Presence Of Exogenous Cholesterol Research Articles

Exogenous cholesterol acquisition signaling in LH-responsive MA-10 Leydig cells and in adult mice.

MA-10 cells, established 4 decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), the use of a cell-permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through the selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to the regulation of endocytic traffic is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that the use of serum could confound or interfere with trophic stimulation and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Sterol uptake and sterol biosynthesis act coordinately to mediate antifungal resistance in Candidaglabrata under azole and hypoxic stress.

Pathogenic fungi, including Candida glabrata, develop strategies to grow and survive both in vitro and in vivo under azole stress. However, the mechanisms by which yeast cells counteract the inhibitory effects of azoles are not completely understood. In the current study, it was demonstrated that the expression of the ergosterol biosynthetic genes ERG2, ERG3, ERG4, ERG10, and ERG11 was significantly upregulated in C. glabrata following fluconazole treatment. Inhibiting ergosterol biosynthesis using fluconazole also increased the expression of the sterol influx transporter AUS1 and the sterol metabolism regulators SUT1 and UPC2 in fungal cells. The microarray study quantified 35 genes with elevated mRNA levels, including AUS1, TIR3, UPC2, and 8 ERG genes, in a C. glabrata mutant strain lacking ERG1, indicating that sterol importing activity is increased to compensate for defective sterol biosynthesis in cells. Bioinformatic analyses further revealed that those differentially expressed genes were involved in multiple cellular processes and biological functions, such as sterol biosynthesis, lipid localization, and sterol transport. Finally, to assess whether sterol uptake affects yeast susceptibility to azoles, we generated a C. glabrata aus1∆ mutant strain. It was shown that loss of Aus1p in C. glabrata sensitized the pathogen to azoles and enhanced the efficacy of drug exposure under low oxygen tension. In contrast, the presence of exogenous cholesterol or ergosterol in medium rendered the C. glabrata AUS1 wild-type strain highly resistant to fluconazole and voriconazole, suggesting that the sterol importing mechanism is augmented when ergosterol biosynthesis is suppressed in the cell, thus allowing C. glabrata to survive under azole pressure. On the basis of these results, it was concluded that sterol uptake and sterol biosynthesis may act coordinately and collaboratively to sustain growth and to mediate antifungal resistance in C. glabrata through dynamic gene expression in response to azole stress and environmental challenges.

Crimean-Congo hemorrhagic fever virus entry and replication is clathrin-, pH- and cholesterol-dependent

To date, the entry pathway and replication mechanisms for members of the family Bunyaviridae, and especially for Crimean-Congo hemorrhagic fever virus (CCHFV), are poorly understood. Considering the severity of disease and the widespread geographical occurrence of CCHFV, investigating viral entry is of great value for development of antivirals. In this study, we have shown that knockdown of clathrin by small interfering RNA significantly reduced CCHFV nucleocapsid protein and viral RNA levels, suggesting that CCHFV utilizes clathrin-dependent endocytosis. In contrast, caveolin-1, an important constituent of caveolae endocytosis, is not important in CCHFV infection. Moreover, treatment with drugs that are known to interfere with the formation of clathrin-coated pits (sucrose and chlorpromazine) or endosome acidification (bafilomycin A1 and NH(4)Cl) also supported a clathrin-dependent pathway in the entry process of CCHFV. Finally, we demonstrated that cholesterol depletion in the cell plasma membrane significantly inhibited CCHFV infection. In the presence of exogenous cholesterol, this process was reversed, suggesting that cholesterol is important in the life cycle of CCHFV.

ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

A Trypsin-sensitive Protein is Required for Utilization of Exogenous Cholesterol for Pregnenolone Synthesis by Placental Mitochondria

The utilization of cholesterol for steroid hormone synthesis by human placental mitochondria is poorly understood. The human placenta does not express the steroidogenic acute regulator protein, which is critical for cholesterol delivery to the cholesterol side chain cleavage system in adrenal and gonadal mitochondria. We explored the mechanism underlying cholesterol transport in human placental mitochondria by measuring its transformation into pregnenolone. Mitochondria of syncytiotrophoblast from human term placenta were isolated by centrifugation through a sucrose gradient. The synthesis of pregnenolone in the presence of exogenous cholesterol was increased two-fold in syncytiotrophoblast mitochondria. Treatment of mitochondria with trypsin prevented the increase in the synthesis of pregnenolone in the presence of exogenous cholesterol. However, when 22-OH cholesterol, a substrate that readily crosses membranes, was added, the trypsin-treated mitochondria synthesized increased amounts of pregnenolone. The trypsin-treated mitochondria were intact, since oxygen consumption, succinate dehydrogenase and the adenine nucleotide translocase activities were not significantly different from in untreated mitochondria. However, activity of NADH cytochrome c oxidoreductase, an outer mitochondrial membrane enzyme, was reduced in the trypsin-treated mitochondria, reflecting the selective degradation of proteins. In addition, SDS-PAGE analysis revealed the loss of a prominent 34kDa band which proved to be a novel porin-like protein that binds to cholesterol. These results support our previous assumption that human placental mitochondria employ a novel protein(s)-mediated the mechanism to take up cholesterol for steroidogenesis.

Determination of hepatic acyl-coenzyme A-cholesterol acyltransferase activity in LPN hamsters: a model for cholesterol gallstone formation.

Acyl-coenzyme A-cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long-chain fatty acyl-coenzyme A derivatives and has been implicated in the development of cholesterol gallstones. In this study we have examined several key components of the hepatic ACAT assay in order to develop a reliable and sensitive ACAT assay for LPN hamsters, a breed of golden Syrian hamster which has been characterized recently by this laboratory as a particularly good model for studying the pathogenesis of cholesterol gallstones. The newly developed ACAT assays were subsequently used to examine whether hepatic ACAT activity is altered in this animal model. Important new methodological findings were: (i) ACAT activity displayed two pH optima, one at 7.0 when assayed using endogenous cholesterol as substrate, and the other at about pH 8.5-9.0 when assayed in the presence of exogenous cholesterol; (ii) ACAT activity increased markedly when exogenous cholesterol was delivered to ACAT in Tween 80 (125-fold) or hydroxypropyl-beta-cyclodextrin (200-fold) in contrast to the use of cholesterol/phosphatidylcholine liposomes (9-fold); (iii) the addition of dithiothreitol, but not reduced glutathione, to the assay mixture resulted in a marked decrease in ACAT activity. Using the optimal assay conditions (exogenous cholesterol added), hepatic ACAT activity was shown to be significantly reduced in hamsters fed a high sucrose lithogenic diet compared with controls (587+/-42 vs 737+/-44 pmol/min per mg; P=0.025). In contrast, ACAT activity measured using endogenous cholesterol as a substrate was greater in sucrose-fed hamsters compared with controls (22.3+/-2.5 vs 13.2+/-2.9 pmol/min per mg; P= 0.030). These results highlight the importance of using an ACAT activity assay which has been well characterized and supports the hypothesis that the pathogenesis of cholesterol gallstones in LPN hamsters is related to an altered hepatic cholesterol metabolism.

Glucocorticoids enhance the cholesterol side-chain cleavage activity of ovine adrenocortical mitochondria

We have shown previously that a chronic treatment with glucocorticoids enhances cAMP- or ACTH-induced steroidogenesis of cultured ovine adrenocortical cells. This effect appears to involve a greater amount of cholesterol in mitochondria. Hence, the present study aimed to define the role of glucocorticoids in cholesterol metabolism by these cells. 2-day-old cultures were exposed to different hormones or inhibitors (10 −6 M ACTH, 10 −5 M metyrapone) for 28–48 h. At the end of the treatment period, the cells were stimulated for 2 h with 10 −3 M 8Br-cAMP, in the presence of 10 −3 M aminoglutethimide (in order to load mitochondria with cholesterol). Mitochondria were then isolated and incubated without or with 100 μM cholesterol either in the presence or absence of 10 −3 M CaCl 2, or with 25 μM 22R-hydroxycholesterol. Mitochondria isolated from dexamethasone-treated cells produced consistently more pregnenolone than mitochondria from control cells, suggesting that at least part of the additional cholesterol present in these mitochondria was available for steroidogenesis. However, similar differences were obtained when mitochondria were incubated in the presence of exogenous cholesterol, both with or without calcium, or in the presence of 22R-hydroxycholesterol. Pregnenolone production under these latter conditions was much higher than when endogenous cholesterol was the only substrate. Conversely, metyrapone treatment of the cells resulted in lower production of pregnenolone from 22R-hydroxycholesterol by their mitochondria. Likewise ACTH treatment enhanced pregnenolone production by isolated mitochondria irrespective of the incubation conditions. These effects of dexamethasone and ACTH were not related to higher amounts of adrenodoxin, adrenodoxin reductase or cytochrome P450scc. These results indicate that exposure of ovine adrenocortical cells to glucocorticoids or ACTH enhances their steroidogenic potency not only by increasing the amount of cholesterol available for steroidogenesis but also by enhancing some step(s) involved in the transformation of cholesterol into pregnenolone.

Human mesangial cell production of monocyte chemoattractant protein-1: Modulation by lovastatin

Macrophages play a critical role in the progression of clinical and experimental glomerular injury. Serum-stimulated human fetal mesangial cells in culture produce a chemotactic factor that is monocyte-selective. This chemotactic factor is most likely monocyte chemoattractant protein-1 (MCP-1) as a monoclonal antibody directed against MCP-1, but not an irrelevant antibody, suppressed the mesangial cell-derived chemotactic activity. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by lovastatin resulted in a reduction of the mesangial cell-derived chemotactic activity as well as MCP-1 mRNA expression. The inhibitory effects of lovastatin in the presence of exogenous cholesterol were reversed by mevalonate, suggesting a role for isoprenoid intermediates of the mevalonate pathway and/or isoprenylated proteins in mesangial cell MCP-1 regulation. These findings suggest an additional mechanism by which HMG-CoA reductase inhibition in vivo may reduce glomerular injury.

Regulation of the threshold for lipoprotein-induced acyl-CoA:cholesterol O-acyltransferase stimulation in macrophages by cellular sphingomyelin content.

Macrophage acyl-CoA:cholesterol O-acyltransferase (ACAT), a key enzyme in atheroma foam cell formation, is stimulated by lipoproteins only after a "threshold" amount of cholesterol has accumulated in the cell. The present study explores the hypothesis that cellular sphingomyelin, by increasing the capacity of the cell to accommodate excess cholesterol, can influence the threshold of ACAT stimulation by lipoproteins. When the sphingomyelin content of macrophages was increased by either incubation with exogenous sphingomyelin or ceramide (a stimulator of endogenous sphingomyelin synthesis), the ability of acetyl-LDL to stimulate whole-cell ACAT activity was substantially reduced despite similar lipoprotein uptake and total cholesterol accumulation as in control cells. When the sphingomyelin content of macrophages was decreased by sphingomyelinase treatment, the ability of acetyl-LDL to stimulate whole-cell ACAT activity was enhanced despite no change in lipoprotein uptake. Importantly, microsomes isolated from control, sphingomyelin-, or sphingomyelinase-treated macrophages showed no difference in ACAT activity when assayed in vitro in the presence of exogenous cholesterol, suggesting that these treatments affected cholesterol trafficking. Lastly, a corollary of the hypothesis, that cells might adapt to a large increase in free cholesterol by increasing their sphingomyelin content, was supported by showing that the sphingomyelin content of macrophages increased 2.6-fold when the cells were induced to accumulate free cholesterol by incubation with acetyl-LDL plus an ACAT inhibitor. Thus, the sphingomyelin content of macrophages can influence the threshold at which ACAT is stimulated by lipoprotein delivery of cholesterol, and the cholesterol content of macrophages can influence the sphingomyelin content of the cell. These findings are consistent with a model in which cellular sphingomyelin plays an important role in accommodating pools of cellular cholesterol that result from the uptake of atherogenic lipoproteins by macrophages.

Cholesterol metabolism in human gallbladder mucosa: relationship to cholesterol gallstone disease and effects of chenodeoxycholic acid and ursodeoxycholic acid treatment.

The objective of this study was to investigate cholesterol metabolism in human gallbladder mucosa, especially in relation to hepatic cholesterol metabolism, gallstone disease and treatment with bile acids. Gallbladder mucosa and liver tissue samples were collected in 44 patients undergoing cholecystectomy; 30 had cholesterol gallstones and the rest were stone free. Ten of the gallstone patients were treated with chenodeoxycholic acid and eight received ursodeoxycholic acid, with a daily dose of 15 mg/kg body wt, for 3 wk before surgery. The 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, governing cholesterol synthesis, was considerably lower in the gallbladder mucosa than in liver tissue (28 +/- 6 and 120 +/- 40 pmol/min/mg protein). The acyl coenzyme A:acyltransferase activity in the gallbladder mucosa catalyzing the esterification of cholesterol was, on the other hand, several times higher than corresponding activity in the liver (92 +/- 23 and 11 +/- 2 pmol/min/mg protein). In the presence of exogenous cholesterol, the acyl coenzyme A:acyltransferase activity increased about twofold in the gallbladder mucosa. The acyl coenzyme A:acyltransferase activity of the gallbladder mucosa from untreated gallstone patients was not stimulated further by the addition of exogenous cholesterol. Otherwise, there were no significant differences in acyl coenzyme A:acyltransferase and 3-hydroxy-3-methylglutaryl coenzyme A reductase activities in the gallbladder mucosa of gallstone patients compared with gallstone-free controls. Treatment with chenodeoxycholic and ursodeoxycholic acids did not affect the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of the gallbladder mucosa but reduced the acyl coenzyme A:acyltransferase activity by 60% to 65%.(ABSTRACT TRUNCATED AT 250 WORDS)

In search of abnormal enzyme activities of hepatic cholesterol metabolism in patients with cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 ± 13 vs. 75 ± 5%, P < 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 ± 11 vs. and 109 ± 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 α-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 ± 1.1 vs. 8.0 ± 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A: cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 ± 0.4 vs. 6.7 ± 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.

Regulation of ACAT activity by a cholesterol substrate pool during the progression and regression phases of atherosclerosis: implications for drug discovery

The regulation of aortic ACAT by a cholesterol substrate pool (CSP) was investigated in a rabbit progression/ regression model of dietary-induced atherosclerosis. ACAT activity increased 25-fold during the 10-week progression phase of the study. ACAT activity decreased 8-fold during the 24-week regression phase of the study, however, it was still 14-fold greater than in normal aortas. ACAT activity assayed in the absence vs. the presence of exogenous cholesterol was used as a qualitative measure of the amount of cholesterol in the CSP. The CSP was filled to 28% of capacity in normal aortas, this increased to 75% during the progression phase. By the end of the regression phase, the CSP was filled to 100% of capacity even though serum cholesterol levels had returned to normal. The data are discussed in terms of emerging concepts of intracellular cholesterol trafficking, ACAT inhibitors, and the types of atherosclerotic lesions which may be subject to amelioration by ACAT inhibitors.

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

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 glomerulosa-like 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.

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [ 3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.

Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

We studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol (r=0.65 p<0.0003). These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans.

The influence of estradiol on cholesterol processing by the corpus luteum.

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.

Hepatic acyl-CoA: cholesterol acyltransferase. Development of a standard assay and determination in patients with cholesterol gallstones

A standard assay was developed for human liver acyl-CoA: cholesterol acyltransferase (ACAT, EC 2.3.2.26) which is more sensitive than previous methods and allows accurate activity determinations on crude microsomal fractions. ACAT activity was measured in microsomes from livers of four gallstone patients and five controls. Preincubation with exogenous cholesterol produced an increase in ACAT activity in all liver samples: gallstone samples showed a mean increase of 1.8-fold, whereas non-gallstone samples showed a mean increase of 8.2-fold. The mean ACAT activity measured in the presence of exogenous cholesterol was 52.8 ± 22.8 ( n = 4) pmol · min −1 · mg −1 for gallstone samples and 82.8 ± 13.5 ( n = 4) pmol · min −1 · m g −1 for non-gallstone samples. These results suggest that patients suffering from cholesterol gallstones have a reduced ability to esterify potentially harmful free cholesterol compared with controls. They support the proposition that cholesterol gallstone formation is related to altered hepatic cholesterol metabolism.

Two major regulatory steps in cholesterol synthesis by human renal cancer cells

Two major mechanisms regulating cholesterol biosynthesis exist in a human renal cancer cell line, Caki-1. Caki-1 is a newly established cell line whose characteristics of rapid growth and active cholesterol synthesis qualify it as a potentially valuable tool for elucidation of regulatory mechanism of cholesterol synthesis and transport. In the absence of exogenous cholesterol, cholesterol is the dominant sterol arising from labeled acetate and mevalonate. As expected, in the presence of exogenous cholesterol, the conversion of acetate to cholesterol and the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) is markedly reduced and this inhibition is released when cholesterol is removed from the medium. An unexpected and possibly unique finding is the inhibition of the conversion of mevalonate to cholesterol in the presence of exogenous cholesterol. This second major control process results in the accumulation of squalene and may involve additional late steps in cholesterol biosynthesis or metabolism. The occurrence of two major mechanisms regulating cholesterol synthesis may be a unique property of renal cancer cells or a previously unrecognized characteristic of a variety of cultured cells.