INTRODUCTION

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THE LIVER PLAYS A CENTRAL role in both the maintenance of whole body cholesterol homeostasis and the regulation of plasma lipoprotein concentrations. Whereas de novo cholesterol synthesis and uptake of dietary cholesterol (as lipoproteins) represent the two input pathways, conversion of hepatic cholesterol to bile acids and biliary secretion of cholesterol are the only significant output pathways. In response to changes in cholesterol influx or efflux, sterol balance across the hepatocyte is maintained by altering the flux of cholesterol through 1) endogenous cholesterol synthesis, 2) lipoprotein uptake, synthesis, and secretion, 3) conversion of cholesterol to bile acids, and 4) reversible conversion of excess cholesterol to cholesteryl esters. Whereas hepatic free cholesterol and cholesteryl esters are maintained in dynamic equilibrium, a regulated flux of cholesterol through these hepatic pools not only maintains free cholesterol levels within the hepatocyte but also influences the secretion of cholesteryl esters as components of plasma lipoproteins. Neutral cholesteryl ester hydrolase (CEH) is the key enzyme required for releasing the pool of metabolically active free cholesterol from intracellular stores of cholesterol esters, providing substrate for bile acid synthesis and for biliary secretion of cholesterol. A second free cholesterol pool is derived from the de novo synthesis of cholesterol, which is regulated at the level of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR). It has been suggested that newly synthesized cholesterol is the preferred substrate for cholesterol 7-hydroxylase (C7H), whereas free cholesterol released by the hydrolysis of cholesteryl esters is the proximal source of biliary cholesterol (31, 24). Treatments that affect cholesterol flux through the liver (e.g., cholesterol, bile acid, or cholestyramine feeding or lovastatin infusion) lead to compensatory changes in the expression of C7H and HMGCoAR (16, 27, 35). Although C7H appears to be regulated largely by changes in the mRNA levels and the contribution of posttranscriptional mechanisms is minimal (30), regulation of HMGCoAR is known to occur at multiple levels (15). However, few data are available concerning the regulation of CEH expression by changes in the cholesterol flux. Indeed, cholesterol-enriched diets decreased CEH activity in rats (16), and a consistent increase in activity is observed in response to cholestyramine feeding (13). CEH activity is also shown to be sensitive to hormonal stimuli in adrenal cortex (33), testis (10), corpus luteum (2), and aorta (17). Although hormonal regulation of hepatic enzymes involved in glycogen and glucose metabolism is widely studied (4), limited information is available on the modulation of hepatic CEH by hormones. Gandarias et al. (11) correlated increases in hepatic cholesterol content in response to estradiol treatment with decreases in hepatic neutral CEH activity after a single injection of estradiol. Thyroid hormone effectively lowers serum cholesterol levels (34), and Day et al. (9) correlated this reduction in plasma cholesterol with enhanced biliary secretion of cholesterol. These studies suggest a shift in the balance between free cholesterol and cholesterol esters by thyroxine, probably mediated by CEH. However, a direct effect(s) of L-thyroxine on hepatic CEH has not been reported.

Because there is little published information on the regulation of hepatic CEH, we have examined the regulation of CEH expression (activity, protein mass, and mRNA levels) in rats in response to cholesterol feeding and drugs or metabolites that perturb cholesterol biosynthesis. Hepatic cholesterol flux was also altered by perturbing bile acid metabolism with chronic biliary diversion or bile acid feeding. To confirm and simplify interpretation of results from in vivo studies, we have also examined the direct effects of hormones and signal transduction pathways on CEH mRNA levels in primary rat hepatocyte cultures. Our results indicate complex regulation of CEH at multiple levels.

	EXPERIMENTAL PROCEDURES

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Materials. Cholesterol, cholesteryl oleate, chenodeoxycholic acid, taurocholic acid, tauroursodeoxycholic acid, mevalonolactone, L-thyroxine, dexamethasone, dextran sulfate, phorbol 12-myristate 13-acetate (PMA), 4-PMA, and N-lauroylsarcosine were purchased from Sigma Chemical (St. Louis, MO). Lovastatin was a generous gift from Merck Sharp & Dohme (Rahway, NJ). Guanidine isothiocyanate and cesium chloride were purchased from Fisher Scientific (Springfield, NJ). Williams' medium E and the nick-translation kit were obtained from Gibco-BRL (Gaithersburg, MD). GeneScreen membrane and radioisotopes ([-³²P]dCTP and cholesteryl-[1-¹⁴C]oleate) were purchased from NEN (Boston, MA). All other reagents were of the highest quality available commercially.

Experimental design. Male Sprague-Dawley rats (Charles River, Cambridge, MA) weighing 250-350 g were housed under controlled lighting conditions on a 12:12-h light-dark cycle (0600-1800 light phase). Groups of age- and weight-matched animals were used for all studies. The treatments were divided into two categories. 1) To change intracellular cholesterol levels, rats were either fed 2% cholesterol mixed with powdered lab chow or infused with mevalonate (180 µmol/h for 48 h) or lovastatin (2 mg · kg¹ · h¹ for 24 h) intravenously. 2) To increase cholesterol efflux and perturb bile acid metabolism, rats either had biliary diversion as described below or were fed 1% chenodeoxycholic acid mixed with powdered chow. Animals were pair fed with modified diets as described above for 14 days. Rats were killed by decapitation, and blood was collected for measurement of serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels as indicators of normal liver function. Livers were harvested, and two 1-g pieces were removed. One piece was used to prepare cytosol (12) and the other to isolate RNA.

Chronic biliary diverted rat model. Under brief methoxyflurane anesthesia, biliary fistula and intraduodenal cannulas were placed as described previously (19). After surgery, rats were placed in individual metabolic cages with free access to water and Purina laboratory chow. All animals received continuous intraduodenal infusion of glucose-electrolyte replacement solution. Dietary intake, activity, and bile flow of the rats were carefully monitored. After 72 h of chronic biliary diversion, rats were killed by decapitation and blood was collected to assess the normal liver function as described above. Livers were harvested and processed as described above for the preparation of RNA and cytosol.

Isolation and culture of primary rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats (250- 300 g) using the collagenase perfusion technique of Bissell and Guzelian (3). Before plating, cells were judged to be >90% viable using trypan blue exclusion. Parenchymal cells (2.5 × 10⁷) were plated onto 150-mm petri dishes previously coated with rat tail collagen. Cells were incubated in Williams' medium E (20 ml/plate) supplemented with insulin (0.25 U/ml) and penicillin (100 U/ml) at 37°C in a 5% CO₂ atmosphere. The medium was changed every 24 h and supplemented with various hormones where indicated. Cells were routinely harvested after 48-72 h.

Measurement of CEH activity and protein. CEH activity was measured in the cytosol by the radiometric assay described previously (12). The specific activity is expressed as nanomoles of oleate released per hour per milligram of protein. Relative CEH protein mass was determined by densitometric analysis of the Western blots (23). Protein was estimated using the Pierce bicinchoninic acid protein assay kit (Rockford, IL).

Preparation of total RNA. A one-gram piece of liver was homogenized using a Dounce homogenizer in a solution of 4 M guanidine isothiocyanate, 10 mM tris(hydroxymethyl)aminomethane · HCl, pH 7.4, and 7% 2-mercaptoethanol. N-lauroylsarcosine was added to a final concentration of 2%, and the homogenate was passed through a 23-gauge needle. This suspension was passed through a 23-gauge needle and underlaid with 5.7 M cesium chloride containing 10 mM EDTA (pH 7.4). The total RNA pellet obtained after centrifugation at 100,000 g for 16 h was washed twice with 100% ethanol, dissolved in diethyl pyrocarbonate (DEPC)-treated water, and precipitated with 2 vol of 100% ethanol in the presence of 0.10 vol of 3 M sodium acetate, pH 5.0, and stored at 20°C for overnight precipitation (5). Total RNA was pelleted by centrifugation at 12,000 g for 30 min, redissolved in DEPC-treated water, and quantified by measuring the absorbance at 260 nm. For RNA preparation from cultured hepatocytes after incubation, the media were aspirated and cells washed with 5 ml of phosphate-buffered saline (PBS). After the last traces of PBS were removed, the cells were harvested in 7.5 ml of guanidine isothiocyanate solution, and N-lauroylsarcosine was added to a final concentration of 2%. The mixture was vortexed to lyse the cells completely, passed through a 23-gauge needle, and processed as described above.

Determination of CEH mRNA levels. The methods for determination of CEH mRNA levels have been described previously (23). In brief, 10 µg of total RNA were electrophoresed on a 1% agarose gel in the presence of formaldehyde, transferred to GeneScreen membrane, and hybridized with ³²P-labeled full-length cDNA probe for hepatic CEH according to the manufacturer's instructions. The blots were washed under high stringencies (0.2× SSC plus 0.1% SDS at 65°C; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Positive hybridization was detected by exposure to Kodak XAR-2 films for 18 h at 70°C (14). Radioactivity associated with each band was quantified using the personal densitometer from Molecular Dynamics. Rat cyclophilin was used as the internal control (8).

Statistical analyses. Data were analyzed by Student's t-test. P < 0.05 was considered significant.

	RESULTS

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Effects of treatments known to change intracellular cholesterol levels. Cellular cholesterol metabolism was perturbed by cholesterol feeding and by intravenous infusion of mevalonate or lovastatin. As shown in Fig. 1, cholesterol feeding significantly decreased CEH activity (56 ± 2%, P < 0.001), protein mass (44 ± 2%, P < 0.001), and mRNA levels (14 ± 3%, P < 0.005) compared with the pair-fed controls. Intravenous infusion of mevalonate, which increases cholesterol biosynthesis, led to a compensatory decrease in CEH activity (42 ± 6%, P < 0.005) and protein mass (74 ± 3%, P < 0.001). The mRNA levels also showed a similar decreasing trend (22 ± 16%); however, unlike with cholesterol feeding, the changes did not reach statistical significance. Infusion of the HMGCoAR inhibitor lovastatin, on the other hand, significantly increased CEH activity (65 ± 12%, P < 0.001). Although CEH mRNA levels also showed an increasing trend after lovastatin infusion, this difference did not reach statistical significance (31 ± 24%) and no significant change was seen in CEH protein mass (Fig. 1).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Effect of treatments known to change intracellular cholesterol on cholesteryl ester hydrolase (CEH) activity, mRNA levels, and protein mass. CEH activity, protein mass, and mRNA levels were measured in rats receiving the following treatments: 2% cholesterol feeding (2% XOL; n = 5), intravenous infusion of mevalonate (iv MEV; n = 3), and intravenous infusion of lovastatin (iv LOV; n = 6). Data are expressed as %control. Pair-fed and sham-operated rats were used as controls for feeding and infusion studies, respectively. * Significant (P < 0.05) change.

Effects of increased cholesterol efflux and bile acid feeding. Chronic biliary diversion, which drains hepatic cholesterol through both biliary cholesterol and bile acid synthesis, increased CEH activity (138 ± 34%, P < 0.025) and mRNA (146 ± 28%, P < 0.05) relative to sham-operated controls (Fig. 2). The CEH protein mass also increased (29 ± 7%, P < 0.025; data not shown). In contrast, chenodeoxycholate (fed as 1% of diet) decreased CEH activity by 46 ± 6% (P < 0.005). Although CEH mRNA levels were also decreased by chenodeoxycholate (26 ± 12%), this trend did not reach statistical significance. Cholic acid (1% of diet) and deoxycholic acid (fed at 0.25% to avoid toxicity) did not significantly change CEH activity and mRNA levels (data not shown).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Effect of increased cholesterol efflux and bile acid feeding on CEH activity and mRNA levels. CEH activity and mRNA levels were measured in rats either fed 1% chenodeoxycholic acid (1% CDCA; n = 5) or with chronic biliary diversion (CBD; n = 5 and 2, for activity and mRNA, respectively). Data are expressed as %control. Pair-fed or sham-operated rats were used as controls. * Significant (P < 0.05) change.

Effects of L-thyroxine and dexamethasone on CEH mRNA levels in primary hepatocytes. Steady-state CEH mRNA levels were initially measured as a function of culture age in a chemically defined medium without added serum. In the absence of dexamethasone and L-thyroxine, a large decrease (98%) in CEH mRNA was observed compared with the levels in freshly isolated hepatocytes (Fig. 3). Addition of both hormones stabilized mRNA levels near those of fresh hepatocytes (Fig. 3). In the presence of 0.1 µM dexamethasone, L-thyroxine yielded a maximum response at concentrations between 1 and 10 µM, whereas 100 µM L-thyroxine reduced mRNA levels to 29% of controls (Fig. 4). In the presence of 1 µM L-thyroxine, a maximum response was seen with 0.1 µM dexamethasone (Fig. 5). Although the specific activity of CEH in freshly isolated hepatocytes is only 50% of that in liver cytosol, activity of CEH could not be measured in cultured hepatocytes, even in the presence of L-thyroxine and dexamethasone (data not shown).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Effect of culture age on steady-state CEH mRNA levels. Primary hepatocytes were plated in the presence or absence of 0.1 µM dexamethasone (Dex) and 1 µM L-thyroxine (T4). Total RNA was isolated from cells at indicated times, and CEH mRNA levels were determined by densitometric analysis of Northern blot as described in EXPERIMENTAL PROCEDURES. A: values are means ± SE; n = 3. Control, mRNA level of corresponding freshly isolated hepatocytes. B: a representative blot probed for CEH and the housekeeping gene cyclophilin (CYC).

View larger version (K): [in this window] [in a new window]   Fig. 4.   Effect of T4 concentration on CEH mRNA levels. Culture medium contained 0.1 µM dexamethasone with indicated T4 concentrations added at time 0. Total RNA was isolated 72 h after plating, and CEH mRNA levels were determined by densitometric analysis of Northern blots. A: values are means ± SE; n = 5. Control, mRNA in hepatocytes with dexamethasone alone. B: representative blot probed for CEH and CYC. Lane 1, dexamethasone alone; lanes 2-7, +0.001-100 µM T4.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Effect of dexamethasone concentration on CEH mRNA levels. Culture medium contained 1 µM T4 with indicated dexamethasone concentrations added at time 0. Total RNA was isolated 72 h after plating, and CEH mRNA levels were determined by densitometric analysis of Northern blots. A: values are means ± SE; n = 6. Control, mRNA in hepatocytes cultured with T4 alone. B: representative blot probed for CEH and CYC. Lane 1, no additions; lane 2, T4 alone; lanes 3-7, +0.001-10 µM dexamethasone.

Effects of modulators of signal transduction pathways on CEH mRNA levels in hepatocytes. The effects of dibutyryl-cAMP (DBcAMP), glucagon, and PMA on CEH mRNA levels were determined under optimal culture conditions (in the presence of insulin, 0.1 µM dexamethasone, and 1 µM L-thyroxine). As shown in Table 1, although addition of glucagon or DBcAMP had no effect on CEH mRNA levels, PMA decreased CEH mRNA levels by 49 ± 13% (P < 0.01). The response to PMA was time dependent with a maximum at 6 h (data not shown). The effects of PMA were specific to 4-PMA, because incubation with the 4-analog of PMA had no significant effect on CEH mRNA (91% of control).

Effects of agents that perturb cholesterol metabolism on CEH mRNA levels in hepatocytes. Hepatic free cholesterol levels are coordinately regulated by HMGCoAR, C7H, acyl-CoA:cholesterol acyltransferase, and CEH. To evaluate the molecular mechanisms underlying the compensatory role of CEH in cholesterol homeostasis in vivo, hepatocytes were treated with lovastatin, a competitive inhibitor of HMGCoAR, or taurocholate, a repressor of C7H and the primary bile acid in rat, before measurement of CEH mRNA levels. Lovastatin increased CEH mRNA levels by 103 ± 15% (P < 0.001). Suppression of CEH mRNA levels by taurocholate was both time (Fig. 6A) and concentration dependent (Fig. 6B). The hydrophilic bile acid tauroursodeoxycholate (50 µM), which does not suppress bile acid synthesis (27), had no effect on CEH mRNA levels (105% of controls with no additions).

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