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Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P

We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

Introduction: Apolipoprotein A-I (apoA-I) mimetic peptides present an exciting avenue in the clinical development of synthetic HDL (sHDL), offering a more feasible and economical approach in sHDL production. One such example is ETC-642, or 22A—a 22 amino acid ApoA-I mimetic peptide tested in single and multiple dose clinical trials. The goal of this study is to understand how specific amino acid substitutions affect physico-chemical properties of 22-mer peptides and correlate them with the ability to form stable nano-sized sHDL particles and promote LCAT activity. Methods/Results: For this study, we used the 22A peptide (PVLDLFNELLNELLEALKQKLK or ETC-642) and designed 10 variants comprised of a single amino acid substitution, denoted 22A-1-10. We used computational programs FMAP (Folding of Membrane-Associated Peptides), PPM2.0 and FRAMEWORK to select peptides that are predicted to form α-helices, have good membrane penetration (>7.8 Å) with low transfer energy to membranes (ΔG transfer helix helix varied from -18.4 to -24.4 kcal/mol, and G transfer varied from -11.1 to -17.2 kcal/mol for the synthesized peptides. The percent peptide α-helicity in lipid bound form was measured by circular dichroism spectroscopy and was found to vary dramatically between 19 and 82%. The lipid solubilization kinetics were determined using UV absorbance to measure turbidity. The particle size and stability of resulting HDL particles were measured by dynamic light scattering and gel permeation chromatography. The peptides with low predicted energy of lipid binding exhibited high helicity and formed stable HDL particles with sizes less than 20 nm. The HDLs formed from apoA-I peptides with high helicity were also able to activate LCAT and facilitate sterol esterification with a K cat of 5 to 30 min -1 . Conclusions: Our data shows that that minimalistic changes in primary structure of ApoA-1 peptide can lead significant changes in physico-chemical properties. Peptides with degree of α-helicity exhibited superior lipid binding and formed stable HDL particles of small size able to interact with LCAT.

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p–Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1–1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.

Scavenger receptor BI influences multiple aspects of cellular sterol metabolism. In this series of studies, we evaluated the effect of scavenger receptor BI expression on the distribution and movement of sterol between the plasma membrane and the endoplasmic reticulum in macrophages, by comparing control J774 cells to J774 cells in which SR-BI expression was constitutively increased 3-fold. J774 cells with increased expression of SR-BI (J774-SRBI cells) esterified plasma membrane cholesterol more rapidly as compared to control cells. The esterification of endogenously synthesized cholesterol was also more rapid in cells with increased SR-BI expression; this could be partially suppressed by removing cholesterol from the plasma membrane. The increased plasma membrane sterol esterification in J774-SRBI cells was not due to increased acyl-coA:cholesterol acyltransferase activity and was observed even though J774-SRBI cells manifested a smaller free cholesterol pool in the endoplasmic reticulum. Cholesterol ester hydrolysis was also more rapid in J774-SRBI cells. Increased expression of SR-BI also facilitated the clearance of cellular cholesterol ester to HDL(3). This latter observation, combined with the measurement of the smaller ER free cholesterol pool in J774-SRBI cells, suggests that the free cholesterol derived from the hydrolysis of cholesterol ester was rapidly transported back to the plasma membrane. It is concluded that expression of SR-BI in macrophages increases the rate of free cholesterol transport, and modulates free cholesterol distribution between the plasma membrane and the internal membrane compartments in macrophages.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.

Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2 , that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2 are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis. Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs. Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity. A serine-to-leucine mutation in the (H/Y)SF motif (residues 338–340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p. Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523–529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completely abolished activity. In human ACAT1, mutagenesis of the corresponding motifs (residues 268–270, and 403–409, respectively) also nullified enzymatic activity. On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes. —Guo, Z., D. Cromley, J. T. Billheimer, and S. L. Sturley. Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.

The enzyme acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1) mediates sterol esterification, a crucial component of intracellular lipid homeostasis. Two enzymes catalyze this activity in Saccharomyces cerevisiae (yeast), and several lines of evidence suggest multigene families may also exist in mammals. Using the human ACAT1 sequence to screen data bases of expressed sequence tags, we identified two novel and distinct partial human cDNAs. Full-length cDNA clones for these ACAT related gene products (ARGP) 1 and 2 were isolated from a hepatocyte (HepG2) cDNA library. ARGP1 was expressed in numerous human adult tissues and tissue culture cell lines, whereas expression of ARGP2 was more restricted. In vitro microsomal assays in a yeast strain deleted for both esterification genes and completely deficient in sterol esterification indicated that ARGP2 esterified cholesterol while ARGP1 did not. In contrast to ACAT1 and similar to liver esterification, the activity of ARGP2 was relatively resistant to a histidine active site modifier. ARGP2 is therefore a tissue-specific sterol esterification enzyme which we thus designated ACAT2. We speculate that ARGP1 participates in the coenzyme A-dependent acylation of substrate(s) other than cholesterol. Consistent with this hypothesis, ARGP1, unlike any other member of this multigene family, possesses a predicted diacylglycerol binding motif suggesting that it may perform the last acylation in triglyceride biosynthesis.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) is an enzyme, located in the endoplasmic reticulum of many types of cells, that catalyzes cholesterol ester formation from cholesterol and fatty acyl CoA substrates. Sterol esterification by ACAT or homologous enzymes is conserved in evolution dating back to yeast. The recent cloning of a human cDNA for ACAT, together with genome sequencing projects, has led to the identification of an ACAT gene family and provided molecular tools for determining ACAT's functions in vivo. Summarized here is the current knowledge concerning the molecular genetics of ACAT.

The conversion of cholesterol to an esterified storage form by the enzyme acyl-coenzyme A: cholesterol acyltransferase, is a critical component of sterol and membrane homeostasis and represents a significant step in atherogenesis. The isolation in 1993 of the first molecular probe of acyl-coenzyme A: cholesterol acyltransferase provided a key to understanding this reaction and its regulation. The sequence is apparently ubiquitous in eukaryotes, often with multiple sequence homologs within an organism. Presented here is a review of the known molecular events that lead to intracellular sterol esterification.

Two temperature-conditional secretory mutations, sec1 and sec5, cause the accumulation of post-Golgi vesicles when strains containing these mutations are grown at 37 degrees C. In addition to accumulating vesicles, the mutants do not esterify free sterol on rich media at the restrictive temperature. It is the high level of inositol in the media that causes this condition in the yeast Saccharomyces cerevisiae, not a defective steryl ester synthase or lack of substrates. When strains containing the sec1 or sec5 mutation were transformed separately with a plasmid carrying SEC1 and SEC5, the esterification and secretory defects were alleviated. Double mutants containing sec6, sec14 or sec18 with either a sec1 or sec5 mutation have normal esterification levels. Strains with suppressor mutations were isolated that grew at 37 degrees C, esterified sterols and had diminished accumulation of vesicles, when grown at the restrictive temperature on defined media with additional inositol. Electron microscopy was used to examine vesicle accumulation, the number of lipid droplets, and to further characterize the esterification defect. When grown at 37 degrees C on defined medium, the strains with sec5 or sec1 accumulated the usual secretory vesicles, but when grown under similar conditions with elevated levels of inositol, accumulated an additional vesicular-like body.

We have identified two yeast genes with similarity to a human cDNA encoding acyl-coenzyme A:cholesterol acyltransferase (ACAT). Deletion of both yeast genes results in a viable cell with undetectable esterified sterol (Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T., Rothstein, R., and Sturley, S. L. (1996) Science 272, 1353-1356). Here, we expressed the human cDNA in the yeast double mutant, resulting in high level production of ACAT protein, but low in vivo esterification of ergosterol, the predominant yeast sterol. The activity of the human enzyme was increased by incubation of these cells with 25-hydroxy, cholesterol, an established positive regulator of mammalian sterol esterification. In contrast, the yeast enzymes were unaffected by this reagent. In vitro microsomal assays indicated no sterol esterification in extracts from the double mutant. However, significant activity was detected from strains expressing human ACAT when cholesterol was equilibrated with the microsomal membranes. The human enzyme in yeast utilized cholesterol as the preferred sterol and was sensitive to competitive (S58035) and non-competitive (DuP 128) ACAT inhibitors. The yeast esterifying enzymes exhibited a diminished sterol substrate preference and were sensitive only to S58035. Human ACAT had a broad acyl-CoA substrate specificity, the other substrate for this reaction. By contrast, the yeast enzymes had a marked preference for specific acyl-CoAs, particularly unsaturated C18 forms. These results confirm the yeast genes as functional homologs of the human gene and demonstrate that the enzymes confer substrate specificity to the esterification reaction in both organisms.

Unesterified sterol modulates the function of eukaryotic membranes. In human cells, sterol is esterified to a storage form by acyl-coenzyme A (CoA): cholesterol acyl transferase (ACAT). Here, two genes are identified, ARE1 and ARE2, that encode ACAT-related enzymes in yeast. The yeast enzymes are 49 percent identical to each other and exhibit 23 percent identity to human ACAT. Deletion of ARE2 reduced sterol ester levels to approximately 25 percent of normal levels, whereas disruption of ARE1 did not affect sterol ester biosynthesis. Deletion of both genes resulted in a viable cell with undetectable esterified sterol. Measurements of [14C]acetate incorporation into saponified lipids indicated down-regulation of sterol biosynthesis in the are1 are2 mutant cells. With the use of a consensus sequence to the yeast and human genes, an additional number of the ACAT gene family was identified in humans.

The tobacco mutant LAB 1–4 overproduces sterols, most of which are esterified and stored as lipid droplets in the cytoplasm (P. Maillot-Vernier, L. Gondet, H. Schaller, P. Benveniste and G. Belliard, Mol. Gen. Genet. 231 (1991) 33–40). We studied the enzymes involved in the esterification of sterols and hydrolysis of steryl esters in tobacco calli and leaves. These activities were shown to be bound to microsomal membranes. The microsomes were delipidated by acetone treatment so as to discard endogenous sterols and steryl esters; optimal conditions were set up which allowed the selective determination of either the sterol acyl transferase (SATase) activity or the steryl ester hydrolase (SEHase) activity. The SATase activity of the LAB 1–4 mutant calli was shown to be twice that of the wild type calli whereas the activities were the same in the leaves of both genotypes. The SEHase activity of LAB 1–4 calli and leaves was only slightly higher than that of the wild type tissues. The results strongly suggest that the activity of the SATase is increased when the ratio sterols/enzyme exceeds a certain threshold. Both enzymes might be involved in the control of the free sterol content of the sterol-overproducing mutant and, more generally, in the homeostasis of free sterols in the plant cells.

A significant accumulation of cellular free cholesterol and steryl esters is observed in J774 macrophages when cells are exposed to low-density lipoproteins (LDL) containing cholesterol 5 beta,6 beta-epoxide. This cellular sterol accumulation is mainly due to the formation of esterified cholesterol and desmosterol. Cellular steryl esters increased to 39.4 and 22.4 micrograms/mg cell protein with 0.8 microM of cholesterol 5 beta,6 beta-epoxide and 3,5-cholestadien-7-one, respectively, whereas hardly detectable levels were observed with the absence of oxysterols. The total cellular sterols increased 45% above the value of control with cholesterol 5 beta,6 beta-epoxide. The uptake of [3H] cholesteryl oleate-LDL was also enhanced by cholesterol 5 beta,6 beta-epoxide. The rapid displacement of desmosterol with cholesterol was observed when cells were treated with cholesterol 5 beta,6 beta-epoxide or 3,5-cholestadien-7-one in the presence of LDL. Cholesterol 5 beta,6 beta-epoxide became associated with LDL in the culture conditions, and its uptake into J774 cells and the cytotoxicity were reduced significantly by the association with LDL. The comparison of selected oxysterols for their ability to stimulate cellular sterol accumulation indicated that cholesterol 5 beta,6 beta-epoxide is the most potent. Cholesterol esterification was enhanced significantly by cholesterol 5 beta,6 beta-epoxide whereas cholesterol 5 alpha,6 alpha-epoxide and 3,5-cholestadien-7-one produced a modest response. In contrast, although cholestantriol, the metabolic hydrolysis product of cholesterol epoxides, also associated with LDL, it showed no stimulating effect on both cellular sterol content and sterol esterification. These results indicate that some oxysterols, such as cholesterol 5 beta,6 beta-epoxide and possibly 3,5-cholestadien-7-one, stimulate cellular sterol accumulation in J774 macrophages and may play an important role in atherogenesis.

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.

Saccharomyces cerevisiae sterol and heme auxotrophs were used to elucidate a role for hemes in sterol esterification. Steryl ester synthase (SES) activity was stimulated on average fourfold in cells supplemented with 50 micrograms/ml delta-aminolevulinic acid (ALA). This stimulation was not dependent on ALA per se, but on the ability of this precursor to effect heme competency. The addition of ALA stimulated SES activity of yeast on either fermentative or respiratory carbon sources. The elevation of SES activity was independent of intracellular free sterol, unsaturated fatty acid, or methionine levels. SES activity increases as the cells enter stationary phase, and this increase is enhanced by heme competency. SES was directly inhibited by the hypocholesterolemic drug lovastatin (mevinolin). The inhibition of SES activity by lovastatin was enhanced in heme-competent cells.

We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media. This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions. Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction. Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2). The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells. One such factor could be the mammalian equivalent of the gene product of UPC2.

Arange of analytical techniques was used to investigate the composition of the steryl fatty acyl esters in a cell suspension culture of celery (Apium graveolens). Gas chromatography (GC) and GC-mass spectrometry (GC-MS), using electron ionization (EI) and negative ion chemical ionization (NICI), were employed to characterize the intact steryl esters. Assignments were supported by analysis of the sterol and fatty acid moieties released from the intact molecular species by alkaline hydrolysis. A selectivity for sterol esterification was noted, with the major free sterol, stigmasterol, occurring only in a very small amount in the esterified form. Instead, the precursors to Δ5-phytosterols, particularly cycloartenol, predominated in the ester fraction. The pentacyclic triterpene, β-amyrin, was also found as the palmitate and linoleate esters. Changes in composition and abundance of the steryl esters during the different growth phases of a celery cell suspension culture were investigated. The total amount of esterified sterols exceeded that of free sterols throughout the growth cycle. The changes observed during growth highlighted differences between the esters of precursor sterols and those of the 4-desmethyl-sterols, and it is postulated that the various steryl esters perform different functions in cell metabolism.

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.

Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT1 and ACAT2 (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67 percent and in tissues by ACAT was 64 percent. Esterification of nine sterols, (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined.(The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82 respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74 respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29 percent and decreased the esterification by LCAT by 5.9 percent. Esterification by ACAT and LCAT was reduced respectively by 25 percent and 11 percent by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.

The mechanisms responsible for reocclusion after percutaneous transluminal angioplasty are still poorly understood. The effects of angioplasty on arterial morphology, deoxyribonucleic acid (DNA) synthesis (3H-thymidine incorporation) and lipid metabolism (14C-oleate incorporation) were studied in renal arteries of 24 male mongrel dogs. Balloondilated (identified by Evans blue dye accumulation) and adjacent normal arterial segments were collected 90 min and 2, 5 and 14 days after the procedure. The immediate vascular response was endothelial cell denudation and platelet accumulation. Two weeks after angioplasty, healing of the luminal surface by “endothelial-like” cells, mild smooth muscle cell proliferation and an angiogenic response with capillary growth into the media were observed. DNA synthesis was increased in balloon-dilated segments at day 5 compared with adjacent nonballoon-dilated artery. This increase in DNA synthesis persisted in the 2 week postangioplasty segments. Additionally, angioplasty produced both quantitative and qualitative changes in arterial lipid synthesis. The most dramatic change was an increase in sterol esterification that was apparent as early as 90 min after angioplasty; the change persisted through day 5 but diminished toward baseline by day 14. Angioplasty-induced alterations of arterial metabolism parallel aspects of the atherogenic process and may be involved in the pathogenesis of postangioplasty reocclusion, particularly in the presence of additional risk factors, such as hyperlipemia.

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.

ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sites: a substrate-binding site and an allosteric-activator site. In the present work, we investigated the structural features of various sterols as substrates and/or activators in vitro. The results show that without cholesterol, the plant sterol sitosterol is a poor substrate for ACAT. In the presence of cholesterol, ACAT1-mediated esterification of sitosterol is highly activated while ACAT2-mediated esterification of sitosterol is only moderately activated. For ACAT1, we show that the stereochemistry of the 3-hydroxy group at steroid ring A is a critical structural feature for a sterol to serve as a substrate, but less critical for activation. Additionally, enantiomeric cholesterol, which has the same biophysical properties as cholesterol in membranes, fails to activate ACAT1. Thus ACAT1 activation by cholesterol is the result of stereo-specific interactions between cholesterol and ACAT1, and is not related to the biophysical properties of phospholipid membranes. To demonstrate the relevance of the ACAT1 allosteric model in intact cells, we showed that sitosterol esterification in human macrophages is activated upon cholesterol loading. We further show that the activation is not due to an increase in ACAT1 protein content, but is partly due to an increase in the cholesterol content in the endoplasmic reticulum where ACAT1 resides. Together, our results support the existence of a distinct sterol-activator site in addition to the sterol-substrate site of ACAT1 and demonstrate the applicability of the ACAT1 allosteric model in intact cells.

We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 ( CgCDR1 ) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11 , which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2 . In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes.

Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17α-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

The formation of sterol esters from free sterols and fatty acyl CoAs is a fundamental pathway in lipid metabolism of eukaryotic cells. In vertebrates, the sterol esterification reaction is catalyzed by acyl CoA:cholesterol acytransferase (ACAT; EC 2.3.1.26), an enzyme located primarily in the endoplasmic reticulum. In addition to its role in cellular cholesterol homeostasis, ACAT has been hypothesized to participate in a number of processes involving mammalian cholesterol metabolism. Recent studies have provided evidence that more than one ACAT enzyme exists in mammals. Here we review the recent research that has led to the identification and characterization of two mammalian ACAT enzymes, ACAT-1 and ACAT-2.

The intracellular esterification of sterols by all eukaryotes represents a critical homeostatic response to excess sterol. This process is mediated by a multigene family in several organisms, including the unicellular eukaryote, Saccharomyces cerevisiae (yeast). We describe here our recent efforts to identify molecular components of sterol esterification and sterol transport in this model system. This includes homologs to the acyl-CoA cholesterol acyl transferase (ACAT) gene, a putative homolog to the Niemann Pick C (NPC) gene, and novel yeast genes with human counterparts.

Esterification of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) is a key element in maintaining cholesterol homeostasis in cells of higher animals. In the budding yeast, Saccharomyces cerevisiae, accumulation of ergosteryl esters accompanies entry into stationary phase and sporulation. We have determined that two genes in yeast, SAT1 and SAT2, encode isozymes of acyl-CoA:sterol acyltransferase (ASAT) which are functionally related to ACAT. The SAT1 isozyme is the major catalytic isoform, accounting for at least 65-75% of total ASAT activity. Targeted deletions of one or both genes do not compromise mitotic cell growth or spore germination. However, diploids that are homozygous for a SAT1 null mutation exhibit significantly reduced sporulation efficiency. Furthermore, a larger fraction of the sporulating diploids arrest after the first meiotic division. Human ACAT expressed in sat1 sat2 mutant cells can catalyze esterification of cholesterol and, to a lesser extent, ergosterol in vitro, but restores ergosteryl oleate formation in vivo to only approximately 8% of that catalyzed by yeast ASAT in wild-type cells.

Cell cultures have been obtained in vitro from a plant producing ecdysteroids, Chenopodium album. The composition of free and esterified sterols was compared in plant and cell cultures after purification by TLC and identification by GC/MS. Free sterols were the most abundant. A selectivity of sterol esterification was noted for 4,4-dimethylsterols (except in roots) and Δ7-sterols. The 4-desmethylsterols from leaves, roots and cell cultures of Chenopodium album were a mixture of Δ7-sterols (>61%) and Δ5-sterols (>36%). The Δ7-sterols were 24-ethylcholesta-7,22-dien-3β-ol and 24-ethylcholest-7-en-3β-ol. The isolated Δ5-sterols were sitosterol and stigmasterol. Traces of cholesterol and Δ5,7-sterols were also detected. Our attention was also given to ecdysteroid contents, which showed much lower quantity in cell cultures than in plants, confirming however the presence of 20-hydroxyecdysone.

The local anesthetic lidocaine was studied for its effects on lipid metabolism in aortas from normal rats, rabbits, and cholesterol-fed (atherosclerotic) rabbits in vitro. Incubation of aortas in the presence of 3–5 mM lidocaine resulted in a statistically significant reduction in the incorporation of [ 14 C]oleate into cholesteryl esters and phosphatidylcholine. Additionally, significant increases in [ 14 C]oleate incorporation into the diglyceride fraction of atheromatous rabbit aortas was observed with a trend to greater incorporation into the diglyceride fraction of normal rat and rabbit arteries as well. The most significant overall effect of lidocaine was its inhibition (50–90%) of the arterial sterol esterification. Assays of acylCoA : cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes revealed that, in addition to local anesthetics (e.g., lidocaine), other membrane-active agents such as chlorpromazine and methoxyflurane inhibit ACAT; this suggests ACAT may be regulated by alterations in the biophysical properties of its membrane milieu.

Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes and in intact arterial tissue from the rat and cholesterol-fed rabbit in vitro. In isolated rabbit arterial microsomes, CPZ resulted in a concentration-dependent inhibition of ACAT with 50% inhibition of [1-14C]oleoylCoA incorporation into [14C]cholesteryl esters occurring at 0.1 mM CPZ. CPZ also effectively inhibited the incorporation of [14C]oleate into triglycerides without affecting incorporation into diglycerides. Additionally, CPZ altered the pattern of arterial phospholipids synthesized from [1-14C]oleate. Incorporation into phosphatidylcholine was depressed while incorporation into phosphatidylinositol was increased. Since diglyceride synthesis appeared to be unaffected by CPZ, a redirection of phosphatidic acid into the CDP-diglyceride pathway of glycerolipid synthesis does not adequately account for the effect of CPZ on arterial phospholipid and triglyceride synthesis in these experiments.

The adequacy of sterol derivatives containing a blocked 3-hydroxyl group for sustaining the growth of two sterol auxotrophs has been investigated. Mycoplasma capricolum, a cholesterol-requiring bacterium, grows nearly as well on media supplemented with cholesteryl methyl ether or cholesteryl acetate as on free cholesterol. The two derivatives are recovered unchanged from the bacterial cells. Similarly, cholesteryl methyl ether or ergosteryl methyl ether replace cholesterol or ergosterol as sterol sources for a yeast mutant, strain GL7, defective in 2,3-oxidosqualene-lanosterol cyclization. During aerobic or semianaerobic growth, yeast cells demethylate some of the cholesteryl methyl ether to free cholesterol. However, cells growing on cholesterol methyl ether under strict anaerobic conditions do not produce free sterol. The bearing of these results on the postulated requirement of a free sterol hydroxyl group for membrane function is discussed. Sterol esterification does not appear to be essential for the two microbial systems.

Activities of enzymes involved in cholesterol metabolism were measured in ovaries of PMS-hCG-primed immature rats. Microsomal 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in sterol synthesis, was low during the period of maximal steroid production and cholesteryl ester storage. Acyl CoA:cholesterol acyltransferase (ACAT), the microsomal enzyme catalyzing sterol esterification and cytosolic cholesteryl ester hydrolase and mitochondrial cholesterol side chain cleavage activities, paralleled storage of sterol esters and levels of plasma progesterone, being highest on day 7 post hCG. Further experiments demonstrated that HMG-CoA reductase and ACAT are regulated by different mechanisms than the sterol esterase and cholesterol side chain cleavage system. When blood sterol levels were lowered by 4-aminopyrazolo (3,4-d)pyrimidine, plasma progestin concentrations fell and ovaries failed to accumulate sterol esters. HMG-CoA reductase increased more than 10-fold as a result of this treatment, while ACAT was 15% of that measured in controls. In contrast, cholesteryl esterase and the cholesterol side chain cleavage system were not affected. Intravenous infusion of human lipoproteins reversed the effects of 4-aminopyrazolo (3,4-d)pyrimidine. Raising blood levels up to 6-fold with either cholesterol-supplemented diets or intravenous injections of rat lipoproteins had no effect upon ovarian sterol metabolism, suggesting that the process by which blood cholesterol is utilized is saturated at normal blood sterol levels. Intravenous injection of LH on day 7 post hCG increased HMG-CoA reductase and decreased ACAT activity in addition to depleting cholesteryl esters. Prior treatment of rats with aminoglutethimide prevented the effects of LH on both HMG-CoA reductase and ACAT, indicating that changes in these enzymes were a consequence of the steroidogenic stimulus imposed by LH. Treatment with aminoglutethimide alone on day 7 post hCG did not further depress the already low HMG-CoA reductase activity, but ACAT and cholesterol ester storage were stimulated while sterol esterase activity was not altered. Aminoglutethimide also produced elevated ACAT activity and sterol ester storage in animals treated on day 2 post hCG, a time when there is normally little sterol ester accumulation. We conclude that HMG-CoA reductase and ACAT are regulated, in a reciprocal fashion, by ovarian cholesterol balance. When sufficient cholesterol is available, HMG-CoA reductase is suppressed and ACAT increases to facilitate esterification of sterol in excess of cell needs. When exogenous sterol supplies cannot meet ovarian demands, HMG-CoA reductase rises, and there is a concomitant decline in ACAT. Thus, gonadotropic control over these enzymes may be exerted, in part, through modulation of the supply of cholesterol to the ovary and/or through regulation of the rate of sterol utilization for hormone synthesis.