Expression of SOAT1 in Adrenocortical Carcinoma and Response to Mitotane Monotherapy: An ENSAT Multicenter Study.

CONTEXT: Objective response rate to mitotane in advanced adrenocortical carcinoma (ACC) is approximately 20%, and adverse drug effects are frequent. To date, there is no marker established that predicts treatment response. Mitotane has been shown to inhibit sterol-O-acyl transferase 1 (SOAT1), which leads to endoplasmic reticulum stress and cell death in ACC cells. OBJECTIVE: To investigate SOAT1 protein expression as a marker of treatment response to mitotane. PATIENTS: A total of 231 ACC patients treated with single-agent mitotane as adjuvant (n = 158) or advanced disease therapy (n = 73) from 12 ENSAT centers were included. SOAT1 protein expression was determined by immunohistochemistry on formalin-fixed paraffin-embedded specimens. SETTING: Retrospective study at 12 ACC referral centers. MAIN OUTCOME MEASURE: Recurrence-free survival (RFS), progression-free survival (PFS), and disease-specific survival (DSS). RESULTS: Sixty-one of 135 patients (45%) with adjuvant mitotane treatment had recurrences and 45/68 patients (66%) with mitotane treatment for advanced disease had progressive disease. After multivariate adjustment for sex, age, hormone secretion, tumor stage, and Ki67 index, RFS (hazard ratio [HR] = 1.07; 95% confidence interval [CI], 0.61-1.85; P = 0.82), and DSS (HR = 1.30; 95% CI, 0.58-2.93; P = 0.53) in adjuvantly treated ACC patients did not differ significantly between tumors with high and low SOAT1 expression. Similarly, in the advanced stage setting, PFS (HR = 1.34; 95% CI, 0.63-2.84; P = 0.45) and DSS (HR = 0.72; 95% CI, 0.31-1.70; P = 0.45) were comparable and response rates not significantly different. CONCLUSIONS: SOAT1 expression was not correlated with clinical endpoints RFS, PFS, and DSS in ACC patients with mitotane monotherapy. Other factors appear to be relevant for mitotane treatment response and ACC patient survival.

Sterol O-Acyltransferase 2 Contributes to the Yolk Cholesterol Trafficking during Zebrafish Embryogenesis.

To elucidate whether Sterol O-acyltransferase (Soat) mediates the absorption and transportation of yolk lipids to the developing embryo, zebrafish soat1 and soat2 were cloned and studied. In the adult zebrafish, soat1 was detected ubiquitously while soat2 mRNA was detected specifically in the liver, intestine, brain and testis. Whole mount in situ hybridization demonstrated that both soat1 and soat2 expressed in the yolk syncytial layer, hatching gland and developing cardiovascular as well as digestive systems, suggesting that Soats may play important roles in the lipid trafficking and utilization during embryonic development. The enzymatic activity of zebrafish Soat2 was confirmed by Oil Red O staining in the HEK293 cells overexpressing this gene, and could be quenched by Soat2 inhibitor Pyripyropene A (PPPA). The zebrafish embryos injected with PPPA or morpholino oligo against soat2 in the yolk showed significantly larger yolk when compared with wild-type embryos, especially at 72 hpf, indicating a slower rate of yolk consumption. Our result indicated that zebrafish Soat2 is catalytically active in synthesizing cholesteryl esters and contributes to the yolk cholesterol trafficking during zebrafish embryogenesis.

Lecitin: colesterol aciltransferasa (LCAT) en pacientes con diabetes mellitus tipo 2 con macroangiopatía diabética de miembros inferiores / Lecithin-cholesterol acetyltransferase (LCAT) in patients presenting with type 2 diabetes mellitus and diabetic macroangiopathy in lower extremities

Objetivo: describir el comportamiento de la enzima LCAT en pacientes diabéticos tipo 2 con macroangiopatía diabética de los miembros inferiores.Métodos: se estudiaron 110 diabéticos tipo 2, ambulatorios con y sin macroangiopatía diabética de los miembros inferiores; y 30 personas adultas, supuestamente sanas, sin diabetes y enfermedad vascular confirmada. Se cuantificaron las siguientes variables: glicemia, HbA1c, actividad de la enzima LCAT y la velocidad inicial de esterificación del colesterol. Resultados: se encontró un mal control glucémico en los diabéticos con macroangiopatía de tipo ocluido. Se halló un intervalo de confianza para la LCAT de 6,1 a 69,7 nmol/mL/h y para la velocidad inicial de esterificación del colesterol de 1,6 a 15,2 por ciento/h. Se encontró una correlación lineal entre la LCAT y la velocidad de esterificación (r= +0,934; p< 0,05). Los diabéticos con macroangiopatía diabética, independientemente de su tipo, mostraron un aumento (p< 0,05) de las 2 variables. No hubo diferencias significativas entre el grupo de referencia y el de diabéticos sin daño vascular periférico. Se encontró en el grupo con macroangiopatía diabética el 58,3 por ciento con actividad enzimática y velocidad normal, el 86,96 por ciento con actividad y velocidad elevada, y el 75 por ciento con un comportamiento anómalo. Al tener en cuenta el tipo de macroangiopatía se observó que las proporciones de diabéticos fueron similares. Conclusiones: los diabéticos tipo 2 con macroangiopatía diabética de miembros inferiores, independiente de su tipo, presentan un aumento en la actividad de la enzima LCAT, aunque no se pudo establecer la posible unión entre la LCAT y la macroangiopatía diabética de miembros inferiores(AU)

Objective: to describe the behavior of the LCAT enzyme in type 2 diabetic patients presenting with diabetic macroangiopathy of lower extremities.Methods: a total of 110 outpatients suffering diabetes mellitus type 2, with and without diabetic macroangiopathy of lower extremities, and 30 supposedly healthy non-diabetic adults with confirmed vascular disease were studied. The following variables were quantified: glycemia, HbA1c, LCAT enzyme activity and the initial speed of cholesterol esterification. Results: there was poor glycemic control in diabetic patients with occluded macroangiopathy occluded. There was found a CI = 6,1 - 69,7 nmol/m;/h for LCAT and CI=1,6- 15,2 percent /h for the initial speed of cholesterol esterification. The linear correlation between the LCAT and the esterification speed was found (r = + 0.934; p < 0.05). The diabetic patients with diabetic macroangiopathy, regardless of its type, showed an increase (p< 0.05) of both variables. There were no significant differences between the reference group and that of diabetics without peripheral vascular damage. In the group with diabetic macroangiopathy, it was found that 58,3 percent had normal enzymatic activity and speed, 86,96 percent showed high activity and speed and the 75 percent showed anomalous behavior. Taking into account the type of macroangiopathy, the proportions of diabetic patient were similar. Conclusions: the type 2 diabetic patients with diabetic macroangiopathy of lower extremities, regardless of type, have increased activity of LCAT enzyme, although the possible link between LCAT and the diabetic macroangiopathy of lower extremities was not identified(AU)

Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study.

BACKGROUND: Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer. RESULTS: Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition. CONCLUSION: The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

ACAT2 is localized to hepatocytes and is the major cholesterol-esterifying enzyme in human liver.

BACKGROUND: Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification. METHODS AND RESULTS: In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined. CONCLUSIONS: The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.

Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor.

BACKGROUND/AIMS: Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance. METHODS: Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis). RESULTS: In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis. CONCLUSIONS: The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.

Dehydroepiandrosterone, an adrenal androgen, increases human foam cell formation: a potentially pro-atherogenic effect.

OBJECTIVES: We studied the effects of dehydroepiandrosterone (DHEA), an abundant adrenal androgen on two key early events of atherogenenis: 1) human monocyte adhesion to vascular endothelium, and 2) human foam cell formation. BACKGROUND: In the U.S., where DHEA is available without prescription, there has recently been a rapid increase in unsupervised self-administration of DHEA. The vascular biologic effects of DHEA are largely unknown, however. METHODS: Regarding adhesion, human umbilical vein endothelial cells (HUVECs), exposed to either DHEA (42 or 420 nmol/l) or control, were incubated with human monocytes, and adhesion was measured by hemocytometry. Surface expression of endothelial cell adhesion molecules was measured by ELISA. Regarding foam cell formation, studies of lipid loading were performed on macrophages treated with DHEA or control and/or the androgen receptor antagonist hydroxyflutamide (HF) (4 micromol/l). Intracellular cholesterol and cholesteryl esters (CE) were quantified by high-performance liquid chromatography. Expression of foam cell formation-related genes was measured by reverse-transcription polymerase chain reaction. RESULTS: DHEA produced a dose-dependent receptor-mediated increase in the male macrophage CE content (up to 120 +/- 4% of control values, p = 0.015). DHEA upregulated messenger ribonucleic acid expression of the lipoprotein-processing enzymes acyl coenzyme A:cholesterol acyltransferase I and lysosomal acid lipase by 3.4- and 5.3-fold, respectively (p < 0.05 vs. control), but had no effect on scavenger receptor expression (p > 0.2). There was no significant effect of DHEA on monocyte-endothelial adhesion (<10% change in values, p = 0.56) or endothelial cell expression of cell adhesion molecules (p > 0.1). CONCLUSIONS: DHEA increases human macrophage foam cell formation, a potentially pro-atherogenic effect. This effect appears to be mediated via the androgen receptor and involves the upregulation of lipoprotein-processing enzymes.

Cytotoxic cellular cholesterol is selectively removed by apoA-I via ABCA1.

Excess intracellular free cholesterol (FC) is cytotoxic. This study examines prevention of FC-induced cytotoxicity in J774 macrophage foam cells by incubation with apolipoprotein AI (apoA-I). J774 were cholesterol enriched using acetylated low-density lipoprotein and FC/phospholipid (PL) dispersions. Treatment with an acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, in the absence of extracellular acceptors, produced hydrolysis of stored esterified cholesterol (EC) and FC-induced cytotoxicity. Incubation of cells with ACAT inhibitor plus apoA-I resulted in FC efflux (0.39 +/- 0.02%/h) along with a reduction in cytotoxicity (26.30 +/- 5.80%), measured by adenine release. Small unilamellar vesicles (SUV) caused greater FC efflux (0.53 +/- 0.02%/h, P = 0.001), but a modest reduction in cytotoxicity (8.40 +/- 2.70%, P = 0.008). Co-incubation of ACAT inhibitor plus the cholesterol transport inhibitor U18666A or the antioxidant Probucol reduced efflux to apoA-I, but not to SUV. Pre-treatment of J774 foam cells with CTP-cAMP upregulates hormone sensitive lipase (HSL) and further upregulates ATP binding cassette A1 (ABCA1). Using mouse serum as a cholesterol acceptor, CTP-cAMP caused greater protection against FC-induced cytotoxicity compared to cells without pre-treatment, suggesting a role of ABCA1 in removal of cytotoxic FC. We conclude that a cytotoxic pool of FC is located in the plasma membrane, is readily available for efflux to apoA-I, and removal of cytotoxic cholesterol may involve ABCA1.

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida, by enzymatic and serological methods.

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.

Regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) synthesis, degradation, and translocation by high-density lipoprotein(2) at a low concentration.

(,Although plasma HDL(2) cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL(2) at a low concentration (< or = 40 microg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL(2) on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL(2) at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL(2), however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL(2) promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL(2) is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.

Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.

Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum.

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing approximately 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.

The influence of dietary saturated and unsaturated fat on hepatic cholesterol metabolism and the biliary excretion of chylomicron cholesterol in the rat.

The biliary excretion of [3H] cholesterol carried in chylomicrons derived from palm oil (rich in long chain saturated fatty acids), olive oil (rich in monounsaturated fatty acids) or corn oil (rich in n-6 polyunsaturated fatty acids was studied in vivo in rats fed the corresponding oil in the diet for 21 days. The secretion of radioactivity into bile as both bile acids and unesterified cholesterol was significantly slower in the animals fed palm oil as compared to those given olive or corn oil, indicating that dietary saturated fat retards the excretion of cholesterol from the diet as compared to mono- or n-6 polyunsaturated fat. In order to investigate the mechanisms underlying these differences, the influence of the three high fat diets on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis in the liver and on biliary lipid output were also measured. The ratio of cholesterol esterification to cholesteryl ester hydrolysis was markedly raised in the olive and corn oil-fed as compared to palm oil-fed animals. Biliary cholesterol secretion was higher in corn oil-fed rats than in those fed olive or palm oil or a low fat diet, and this was associated with a markedly increased lithogenic index in these animals. The activity of cholesterol 7alpha hydroxylase was higher in the olive and corn oil-fed than in the palm oil-fed animals, although the expression of mRNA for the enzyme was increased only in the olive oil diet group. After 20 h biliary drainage, the rate of bile acid secretion into bile was increased in the rats fed olive and corn oil rather than to palm oil. These findings indicate that feeding rats mono- or n-6 polyunsaturated as compared to saturated fat in the diet promotes the storage of cholesteryl ester in the liver and leads to increased bile acid synthesis, resulting in the more rapid excretion of cholesterol originating from the diet via the bile.

Ontogeny and location of HMG-CoA reductase, ACAT, and MGAT in human small intestine.

Because a few enzymes are in most tissues, enabling them to produce lipids necessary for growth and differentiation, the development of their activity in the intestine, an important organ of fat transport and metabolism, is of great interest. In this investigation, the ontogeny and location of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (the key regulatory enzyme in the cholesterol pathway), acyl-CoA:cholesterol acyltransferase (ACAT; responsible for cholesterol esterification), and monoacylglycerol acyltransferase (MGAT; the more representative enzyme of the neutral lipid pathway) were examined in the human fetal small intestine. The developing gut exhibited high levels (pmol.mg-1.min-1) of HMG-CoA reductase (7.65 +/- 0.35), ACAT (16.98 +/- 1.12), and MGAT (689.74 +/- 37.54). Significant positive correlations were recorded between fetal age (8-22 wk) and the enzyme activities of HMG-CoA reductase in the proximal (P < 0.005) and middle (P < 0.01) segments, ACAT in the distal segment (P < 0.03), and MGAT in the proximal segment (P < 0.03) of the gut. Age-specific changes were found in the location of the three enzymes in the contiguous intestinal segments that were investigated. We concluded that the fetal small intestine has substantial HMG-CoA reductase, ACAT, and MGAT activity, which displays specific patterns during development.

Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts.

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted approximately 0.1-2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Right hepatic lobectomy in elderly patients with hepatocellular carcinoma.

BACKGROUND/AIMS: The outcome of hepatectomy in elderly patients with hepatocellular carcinoma have been reported, however neither the morphological nor functional hepatic regeneration in elderly patients have been fully investigated. MATERIALS AND METHODS: Fifty-six patients with hepatocellular carcinoma, who underwent a right hepatic lobectomy over an 8-year period, were classified into three groups according to their age; group 1 (n = 7), more than 70 years of age; group 2 (n = 40), patients from 50 to 69 years of age and group 3 (n = 9), under 50 years of age. There were no significant differences regarding backgrounds or intra-operative parameters among the three groups. The perioperative hepatic function, postoperative complications and the regeneration rate of the remnant left lobe at 1 month after operation were compared. RESULTS: No differences were found in the regeneration rate, however, the levels of the hepaplastin test and lecithin:cholesterol acyltransferase at 7 days after hepatectomy in group 1 (31.3%, 8.8 U) were significantly lower than those in groups 2 and 3 (37.4%, 18.4 U; 47.9%, 29.4 U, respectively). The incidence of hospital death due to hepatic failure in group 1 (42.9%) was also significantly higher than that of group 2 (5.0%) or group 3 (0%). CONCLUSION: The decline of postoperative protein synthesis regardless of the voluminal regeneration is a characteristic of the elderly. This phenomenon might thus be an important promoter of postoperative hepatic failure which remains unpredictable using any type of examination. Therefore, at this time, a major hepatectomy is not recommended as a viable treatment alternative in the elderly.

Effect of estrogen and progesterone on the expression of hepatic and extrahepatic sterol 27-hydroxylase in baboons (Papio sp).

Sterol 27-hydroxylase plays an important role in cholesterol metabolism in hepatic and extrahepatic tissues. To determine whether female sex steroid hormones influence its expression, we measured plasma and hepatic 27-hydroxycholesterol, hepatic mRNA levels, activity of sterol 27-hydroxylase, and adrenal mRNA levels of this enzyme in baboons (n = 6 per group) treated with placebo, estrogen, estrogen + progesterone, and progesterone. We also measured hepatic cholesterol concentration and hepatic acyl coenzyme A:cholesterol acyltransferase (ACAT) activity to determine their relationship with hepatic sterol 27-hydroxylase activity. Plasma 27-hydroxycholesterol concentration was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P = .090) and LDL (P = .026) cholesterol concentrations. Similarly, hepatic sterol 27-hydroxylase activity was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P = .056) and LDL (P = .052) cholesterol concentrations but was positively correlated with hepatic and plasma 27-hydroxycholesterol concentrations (P < .001). Hepatic ACAT activity was increased by progesterone (P < .004) and was positively correlated with plasma (P = .002) and LDL (P = .009) cholesterol concentrations but was negatively correlated with hepatic sterol 27-hydroxylase activity (P = .035). Hepatic and adrenal gland mRNA levels for sterol 27-hydroxylase were increased by estrogen alone or in combination with progesterone (P < .05). Hepatic sterol 27-hydroxylase activity was positively correlated with hepatic mRNA levels (P < .001), an observation suggesting that estrogen increases the activity of sterol 27-hydroxylase by increasing its synthesis. Hepatic cholesterol concentration was not influenced by the hormone treatment. These observations suggest that estrogen alone or in combination with progesterone increases the synthesis of sterol 27-hydroxylase in hepatic and extrahepatic tissues, and the increased activity of hepatic sterol 27-hydroxylase resulting from the increased synthesis is associated with a hypolipidemic effect on plasma LDL levels. Furthermore, progesterone alone increases the hepatic ACAT activity, but given in combination with estrogen progesterone does not have the same effect on hepatic ACAT activity. The effect of estrogen on hepatic ACAT activity may be mediated by sterol 27-hydroxylase and its effect on cholesterol metabolism (decreased cholesterol synthesis and increased output of cholesterol in the bile) in liver.

Regulation and immunolocalization of acyl-coenzyme A: cholesterol acyltransferase in mammalian cells as studied with specific antibodies.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Developmental expression of elements of hepatic cholesterol metabolism in the rat.

The expression of several key enzymes and receptors of rat hepatic cholesterol metabolism was studied during development. Among major findings were: acyl coenzyme A:cholesterol acyltransferase, the cholesteryl ester hydrolases, cholesterol-7 alpha-hydroxylase and the alpha 2-macroglobulin receptor (LRP) were very low in fetal livers, but all were induced shortly before birth, suggesting that these elements are important for extrauterine life. Although the other elements continued to increase, by day 6 of postnatal life, cholesterol-7 alpha-hydroxylase had reached undetectable levels. It reappeared by day 12 of suckling, placing it in the group of late-appearing activities necessary for the fully mature hepatic phenotype. Changes in acyl coenzyme A:cholesterol acyltransferase activity appeared due predominantly to changes in amount of active protein. The cholesteryl ester hydrolase (CEH) activities all showed different developmental patterns, suggesting that each was a unique activity. The bile salt-dependent CEH activity was much higher in the suckling period than in the adult where it was almost undetectable, suggesting that this CEH may have its major importance in the suckling period of development. Low density lipoprotein receptors exhibited a pattern very different from that of the alpha 2-macroglobulin receptors and did not show consistent correlation with any other elements. At some developmental time points, the relationships amongst the elements differed significantly from the adult pattern. These studies provide for the first time an integrated picture of developmental expression of key elements of hepatic cholesterol metabolism and set the stage for further studies on their modes of regulation.