Bioscience Reports, Vol. I2, No. 5, 1992
Effect of H D L 1 Infusion on Biliary Secretion in Perfused Rat Liver Roberto Rivabene, ~ Alfredo Cantafora, ~ Chong Chao Yah,: Flavia Castellano, 2 Giovannella Bruscalupi, 2 and Elena Bravo ~'3 Received August 2, 1992 The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria ph:s lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant. KEY WORDS: perfused rat liver; bile secretion; HDL1; cholesterol.
INTRODUCTION Hepatic cells are the main site of lipoprotein cholesterol uptake and secretion and have the unique ability of catabolizing cholesterol into bile acids. The relationship between hepatic lipoprotein uptake and bile lipid secretion is poorly understood. Cholesterol carried by H D L appears to be preferentially secreted into bile [1, 2]. Rat plasma normally contains low amounts of a lipoprotein rich in ApoE and cholesteryl ester with a density ranging between 1.04 and 1.08 g/ml called HDL1 [3]. Its concentration increases up aging [4] and particularly during cholesterol feeding [5]. It has been hypothesized that this lipoprotein has an important role in transporting "excess" cholesterol from peripheral tissue to liver for elimination from the body via biliary secretion [6, 7]. In a previous study, we observed that administration of small amounts of H D L cholesterol to perfused rat liver induced a transient but significant increase I Istituto Superiore di Sanita', Laboratory of Metabolism and Pathological Biochemistry, Viale Regina Elena 299, 00161 Rome, Italy. 2 Department of Cell and Developmental Biology, University La Sapienza, Rome, Italy. 3 To whom correspondence should be addressed. 425
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in bile flow with a consequent temporary increase in biliary steroid output. A significant increase in both the cholesterol/phospholipid molar ratio in liver microsomes and a decrease of 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGCoA-red) activity were also observed [8]. These effects were in agreement with the role of HDL in reverse cholesterol transport [9]. In the present study, we investigated the effects induced by a cholesterol load, carried by HDL1, on bilary lipid secretion and hepatic membrane subfraction composition in the in vitro model of the perfused rat liver. The aim of this study was to investigate the hypothesis that this lipoprotein fraction may have an important role in removing the "excess" cholesterol from peripheral tissues via bile secretion [6, 7].
MATERIAL AND METHODS Sodium taurocholate was purchased from Calbiochem (La Jolla, CA), [14C]-HMGCoA and [3H]-mevalonate were obtained from Amity PG (Milan, Italy). All the chemicals used were of analytical grade and were purchased from Sigma Chemicals (St. Louis, Mo). Analytical grade solvents (Merck, Darmstad, Germany) were used throughout the study. The enzymatic kits for the determination of cholesterol and triglycerides were purchased from Boehringer Mannheim (Milan, Italy). Male Wistar rats (Charles River, Como, Italy) (250+20 g) housed under standard lighting regime were used.
Lipoprotein Perparation Atherogenic diet with 5% lard, 0.3% sodium taurocholate, 1% cholesterol and 0.1% propylthiouracil were fed ad libitum to 15 rats. After 3 weeks, their blood was collected in EDTA (4mg/ml) and cholesterol-rich HDL1 were isolated by sequential ultracentrifugation ( d = 1.04 to 1.08g/ml) [5]. The lipoprotein fraction was then dialyzed, concentrated and analyzed, as previously described [10].
Liver Perfusion Rats were anaesthetised with an intraperitoneal injection of Farmotal (100 mg/kg b.w.). The livers (11.6 + 1.6 g) were isolated by a standard surgical technique [11] and the bilary duct was cannulated with a PE50 catheter (Clay Adams, Parsippany, N.Y.). Liver perfusion was performed as previously reported [8]. Perfusion medium contained 1/3 freshly prepared bovine erythrocytes and 2/3 Krebs-Ringer bicarbonate buffer (pH 7.4) together with 4% BSA, 0.1% glucose and 40 #M sodium taurocholate [8].
Experimental Protocol Equilibration of liver with sodium taurocholate was obtained with the perfusion of 50 ml of medium for 30 min during which the basal bile sample was
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collected. The medium was changed and HDL1 was infused as a single bolus and recirculated for 10 min. After a new change of the medium, the perfusion was continued for different intervals of time following the lipoprotein infusion: 15, 30 and 120 minutes. In total, five groups of perfusions were performed. Three perfusions were stopped just before lipoprotein infusion (Group 1): only one sample of bile was obtained from these experiments. In the second group (Group 2), the perfusions were stopped immediately after tipoprotein infusion: two samples of bile were collected in these experiments. The third, fourth and fifth groups (Groups 3, 4, and 5 respectively), were stopped 15, 30 and 120 minutes after the HDL1 administration, respectively. Three bile samples were collected during these last three groups of experiments. For each group, three rat liver perfusions were conducted. Liver Membrane Isolation HMGCoA-reductase activities were determined in microsomes prepared from 1 gram of liver according to Erickson et al. [12]. The remaining liver was homogenized in four volumes (w/v) of NaHCO3 1 mM and centrifugated at 600 x g. Liver Plasma Membranes (LPM) were isolated from the nuclear pellet of liver homogenate according to Boyer et al. [13]. Nuclear supernatant was diluted with buffer in order to obtain a final concentration of 250 mM sucrose, 5 mM MgC12 and 1 mM NaHCO3 (pH 7.5). The mitochondrial/lysosomal and microsoreal fractions were pelletted with two consecutive centrifugations of nuclear supernatant at 10,000 x g for 20 rain and 100,000 x g for 60 min, using an L-70 centrifuge equipped with a 60 Ti rotor (Beckman Instrument, Fullterton, Ca). The enrichment of the isolated subfractions was ascertained by the following marker activities: 5-nucleotidase [14], succinate deydrogenase [15], glucose-6phosphatase [16] and aryl sulphatase-A [17] for plasma membranes, microsomes and mitochondria/lysosomes, respectively. Analytical Procedures The assay of HMG-CoA reductase activity, based on the formation of labelled mevalonic acid from [3-14C] H M G CoA, was conducted as described by Erickson et al. [12] with minor modifications introduced in our laboratory [18]. The protein content in lipoprotein and microsome preparations was determined by the method of Lowry [19] using bovine serum albumin as standard. Free and total cholesterol and triglycerides were determined in lipoprotein fractions by the use of commercial enzymatic kits. Free and total cholesterol were assayed in lipid extracts by a modification of the enzymatic method previously described [20]. Phospholipids were measured by a lipid phosphorus assay according to Bartlett [21]. The per cent distribution of apoprotein was determined by sodium dodecyl sulfate-poyacrylamide gel electrophoresis as previously described [22]. Bile salts were determined by HPLC after addition of an internal standard as described by Cantafora et al. [8, 23].
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RESULTS The HDL1 fractions used in these experiments contained 4.33 5: 4.3% of cholesterol and up to 49.8 • 7.9% of its apoprotein content was apoE. The percentages of apo AI, AIV and apo C were 16 • 5%, 7 • 3% and 28 • 5%, respectively. Albumin was present only in trace amounts. In each perfusion 10/~moles of total cholesterol were infused of which about 70% was esterified. Lipoprotein uptake was evaluated by triple measurements of triglyceride and free and total cholesterol on medium perfused to livers during the HDL1 infusion. Liver uptake ranged from 29 • 6 to 42 • 5% for triglyceride and free cholesterol, respectively, and 35 + 7% for total cholesterol. In average, 2.5 to 3.9/~moles of HDL1 total cholesterol were taken up by perfused liver during the 10 minutes of lipoprotein administration. This quantity roughly corresponded to 3-6% of total cholesterol present in a rat liver. However, total cholesterol liver content did not significantly change from basal values of 68.4 + 10.3 ~mol after the lipoprotein administration in any perfusion group studied. Enzyme "marker" profile in isolated subcellular compartments is reported in Table 1. Acceptable enrichment of subfractions, in agreement with similar methods of isolation [24], for mitochondria/lysosomes, plasma membranes and microsomes was achieved. Values of glucose 6-phosphatase activity found in microsomes prepared according to Erickson [12] are not reported, but similar
Table 1. Enzyme marker profile in mitochondria + lysosomes and liver plasma membranes (LPM) subfractions isolated from during (Group 2), and 15, 30 a n d 120 minutes after (Group 3, H D L 1 fraction. Data reported are mean + standard deviation of group Perf
Group
Membrane fraction
5' nucleotidase Enr 1
%Rec z
Glucose-6 phosphatase Enr t %Rec 2
(MIT + LYS), microsomes (MICR)
rat perfused liver before (Group 1), 4 a n d 5 respectively) the infusion of three experiments for each perfusion Succinate dehydrogenase
EnP
%Rec 2
Arylsulphatase Enr 1
%Rec 2
1
MIT+LYS MICR LPM
1.6+0.1 2.15:0.6 25.55:8.2
16.3+2.3 6.1+1.1 7.15:1.9
1.35:0.3 3.05:1.6 2.65:0.3 19.55:2.1 1.05:0.5 0 . 1 5 : 0 . 0
2.35:0.2 44.15:9.9 0.45:0.0 1.85:0.8 0 . 1 5 : 0 . 0 0.15:0.0
1.45:0,5 0.85:0.4 0.05:0.0
22.05:11.9 4.05:1.9 0.05:0.0
2
MIT+LYS MICR LPM
1,55:0.1 2.35:0.6 10.05:5.7
10.1• 4.85:1,4 6.95:l.3
1.0+0.4 3.1+1.1 2 . 5 5 : 0 . 6 21.05:8.9 0.75:0.1 0.25:0.1
2.05:0.2 48.25:15.5 1.95:0.5 0.45:0.1 1.75:0.1 0 . 5 5 : 0 . 2 0.25:0.1 0.05:0.0 0.15:0.0
23.05:10.9 1.15:0.5 0.05:0,0
3
MIT+LYS MICR LPM
1.45:0.4 2.55:0.4 15.95:5.1
18.15:6.8 10.15:0.9 9.25:0.9
1.15_0.2 2 . 7 + 1 . 1 2 . 9 5 : 0 . 4 22.15:7.9 0.55:0.1 0 . 2 5 : 0 . 0
1.85:0,3 41.55:7.7 0.45:0.1 2.35:0.7 0 . 1 5 : 0 . 0 0.05:0.0
1.55:0.8 0.45:0.2 0.15:0.0
40.55:18.1 2.35:1.1 0.05:0.0
4
MIT+LYS MICR LPM
1.65:0.2 2.45:0.3 ll.4+:1.4
18.35:2.5 5.3:1:0.3 5.9+0.5
1.25:0.3 3 . 3 5 : 1 . 4 3.55:0.8 23.05:5.8 0.45:0.2 0.05:0.0
2.15:0.5 49.25:10.1 1.25:0.1 0.35:0,1 1.75:0.6 0 . 4 5 : 0 . 2 0 . 1 5 : 0 . 0 0.05:0.0 0 . 1 5 : 0 . 0
26.85:6.5 2.25:1.0 0.05:0.0
5
MIT+LYS MICR LPM
1.85:0.2 2.65:0.9 18.95:3.1
16.45:8.5 5,95:1.7 10.15:2.0
1.15:0.5 3.15:1,4 1.95:0.4 46.15:7.5 2 . 7 5 : 0 . 8 30.05:8,9 0,45:0.1 1.65:0.3 0.95:0.4 0.35:0.2 0.05:0.0 0 . 0 5 : 0 . 0
1.45:0.4 0.35:0.1 0.15:0.0
32.45:12.0 1.75:0.6 0.05:0.0
1 E = Enrichment of the enzymatic activity in the membrane fraction (ratio between the activity in the
fraction and the homogenate both expressed as nmol/min/mg protein) 2 % R E C = % recovery of the enzymatic activity (ratio between the activity in the fraction and the
homogenate both expressed as nmol/min/g liver).
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Table 2. Cholesterol to phospholipid molar ratio in hepatic membrane subfractions isolated from perfused liver before (Group 1), during (Group 2) and 15, 30, and 120 minutes after (Group 3, 4, and 5 respectively) HDL1 infusion. Data reported are mean 5: standard deviation of three experiments for each perfusion group CH/PL molar ratio Perfusion grot~p
Whole liver
Mitochondria • lysomes
I
0.163 + 0.011
0.123 5:0.010
0.169 5:0.038 *0.129 5:0.025
0.297 5:0.093
2
0.167 5:0.030
0.104 5:0.004
0.209 • 0,079 "0.156 • 0.025
0.236 5:0.009
3
0.158 5:0.021
0.106 5:0.017
0.189 5:0.014 "0.139 5:0.009
0.258 5:0.077
4
0.159 5:0.024
0.110 5:0.018
0.21 ~ 5:0.035 *0.153 5:0.008
0.307 5:0.107
5
0.163 5:0.031
0.128 5:0.019
0.209 5:0.017 "0.152 5:0:025
0.381 5:0.039
Microsomes
Plasma membranes
* Microsomes prepared according to Erickson [12].
degrees of enrichment and recovery were also obtained in this different microsomal preparation. Cholesterol/phospholipid molar ratio values in isolated hepatic subfractions are reported in Table 2. For microsomes, values obtained for both preparations of microsomes are reported. Absolute values of this ratio are different, but the relative behavior is similar in the five groups of perfusions performed. As a consequence of HDL1 administration, cholesterol to phospholipid molar ratio did not significantly change either in whole liver or in the cellular subfractions studied. Only an insignificant increase of about 30% in Cholesterol to phospholipid molar ratio, with respect to the basal value, was found in microsomal fractions after HDL1 administration (Table 2, Group 2 to 5). HMGCoA-red activity decreased after 15 rain of infusion of HDL1 (Group 3): its activity was 74% of the basal value (0.379 + 0.085 and 0.282 5:0.079 nmol/ min/mg protein, respectively) and remained constant after 30 and 120 minutes of HDL1 infusion (0.273 :t:0.090 and 0.2915: 0.074nmol/min/mg protein, respectively). Data relative to biliary lipid secretion (concentration, output and molar ratio) in the five groups of liver perfusions are shown in Table 3. In general, the HDL1 load to perfused rat liver increased both the concentration and the output of biliary lipid, but only for a short interval of time after lipoprotein infusion. In fact, in Group 5 (120 min post-infusion) all biliary parameters studied were significantly decreased (e.g., bile flow, bile salt concentration and biliary lipid output) or similar to basal values (Table 3). Cholesterol and bile acid secretion was significantly increased during lipoprotein infusion (Group 2) because of the rise in bile flow that is 0.51+0.13/B/min/g liver in the basal sample and 0.88 + 0.29 in bile collected during the infusion of HDL1 to perfused rat liver. No significant change in bile salts and phospholipid concentration was found during these experiments (Table 3) while the concentration of biliary cholesterol significantly increased (p