HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995 A A S L D A B S T R A C T S 239A

529 ROLE OF APOLIPOPROTE1N E (ApoE) IN INTRACELLULAR LIPID METABOLISM AND VERY LOW DENSITY LIPOPROTEIN (VLDL) SECRETION BY MOUSE HEPATOCYTES. F Kuipers, R Havinga, MH Hotker**, RJ Vonk, HMG Princcn*, LM Havekes*. Grnningen Institute for Drug Studies, Laboratory of Nutrition & Metabolism, Groningen, *TNO-PG, Gaubius Laboratory, Leiden, **MGC-Department of Human Genetics, University of Leiden, Leiden, the Netherlands.

ApoE, a 34 kD apolipoprotein, is an important constituent of a number of plasma lipoproteins, including VLDL, chylomicrons and their remnants. ApoE is essential for recognition and uptake of these triglyeeride (TG)-rieh lipoproteins by specific receptor systems. ApoE is synthesized primarily, but not exclusively, in hepatocytes. Mice in which the apoE gene is inactivated by homologous recombination develop severe hypercholesterolemia when fed normal lab chow, whereas TG levels are only slightly increased [van Ree et eL, Atherosclerosis 111: 25, 1994]. In the present study, we have investigated hepatic lipid metabolism and secretion in homozygous apoE-deficient mice (apoE -/-), both in rive and in vitro. In vitro experiments were performed with 24-h cultured hepatocytes. Hepatic free cholesterol (+56%) and TG (+232%) contents were significantly increased in chow-fed apoE (-/-) mice when compared to controls (+/+), whereas those of cholesterylester and of phospholipids were similar in both groups. Lipid accumulated predominantly in periportal areas of apoE (-/-) livers. Hepatic HMG-CoA reductase mRNA levels were lower in apoE (-/-) than in (+/+) mice and enzyme activity was reduced by 50%. ACAT activity was unaffected. The changes in cholesterol metabolism did not alter removal of the sterol via the hepatobiliary pathway; both biliary cholesterol secretion and bile acid synthesis were unaffected in the apoE (-/-) mice. In contrast, the secretion of VLDL-associated 3H-TG, synthesized from 3H-glycerol, was reduced by 50-60% in cultured apoE (-/-) hepatocytes during 3-h incubations, both under control conditions and in the presence of 1 mM oleic acid to stimulate TG synthesis and secretion. Cellular content of newly synthesized 3H-TG, on the other hand, was approximately doubled in apoE (-/-)hepatocytes under these conditions. In cundusion~ apoE deficiency strongly affects hepatic cholesterol and TG metabolism, both in viva and in vitro. Modulation of intraccUular TG metabolism by apoE may play a role in the regulation of VLDL secretion by liver cells.

530 POLYSPECIFIC STEROID AND DRUG TRANSPORT BY AN ORGANIC ANION TRANSPORTER OF HUMAN LIVER. X Bossuvt*, A. Schroeder*, M Moiler +, B Ha~enbuch*, and PJ Meier_~*. *Division of Clinical Pharmacology and Toxicology, Departement of Medicine, University Hospital, CH-8091 Zurich/Switzerland. +Department of Gastroenterology and Hepatology, University Hospital, Groningen, The Netherlands

The first step in overall hepatic drug clearance is generally thought to be mediated by class-specific and charge-selective hepatocellular uptake systems for amphipathic organic anions, neutral compounds and organic cations. However, we have recently shown that a single sinusoidal organic anion transporting polypeptide (oatp) can mediate charge-independent amphipathic substrate transport in rat liver (J. Hepatology 1995, in press). In this study we investigated the substrate specificity of the corresponding human organic anion transporting polypeptide (OATP) that has been previously shown to mediate sodium-independent uptake of sulfobromophthalein and bile acids (Gastroenterology 1995, in press). When expressed in Xenopus laevis oocytes, OATP also mediated uptake of the neutral compounds ouabain (Km ~

5.5 mM) and leukotriene B4, the anionic substrate estrone-3-sulfate (Kin~ 59p.M) and the pemmnently charged derivative of the cationic drug N- propylajmaline (N-(4,4-azo-n-pentyl)-2t deoxyajmalinium). For all these compounds OATP-mediated transport could be inhibited by conjugated bile salts and was dependent on the amounts of eRNA injected. This charge- independent substrate specificity of OATP was further supported in stably transfected CHO-cells. No OATP-mediated transport was found for p- aminohippurate, aspartate, tetraethylammonium and dinitrophenylglutathione. Conclusions: These data demonstrate that, similar to the rat oatp, the human OATP can also account for multispecific organic anion, neutral steroid and organic cation transport. Hence, a single sinusoidal (or basolateral) transporting polypeptide can account, at least in part, for charge-independent steroid and drug clearance in mammalian liver. Since OATP is also expressed in brain, lung, kidney and testis, OATP-mediated drug (and drug conjugate) transport may be an important transport function in extrahepatic ceils as well.

531 PROTEIN KINASE C ACTIVATION/CELLULAR REDISTRIBUTION AND CHOLESTEROL 7=-HYDROXYLASE mRNA REPRESSION BY BILE ACIDS IN RAT HEPATOCYTES. RT Stravitz. Y Rao. ZR Vlahcevic. EC Gurley, and PB Hylcm0n. M~Guim VAMC and Medical College of Virginla/VCU, Richmond, VA.

We have recently shown that taurocholate (TCA) represses cholesterol 7a- hydroxylase (C7¢d-I) transcriptional activity through a protein kinase C (PKC)- dependent mechanism in primary cultures of rat hepatocytes. Objectives. To determine 1) whether bile acids directly activate PKC m vitro and 2) the consequences of PKC activation on hepatocdlular isoform distribution and C7aH mRNA levels. Methods. Soluble PKC was partially purified fi-om rat liver by DEAF-~sepharose chromatography, and specific activity determined using histone I]I-S as substrate. Isolated hepatocytes were cultured for 24-30 h. PKC isozymes were quantified by Western immunoblotting and laser densitometry of ECL exposures, and C7ttH mRNA by RNase protection assay. Results. TCA or tauredeoxyeholate (1 O-1 O0 liM) increased in vitro PKC activity by 3-fuld and 4- fold, with EC~0's of ~35 ~M and 27 gM, respectively. Tanroursodeoxycholate, TCA, taaroehanodeoxycholate, and tanrodnoxycholate (50 ItM) stimulated PKC activity in proportion to their hydrophobieity index (r=0.99). A 15 rain exposure of cultured hepatocytas to TCA (12.5-100 ~M) increased membrane-associated PKCa,5, and e by 1.5-3-fold (EC~0 ~35 laM); PKC[~ a was not affected. Membrane-associated PKC progressively increased, and cytosolic mass decreased, for I h alter the addition of TCA (50 ~M); after 24 h, whole-cell mass of PKCa,5, and e, but not [~n, was down-regulated to 30-50% of untreated cultures. Finally, C7ttH mRNA was repressed 71% by phocbol 12-myristam, 13*acetate (100 nM for 3 h), an activator of PKC~,5, and e. In contrast, the phorhol ester thymeleatoxin (100 nM for 3 h), which increased membrane-associated PKCct >10-fold but had no effect on PKC5 nor ~, decreased C7uH mRNA by only 6%. Conclusions. Bile acids directly activate hepatoceUular PKC, resulting in redistribution and down- regulation of calcinm-depondent (it) and -independent (5,¢) isoforms. The calcium-independent isoforms may be the most important mediators of the repression of C7~H mRNA by bile acids.

532 I N C R E A S I N G H E P A T I C C H O L E S T E R O L 7a - HYDROXYLASE REDUCED PLASMA CHOLESTEROL CONCENTRATIONS IN RABBITS. G XU. G Salen. S Shefer, G Ness. L Nouven. A K Batta. T Chen. GS Tint. VA Medical Center, East Orange, NJ 07018 and South Florida Univ. We investigated the effect of bile acid depletion and replace- ment with glycodeoxyeholic acid (GDCA) on plasma choles- terol concentrations, hepatic LDL receptor-mediated binding, mRNA and protein levels, and hepatic activities and mRNA levels for HMG-CoA reductase and cholesterol 7a-hydroxylase in 19 New Zealand White (NZW) and 15 Watanabe heritable hyperlipidemic (WHHL) rabbits. Bile acid depletion was achieved by bile fistula and replacement, by infusing GDCA. Plasma cholesterol concentrations were 13 times greater, he- patic LDL receptor-mediated binding 26% lower, and choles- terol 7~,-hydroxylase activity and mRNA levels were 62% and 66% less in WHHL than NZW rabbits. After bile drainage, plasma cholesterol concentrations declined 29% (p<0.05) in NZW and 40% (p<0.005) in WHHL rabbits with a 2.1 fold rise in hepatic LDL receptor-mediated binding in the NZW but no change in WHHL rabbits. Cholesterol 7a-hydroxylase activity and mRNA levels rose 3 -4 times in both NZW and WHHL rab- bits. Replacement with GDCA raised plasma cholesterol con- centrations 1.7 times, decreased enhanced cholesterol 7~- hydroxylase activity 54%, mRNA levels 86%, cholic acid syn- thesis 38%, and hepatic LDL receptor-mediated binding 57% in NZW rabbits. Bile acid depletion stimulated cholic acid syn- thesis by upregulating cholesterol 7a-hydroxylase to utilize cholesterol and reduce plasma concentrations substantially in both NZW and WHHL rabbits, although LDL receptors func- tioned only in NZW rabbits. GDCA replacement inhibited elevated cholesterol 7,,-hydroxylase, cholic acid synthesis and hepatic LDL receptor binding to reestablish baseline plasma cholesterol levels in NZW rabbits. Hypercholesterolemia in WHHL rabbits was related to the combination of dysfunctional LDL receptors and inhibited cholesterol 7a-hydroxylase.