of BAC cells were placed in medium depleted of lipoprotein (LPDM) for 24 hours. The medium was then replaced with LPDM supplemented with hLDL (0) or hHDL (0) plus ACTH (50 nM). The control (A) represents cells in LPDM alone. The experiment lasted 24 hours with the media changed after 12 hours. After treatment, cells were washed, harvested, and frozen in buffer B until assay of HMG-CoA reductase. Values represent the mean and range of four determinations on duplicate samples. Statistical comparisons were made with cells treated with ACTH alone. *0.05; **0.01; ***0.001.
This report constitutes the first examination of BAC cell utilization of homologous lipoproteins for steroid hormone production. Our findings demonstrate that bLDL and bHDL can increase the level of cortisol production. This observation may be important when one considers the bovine plasma lipoprotein profile where 8040% of the lipoprotein is HDL.13*14*22 As a measure of steroid production by BAC cells, we examined the release into the medium of cortisol, which represented 60-80% of the total amount of steroid released in response to ACTH by BAC cells when grown under the described conditions.’ We have demonstrated that ACTH increases cortisol release 7- to 20fold in the absence of lipoproteins and that the source of cholesterol for steroidogenesis under such lipoproteindepleted conditions is de novo synthesis of cholesterol.’ Herein we reaffirm this observation showing that ACTH stimulated and maintained cortisol production for the f30-hour period examined. Supplementation of the medium with hLDL increased the ACTH-stimulated production of cortisol by five-fold. These data confirm other reports that addition of hLDL can increase steroid production of BAC cells.2*23The increase in steroid production presumably resulted from the uptake and degradation of the hLDL particle, which released cholesterol for use as additional substrate.2*24 Bovine LDL was not as effective at increasing ACTHstimulated steroid production as hLDL. Kovanen et aL2 previously demonstrated that both hLDL and bLDL bound to the same receptors on BAC cells. Steroids,
1992, vol. 57, April
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Papers
Whereas bLDL was observed to have a higher affinity for the receptor, hLDL was observed to be degraded at a two-fold higher rate than bLDL. This increase in degradation may explain our observation that hLDL increased cortisol production to higher levels than bLDL. Newton and Shawl’ have shown that the bovine adrenal cortex has, in addition to LDL receptors, specific binding sites for HDL. In the present study the addition of bHDL increased the level of cortisol production after ACTH treatment, compared with cells treated in the absence of lipoprotein. Together, these data suggest that bHDL is binding to BAC cell surface receptors, and can supply cholesterol for steroid production. This observation is similar to that reported by Durand et al. using fetal sheep adrenal cells in culture. These cells were able to use sheep HDL as a source of cholesterol for steroidogenesis.” To better understand the sources of cholesterol for steroid production in BAC cells, we examined the level of the enzyme HMG-CoA reductase. HMG-CoA reductase is considered a key regulatory enzyme in the pathway of cholesterol biosynthesis. The levels of this enzyme are also known to change within the adrenal depending on the availability of lipoproteins for steroid hormone production. 9*‘oWhen BAC cells were treated with ACTH for 24 hours the level of HMG-CoA reductase increased by eight-fold. Maintenance of steroid hormone production was dependent on the activity of HMG-CoA reductase. This fact was supported by the ability of the HMG-CoA reductase inhibitor, mevinolin, to inhibit cortisol production. When cells were treated in LPDM containing hLDL, HMG-CoA reductase activity decreased with increasing concentrations of hLDL. Human and bLDL have previously been shown to decrease de novo synthesis of cholesterol in BAC cells.* However, the BAC cells used in these studies did not increase steroid production or HMG-CoA reductase activity in response to ACTH treatment. Thus, the effects generated by lipoprotein addition to the medium were manifested on basal reductase activity. The BAC cells used in the current study were able to maintain steroidogenesis by an increase in cholesterol biosynthesis in lipoprotein-depleted conditions. We demonstrate that both hLDL and bLDL can donate cholesterol and repress the need for an increase in cholesterol biosynthesis. The addition of hHDL to the experimental medium did not affect the ACTH stimulation of HMG-CoA reductase activity in BAC cells. Human HDL addition to culture medium of other cell types also was not observed to decrease reductase activity.2323 Therefore, it was surprising when bHDL slightly suppressed reductase activity of BAC cells. The suppression coincided with the concentration of bHDL, which increased cortisol production to levels observed in the presence of bLDL. The retention of HMG-CoA reductase activity suggested that the BAC cells were using both exogenous cholesterol and cholesterol synthesized de novo in order to maintain steroidogenesis. Our present observations on the requirements of 172
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1992, vol. 57, April
lipoproteins in BAC cell steroidogenesis are indicative of roles for both bLDL and bHDL, as well as cholesterol synthesis de novo. Importantly, bHDL, the major lipoprotein of bovine plasma, may donate cholesterol for steroid synthesis while still permitting the BAC cell to increase its own ability to produce cholesterol. Such information reinforces the view of a close association between exogenous and endogenous sources of cholesterol for substrate in adrenal steroidogenesis.
Acknowledgments The authors appreciate the technical assistance of Ruby Lu, Glenna Lynch and Lakshmeswari Ravi, and the editorial assistance of E. Ann Whisenand. This research was supported by Grants AG-08175 from the National Institutes of Health, Department of Health and Human Services, and American Heart Association Grant 916-082. R.J.R. was a recipient of a grant-in-aid from the Chilton Foundation.
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