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.     

Esterification of cholesterol in high density lipoprotein decreases its ability to support ACTH-stimulated steroidogenesis by rat adrenocortical cells

Addition of high density lipoprotein 3 (HDL3) isolated from human plasma of d greater than 1.125 g/ml which had been preincubated for 24 h at 37 degrees C enhanced steroidogenesis by cultured rat adrenal cells only 38% as well as HDL3 isolated from unincubated plasma. Loss of steroidogenic activity due to preincubation was associated with a decrease in the percent HDL3 cholesterol remaining unesterified. Inhibition of lecithin-cholesterol acyltransferase activity by heating (60 degrees C, 1 h) or addition of dithionitrobenzoic acid (1.4 mM) prevented esterification of cholesterol in HDL and also prevented loss of steroidogenic activity. Although incubation of plasma of d greater than 1.125 g/ml prior to isolation caused cholesterol esterification, there was no change in the ratio of total cholesterol to protein in HDL, size and shape of the HDL particle as assayed by measurement of sedimentation velocity, nor affinity for the putative HDL receptor. Addition of unesterified cholesterol to preincubated HDL restored steroidogenic activity. These results indicate that unesterified cholesterol in HDL is preferentially used as substrate for rat adrenal steroidogenesis. The effects of nonlipoprotein serum proteins on HDL action in the adrenal were also examined. The ability of HDL3 to enhance rat adrenal steroidogenesis was not significantly less in serum-free media than in media supplemented with lipoprotein-poor fetal calf serum or human plasma of d greater than 1.21 g/ml, suggesting that rat adrenal uptake of HDL cholesterol does not depend on participation of plasma enzymes or transport proteins.     

Specific low density lipoprotein receptors in pig Leydig cells. Role of this lipoprotein in cultured Leydig cells steroidogenesis

Freshly prepared and cultured pig Leydig cells were shown to possess specific binding sites for iodinated human low density lipoprotein (LDL). Binding of LDL was followed by internalisation. Both processes were inhibited by unlabelled LDL but not high density lipoprotein (HDL). The number of LDL binding sites was enhanced by prior treatment of cultured cells with hCG. Addition of LDL to the culture medium caused a large enhancement of the rate of steroid secretion (testosterone and dehydroepiandrosterone sulfate) both in the presence or absence of hCG. On the contrary, HDL decreased the steroid output, both in the presence or absence of hCG. We concluded that LDL cholesterol rather than cholesterol synthesized $\text{de novo}$ by the cells, is the major substrate for androgen production by pig Leydig cells in culture.     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Comparison of binding and degradation of high density lipoprotein by intestinal mucosal cells, fibroblasts and adrenal cortical cells in culture

In this study we have compared the binding and degradation of human high density lipoprotein (HDL3), devoid of apolipoprotein E, by rat intestinal (mucosal) and adrenal cells and by human fibroblasts in culture. Binding of HDL3 to adrenal and intestinal cells was characterised by saturable, specific processes whereas skin fibroblasts from normal humans did not convincingly demonstrate saturability and had a lower affinity and capacity compared with adrenal and intestinal cells. Post-receptor events also appeared to differ. Cells from the adrenal cortex and gut showed similar binding affinities for HDL3 but the capacity for binding and for degrading HDL3 was much higher with intestinal cells. The large amounts of HDL degraded by intestinal cells suggest a specific role for the gut in HDL catabolism, and that, in the rat, intestinal cholesterol may be derived from circulating HDL. Finally, it is suggested that rat adrenal cortical and intestinal mucosal cells possess surface receptors for HDL3 which differ from the LDL receptor.     

Lipoprotein geometry. II. Apoprotein exchange in human plasma high density lipoprotein.

The relationships between the apoproteins of intact human serum high density lipoprotein particles, HDL₂ and HDL₃, have been studied by observing the exchange of radioactively labeled apoproteins between one subclass and the other. This exchange process can be inhibited by chemically crosslinking the apoproteins of either the labeled or unlabeled subclass. These results are consistent with a dynamic relationship between HDL₂ and HDL₃ which appears dependent upon the association and perhaps the conformation of the apoprotein components of the lipoprotein particles.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

Uptake of very low density lipoprotein triglyceride by bovine aortic endothelial cells in culture.

Primary monolayers of calf aortic endothelial cells were presented with isolated human very low density lipoproteins that had been labeled with radioactive triglyceride. The cells were observed to take up triglyceride over a 24 hr period; incorporation increased with exogenous lipoprotein concentrations, and up to 60% of the triglyceride taken up was converted to other cell lipids within 24 hr. When [2-3H]glyceryl tri[1-14C]oleate-labeled very low density lipoprotein was used, the 3H/14C ratio in the cell triglyceride was always similar to that of the exogenous lipoprotein triglyceride. Moreover, no significant hydrolysis of the exogenous very low density lipoprotein triglyceride was observed during the time of exposure to the cells. Similar experiments using doubly-labeled triglyceride exposed to endothelial cells in triglyceride-phospholipid liposome preparations also resulted in incorporation of the exogenous triglyceride without evidence of extracellular hydrolysis. The results indicate that primary monolayers of endothelial cells in culture are able to incorporate and metabolize very low density lipoprotein triglyceride. However, triglyceride does not appear to be significantly hydrolyzed during uptake, suggesting an absence of lipoprotein lipase activity in these cells.     

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Nonparenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.