Regulation of apolipoprotein B secretion in hepatocytes from Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia

ATHEROSCLEROSIS

ELSEVIER

Atherosclerosis 114 (1995) 73382

Regulation of apolipoprotein B secretion in hepatocytes from Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia Makoto Tanaka*, Hideo Otani, Masayuki Yokode, Toru Kita Departmenf

of Geriatric

Medicine,

Faculty

of Medicine,

Kyoto

University.

54 Shogoin-Kawahara-cho,

Sakyo-ku.

Kyoto

606, Japan

Received25 July 1994;revision received 31 October 1994;accepted2 November 1994

--Abstract

Regulation of apolipoprotein B-100 (apo B) secretionin the liver in familial hypercholesterolemia(FH) remains largely unknown. In a previous study, we developeda rabbit hepatocyte culture systemand investigateda cellular mechanismwhich regulatesapo B secretionfrom hepatocytesin responseto cellular cholesteryl estercontents [18]. Using this system,in the present study, we investigated regulation of apo B secretionin hepatocytesfrom the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for FH. Incubation with low density lipoproteins (LDL) at concentrationsof 50 or 200 pg/ml, which increasedboth cellular cholesterylester and apo B secretionsignificantly in normal rabbit hepatocytes,did not causesuch increasesin WHHL rabbit hepatocytes. However, when WHHL rabbit hepatocyteswere incubatedwith LDL at a concentration of 500 fig/ml, a significant increasein cellular cholesterylesterand apo B secretionwasobserved.The effect of the increasein cellular level of cholesteryl ester upon apo B secretionin WHHL rabbit hepatocyteswas compatiblewith that in normal rabbit hepatocytes.Indeed, when WHHL rabbit hepatocyteswere incubated with LDL at 1680pug/ml,a concentration comparableto that of WHHL rabbit plasma,the amount of LDL degradation,cellular cholesterylesterlevel, and level of apo B secretionwere the sameas those in normal rabbit hepatocytesthat were incubatedwith LDL at 160 pg/ml, a concentration comparableto that of normal rabbit plasma.In summary,our presentstudy suggeststhat, at a steadystatewith sucha high plasmaconcentrationof LDL, the hepaticcholesterolcontent in WHHL rabbits could be set at the samelevel as in normal rabbits. It wasalso shownthat cellular cholesterylestercontentshad the same regulatory effect on apo B secretionin WHHL rabbit hepatocytesas in normal rabbit hepatocytes.Therefore, we conclude that in the presenceof the genetic defect of the LDL receptor, plasmacholesterolin WHHL rabbit is maintained at a concentration suchthat apo B secretionis similar to that in normal rabbit. Apolipoprotein B; WHHL rabbit; Familial hypercholesterolemia;Low density lipoprotein; HMG-CoA reductaseinhibitor --* Corresponding author.Tel.: 8175 7513460,Fax: 81 757719784. Abbreoiations: apoB,apolipoprotein B;HMG, 3-hydroxy-3-methylglutaryl; VLDL, very lowdensitylipoprotein;LDL, lowdensity lipoprotein;DMEM, Dulbecco’s modifiedEagle’s medium;MEM, minima1 essential medium;PBS,phosphate bufferedsaline;FCS, fetalcalfserum;SDS,sodiumdodecylsulfate;PAGE,polyacrylamide gelelectrophoresis; WHHL, Watanabe heritable hyperlipidemic. Keywords:

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1. Introduction Cholesterol has a wide variety of functions in the body. It is the precursor material for synthesis of steroid and bile acids. Cholesterol also plays essential roles in the cell membrane because of its hydrophobic property. This property, however, has a potentially lethal effect, namely development of atherosclerosis, when it exists in overwhelming concentrations in the plasma. To avoid such an adverse effect, animals control both the intracellular and extracellular cholesterol levels by elaborate mechanisms [l]. Cholesterol synthesized in the liver is delivered into the circulation by very low density lipoprotein (VLDL) containing both apolipoprotein B-100 (apo B) and apolipoprotein E. VLDL is converted into the smaller lipoproteins, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL). IDL and LDL are then removed from the circulation by the LDL receptor [2]. By this mechanism, plasma levels of cholesterol are under delicate homeostatic regulation in the synthesis (or production) and catabolism of these lipoproteins [1,2]. A remarkable example of disruption of this homeostasis is familial hypercholesterolemia (FH). This is one of the most common hereditary diseases, affecting approximately 1 in 500 persons in the general population. This inheritable disease with an autosomal dominant trait is characterized by the elevation of plasma cholesterol level, tendon xanthomas, and early onset of coronary artery disease. The disorder is caused by defects in the gene encoding the cell surface receptor for LDL [2]. Watanabe heritable hyperlipidemic The (WHHL) rabbit is an animal model for FH, producing a mutant LDL receptor, which has little affinity for apo B and is transported to the cell surface at only one-tenth the normal rate. The genetic defect arises from an in-frame deletion of 12 nucleotides, which results in the loss of four amino acids from the ligand binding domain of the LDL receptor [3,4]. Thus far, a defect in the catabolism of LDL has been elucidated both in FH and the WHHL rabbit [5- 111. While it was shown in normal counterparts that approximately two-thirds of LDL was catabolized by the LDL

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receptor pathway and one-third of LDL was catabolized by alternative pathway(s) distinct from the LDL receptor pathway, essentially all of the LDL was displayed to be catabolized via receptorindependent process(es) in FH and the WHHL rabbit [5-l I]. On the other hand, the regulatory mechanisms for production and secretion of VLDL in WHHL rabbits still remain elusive. It was reported that the secretion rate of apo B, a major constitutive apoprotein of VLDL, from the perfused liver of WHHL rabbit was almost equal to that from the normal rabbit liver [12]. Yet, there has been no study which explicated how lipoprotein secretion from the WHHL rabbit liver is regulated at the cellular level. It has been suggested that cellular cholesteryl ester might regulate apo B secretion from hepatocytes [13- 151. It has also been suggested that apo B is constitutively expressed and undergoes post-translational degradation in the cell [15- 171. In the previous study, we developed an experimental system with a primary culture of rabbit hepatocytes and demonstrated that cellular cholesteryl ester regulates apo B secretion by changing the intracellular degradation rate of apo B [18]. The intracellular cholesterol levels are known to be under negative feedback regulation. When the LDL particles are incorporated via the LDL receptor, cholesterol molecules are liberated from the degraded lipoproteins and suppress the activity of 3-hydroxy-3-methylglutaryl-Co-enzyme A (HMG-CoA) reductase, the rate limiting enzyme of cholesterol biosynthesis. Since WHHL rabbits have high plasma LDL levels, it is of interest to examine how metabolism of apo B is regulated by the intracellular cholesterol contents in the hepatocytes of these rabbits. In this study, we studied production and secretion of apo B in WHHL rabbit hepatocytes incubated with various concentrations of LDL. 2. Materials

and methods

2.1. Animals and materials Homozygous WHHL rabbits were raised in Kyoto by mating heterozygous WHHL females with homozygous WHHL males. Japanese white rabbits were purchased from Shimizu Laborato-

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ries (Kyoto, Japan). Rabbits were fed rabbit laboratory chow (Oriental Yeast, Tokyo, Japan). 35S-L,-Methionine (1250 Ci/mmol), mol), L251-Na ( > 17 Cijmg) and [a-32P]UTP ( > 3000 Ci/mmol) were purchased from New England Nuclear. Fetal calf serum (FCS), obtained from HyClone, was inactivated at 56°C for 30 min before use. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui (Tokyo, Japan). LGlutamine and streptomycin/penicillin solutions were from Flow Laboratories and GIBCO, respectively. Collagenase was obtained from Nitta (Osaka, Japan). Pravastatin was kindly provided by Sankyo (Tokyo, Japan). Other chemicals were all reagent grade. 2.2. Prepuration

of cultured rabbit hepatocytes

Male Japanese white rabbits (2.5 kg) or male WHHL rabbits (2.5 kg) were anesthetized with pentobarbital on the morning, and parenchymal hepatocytes were isolated by in situ perfusion of the liver with collagenase, as described by Seglen [19]. The isolated cells were suspended in DMEM with 10% FCS and plated at 1.0 x 106/35 mm dish, or 1.0 x 107/150 mm dish. 2.3. Lipoproteins

Human LDL and rabbit LDL (p = 1.0191.063) were isolated by sequential ultracentrifugation from plasma of healthy human subjects and Japanese white rabbits, respectively. FCSlipoprotein deficient serum (FCS-LPDS, p > 1.2 15) wa.s prepared by ultracentrifugation from FCS. Human LDL was radioiodinated with iodine monochloride [20]. Human LDL was methylated according to the method by Weisgraber et al. [21]. 2.4. Enzyme-linked

immunosorbent

assay of apo B

Enzyme linked immunosorbent assay (ELISA) was performed as described previously [18]. Briefly, rabbit LDL was absorbed to the wells of 96-well plates. Samples and rabbit LDL standards in the range 0.02-2.0 pg/ml were incubated with anti-rabbit apo B monoclonal antibody (final concentration of 0.3 pg/ml) and aliquots ‘were added to the wells. This monoclonal antibody was raised in our laboratory. We

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confirmed that this antibody did not cross-react with human apo B or rabbit apo E. After incubation, the wells were washed and goat antimouse IgG antibody conjugated with horse radish peroxidase (Organ Teknika-Cappel) was added to the wells. Orthophenylenediamine and H,O, were added, and the absorbances were read at 492 nm using an ELISA plate reader (Bio Rad). 2.5. Determination

of cellular lipid content

Cell monolayers were washed four times with buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), and 2 mg/ml of bovine serum albumin (BSA) and once with buffer containing 150 mM NaCl and 50 mM Tris (pH 7.4), and cellular lipid was extracted with hexane/isopropyl alcohol (3:2). After evaporation of the organic solvent, the lipid was resuspended in ethanol and cellular total cholesterol, free cholesterol, phospholipid and triglyceride were measured with an enzymatic kit (Ono Pharmacy and Wako Chemical, Tokyo, Japan). 2.6. “‘I-LDL

degradation assay

Freshly isolated hepatocytes were plated at 1.0 x 106/35 mm dish in DMEM with 5% FCSLPDS. After overnight culture, the cells were incubated with indicated concentrations of ‘251LDL for 4 h. The proteolytic degradation of ‘251-labeled LDL by hepatocytes was measured by assaying the amount of 1251-labeled trichloroacetic acid (TCA) soluble (noniodide) material formed by the cells and excreted into the culture medium [22,23]. 2.7. Immunoprecipitation

Methionine-free minimum essential medium (MEM) and MEM containing 500 PM methionine were generated from minimum essential medium Select-amine kit (GIBCO). Immunoprecipitation was carried out as described previously [18]. Briefly, after completion of the labeling or chase period, medium was harvested and a protease inhibitor mixture was added. ,The cells were washed with phosphate buffered saline (PBS) and solubilized in lysis buffer. Medium or cell lysate were incubated with anti-rabbit apo B

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Fig. 1. Effect of LDL and pravastatin on apolipoprotein B secretion and cellular cholesteryl ester contents in WHHL rabbit hepatocytes. Freshly isolated WHHL rabbit hepatocytes were plated at 1.0 x 106/35 mm dish in DMEM with 5% FCS-LPDS. After overnight culture, each monolayer was washed with PBS and received DMEM with 5% FCS-LPDS containing the indicated concentration of human LDL, none (control) or 10 pg/ml of pravastatin. After incubation for 24 h, the net secretion of apolipoprotein B was measured by enzyme linked immunosorbent assay. After collecting the culture media, the cell monolayers were washed with PBS and cellular lipid was extracted with hexane/isopropyl alcohol (3:2). Cholesteryl ester was measured by enzymatic assays. Values are means of nine dishes from three separate experiments. Panel A: apo B secretion. Panel B: cholesteryl ester contents. From the left: P, cells incubated with 10 pg/ml of pravastatin; C, control cells; L50, L200 and LSOO, cells incubated with 50, 200 and 500 ,cg/ml of LDL, respectively. Each value represents mean f S.D. *P < 0.05; **P < 0.01.

monoclonal antibody, and then rabbit anti-mouse IgG antiserum (Organ Teknika-Cappel) was added. After incubation, protein A-Sepharose CL4B beads (Pharmacia, LKB) were added and the incubation was continued. The protein A beads were collected by centrifugation, washed, and dissolved in sodium dodecyl sulphate (SDS) sample buffer [24]. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5%) and fluorography. The gells were scanned with ImageMaster (Pharmacia LKB). 2.8. Isolation of cellular RNA and RNaseprotection assay

Cellular total RNA was extracted by acid-guanidinium phenol-chloroform method [25]. Three pug of total RNA and the RNA probe (2 x 10’ counts/min) for apo B were hybridized and digested with RNase A, and RNase-resistant fragments were analyzed by 3.5% PAGE containing 8M urea and autoradiography, as described previously [18]. 2.9. Other assays

Protein was determined by the method of Lowry et al. [26].

3. Results 3.1. Efect of exogenous LDL and an HMG-CoA reductase inhibitor on apo B secretion and cellular cholesteryl ester contents in WHHL rabbit hepatocytes

To investigate the effect of exogenous LDL on apo B secretion and cellular cholesteryl ester contents, we incubated WHHL rabbit hepatocytes with increasing concentrations of LDL. As shown in Fig. lA, at concentrations of 50 or 200 ,ug/ml, LDL had no significant effects on apo B secretion in WHHL rabbit hepatocytes. However, a significant increase in apo B secretion was observed when LDL concentration was increased to 500 pg/ml. The addition of pravastatin (10 pug/ml), an HMG-CoA reductase inhibitor, suppressed apo B secretion by 62.3% (P < O.Ol), as shown in Fig. 1A. In spite of those changes in apo B secretion, no change was seen in 35S-albumin or “S-total protein secreted by the cells (data not shown). As shown in Figs. 1A and lB, there were parallel changes in cellular cholesteryl ester contents and amount of apo B secretion. A significant positive correlation was shown between cellular cholesteryl ester contents and apo B secretion in

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II

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Fig. 2. Correlation between cellular cholesteryl ester contents and were integrated and the regression line was given. (B) The data hepatocytes 1181were plotted on the same scattergram. 0, WHHL line for normal rabbit hepatocytes. B: regression line for WHHL

WHHL rabbit hepatocytes (Fig. 2A). When the correlation in WHHL rabbit hepatocytes was compared with that in normal rabbit hepatocytes [18], the points for both cell types fell on almost the same line (Fig. 2B). Correlation coefficients for WHHL rabbit hepatocytes and normal rabbit hepatocytes were 0.806 and 0.802, respectively. There were no significant changes in the cellular level of free cholesterol, triglyceride or phospholipid after incubation either with 500 pg/ml of LDL or 10 pg/ml of pravastatin (data not shown). We also investigated in what form apo B was secreted into culture media in WHHL rabbit hepatocytes and normal rabbit hepatocytes. When examined by high performance liquid chromatography, only VLDL particles were detected in the media of the hepatocytes from the WHHL and normal ra.bbits (data not shown). 3.2. Efect of’ methyl-LDL on cellular cholesteryl ester contents and apo B secretion in normal and WHHL rabbit hepatocytes To determine whether LDL taken up via alternative processes distinct from the LDL receptor pathway is responsible for the increase in cellular cholesteryl ester content and apo B secretion in WHHL rabbit hepatocytes, we incubated the cells with native LDL or methyl LDL at a concentration of 500 pg/ml. As shown in Fig. 3, addition of methyl-LIDL, which is not recognized by the LDL receptor [21], caused a significant increase both in

amount of apo B secretion. (A) The data from Figs. IA and B from WHHL rabbit hepatocytes (Fig. 1A) and normal rabbit rabbit hepatocytes; 0, normal rabbit hepatocytes. A: regression rabbit hepatocytes.

cellular cholesteryl ester contents and apo B secretion in WHHL rabbit hepatocytes. Methyl-LDL increased cellular cholesteryl ester contents and amount of apo B secretion in WHHL rabbit hepatocytes to the same extent as native LDL. The degree of these increases by methyl-LDL in WHHL rabbit hepatocytes was the same as those in normal rabbit hepatocytes. 3.3. Cellular mRNA level and intracellular turnover of apo B in WHHL rabbit hepatocytes To investigate the mechanism whereby cellular cholesteryl ester content regulates apo B secretion in WHHL rabbit hepatocytes, we first examined apo B mRNA levels in WHHL rabbit hepatocytes. An RNase protection assay demonstrated that neither pravastatin nor LDL caused any significant change in cellular levels of mRNA for ape B in WHHL rabbit hepatocytes (Fig. 4). These studies were repeated five times and showed the same results. Cellular levels of mRNA for ,!I-actin did not change either (data not shown). We next investigated intracellular apo B turnover by pulse-chase experiments. As expected, incubation for 24 h with 50 pg/ml of LDL caused no significant changes in the disappearance rate of intracellular ‘5S-apo B. However, disappearance of intracellular jSS-apo B was rapid when WHHL rabbit hepatocytes were incubated with pravastatin and was slow when the cells were incubated with 500 pg/ml of LDL (Fig. 5A,B). Three sepa-

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Fig. 3. Effect of methyl-LDL on apo B secretion and cellular cholesteryl ester contents in WHHL rabbit hepatocytes. Freshly isolated WHHL and normal rabbit hepatocytes were plated at 1.0 x 106/35 mm dish in DMEM with 5% FCS-LPDS. After overnight culture, each monolayer was washed with PBS and incubated with 500 pg/ml of methyl-LDL, 500 pg/ml of native LDL, or none (control) in DMEM with 5% FCS-LPDS for 24 h. Net secretion of apo B (A) and cellular cholesteryl ester contents (B) were measured. From the left: C, control WHHL rabbit hepatocytes; M500, L500, WHHL rabbit hepatocytes incubated with 500 pg/ml of methyl-LDL and 500 pg/ml of native LDL respectively; C, control normal rabbit hepatocytes; M500, L500, normal rabbit hepatocytes incubated with 500 jig/ml of methyl-LDL and 500 lg/ml of native LDL, respectively. *P < 0.05; **P -C 0.01

rate experiments showed that after a 2 h chase, 25.4% _+ 2.3%, 33.8% f 4.2%, 34.0% f 4.8%, and 57.5% + 1.0% (mean f SD.) of newly synthesized apo B remained in cells treated with pravastatin, control cells, cells treated with 50 pgg/ml of LDL, and cells treated with 500 lug/ml of LDL, respectively. Intracellular 35S-apo B levels at 0 h were almost the same among these cells (data not shown). In parallel with these changes in intracellular apo B, secretion of 35S-apo B during a 2 h chase was decreased in the cells incubated with pravastatin and increased in the cells incubated with 500 ,ug/ml of LDL. There were no significant changes in 35S-apo B secretion in the cells incubated with 50 ,~gg/ml of LDL (Fig. 5C,D). It was therefore indicated that addition of pravastatin accelerated intracellular degradation of apo B, thereby decreasing apo B secretion, whereas 500 pg/ml of LDL slowed intracellular apo B degradation rate, thereby increasing apo B secretion. Addition of 50 pg/ml of LDL had no significant effects on intracellular apo B turnover in WHHL rabbit hepatocytes.

It was reported previously that the concentration of LDL apo B in plasma in vivo was 167.5 and 15.7 mg/dl in WHHL rabbits and normal rabbits, respectively [27]. We therefore cultured WHHL rabbit hepatocytes and normal rabbit hepatocytes in the media containing 1251-LDL at concentrations comparable to those in the plasma, namely 1680 and 160 pug protein/ml, respectively, for 24 h. As shown in Fig. 6A, the rate of apo B degradation was similar in WHHL and normal rabbit hepatocytes. We next examined the cellular cholesteryl ester levels and apo B secretion in the same condition. There were no significant differences in apo B secretion or cellular cholesteryl ester contents between WHHL and normal rabbit hepatocytes (Fig. 6B,C). In addition, even in the presence of LDL at a concentration of 1680 pg/ ml, pravastatin decreased apo B secretion significantly in WHHL rabbit hepatocytes (by 22.6%, data not shown).

3.4. LDL degradation, cellular cholesteryl ester level, and apo B secretion in WHHL rabbit hepatocytes incubated with LDL at a concentration comparable to that in the plasma

In the present study, we investigated cholesterol and apo B metabolism in WHHL rabbit hepatocytes. We first examined the effect of exogenous LDL on cellular cholesteryl ester contents and

4. Discussion

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et al. 1 Atherosclerosis

apo B secretion by adding LDL into the culture medium. Although LDL at concentrations of 50 or 200 pg/ml increased both cellular cholesteryl ester contents and apo B secretion significantly in normal rabbit hepatocytes [18], no significant effects were observed either on cellular cholesteryl ester contents or apo B secretion in WHHL rabbit hepatocytes (Fig. 1). However, a high concentration (500 pg/ml) of LDL increased cellular cholesteryl ester contents and apo B secretion significantly in WHHL rabbit hepatocytes (Fig. 1). These results suggest that, when incubated with such a high concentration of LDL, WHHL rabbit hepatocytes could take up a significant amount of LDL, which in turn would increase cellular cholesteryl ester contents and conse-

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Fig. 4. Effect of pravastatin and LDL on cellular apo B mRNA level in WHHL rabbit hepatocytes. Freshly isolated hepatocytes were plated at 1.0 x IO’,‘150 mm dish in DMEM with 5% FCS-LPDS. After overnight culture, each monolayer was washed with PBS and received DMEM with 5% FCS-LPDS containing one of the following additions: 10 pg/ml of pravastatin, none (control), 50 or 500 pg/ml of human LDL. After incubation for 24 h, the cells were washed with PBS, cellular total RNA was extracted and RNase protection assay was performed as described in Materials and methods. P, cells incubated with 10 pg/ml of pravastatin; C, control cells; LSO, L500. cells incubated with 50 pgjml or 500 /rg/ml of LDL, respectively.

Fig. 5. Intracellular turnover and secretion of apolipoprotein B in WHHL rabbit hepatocytes. Freshly isolated WHHL rabbit hepdtocytes were plated at 1.0 x lo6135 mm dish in DMEM with 5% FCS-LPDS. After overnight culture, each monolayer was washed with PBS and incubated with 10 pg/ml of pravastatin, none (control), 50 pg/ml of human LDL or 500 fig/ml of human LDL in DMEM with 5% FCS-LPDS for 24 h. The cells were washed with PBS and pulse-labeled with 125 pCi/ml of %-L-methionine in methionine-free MEM for 30 min. The cells were washed with PBS and chased for the indicated time in MEM containing 500 PM unlabeled methionine with 10 pg/ml of pravastatin, none (control), 50 fig/ml of human LDL or 500 pg/ml of human LDL. After chasing, cell lysate and medium were analyzed by immunoprecipitation, SDS-PAGE and fluorography, as described in Materials and methods. A, intracellular “S-apo B chased for 0, 1, and 2 h. There was no significant difference in intracellular ‘%-apo B at 0 h between each incubation. B: Intracellular 35S-apo B (A) was assessedby densitometric scanning (‘l/u of 0 h). C: 75S-apo B secreted into the medium during the 1 and 2 h chase. D: secreted 75S-apo B (C) was assessed by densitometric scanning. (A,C) P, cells incubated with IO pg/ml of pravastatin; C, control cells; L50, L500, ceils incubated with 50 pg/ml, or 500 pg/mi of LDL respectively. (B,D) a, cells incubated with 10 pg/ml of pravastatin; 0, control cells; 0, cells incubated with 50 pg/ml of LDL: 17, cells incubated with 500 pgg,‘ml of LDL.

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Fig. 6. Degradation of lz51-LDL, cellular cholesteryl ester contents and apo B secretion in WHHL rabbit hepatocytes, incubated with LDL at the concentration comparable to that in the plasma. Freshly isolated WHHL and normal rabbit hepatocytes were plated at 1.0 x 106/35 mm dish in DMEM with 5% FCS-LPDS. (A) After overnight culture, WHHL rabbit hepatocytes and normal rabbit hepatocytes were incubated with 1680 fig/ml and 160 pg/ml of ‘*%LDL, respectively, in DMEM with 5% FCS-LPDS for 4 h and proteolytic degradation was measured as described in Materials and methods. (B,C), WHHL rabbit hepatocytes and normal rabbit hepatocytes were incubated with 1680 fig/ml and 160 pg/ml of human LDL, respectively, in DMEM with 5% FCS-LPDS for 24 h and cellular level of cholesteryl ester and apo B secretion were measured as described in the legends to Figs. IA and 1B. From the left: Hl680, WHHL rabbit hepatocytes incubated with 1680 fig/ml of LDL; N160, normal rabbit hepatocytes incubated with 160 /‘g/ml of LDL.

quently increase apo B secretion significantly. The addition of an HMG-CoA reductase inhibitor suppressed cellular cholesteryl ester contents and apo B secretion in WHHL rabbit hepatocytes as it did in normal rabbit hepatocytes [18]. When apo B secretion was assessed as a function of cholesteryl ester contents in WHHL rabbit hepatocytes, as shown in Fig. 2, the regression line was almost the same as that in normal rabbit hepatocytes [ 181, suggesting that cellular cholesteryl ester has the same regulatory effect on apo B secretion in WHHL rabbit hepatocytes as in normal rabbit hepatocytes. The suppressive effects of HMG-CoA reductase inhibitors on apo B secretion have been demonstrated both in humans and other species [18,28301. In patients with homozygous FH, however, it has been shown that HMG-CoA reductase inhibitors have little effect on plasma LDL concentrations [31]. In contrast, in the present study, it was shown that in WHHL rabbit hepatocytes, pravastatin reduced apo B secretion, even in the presence of a high concentration of LDL. This result was consistent with in vivo studies which showed that pravastatin reduced serum cholesterol levels in the WHHL rabbit [32,33]. Moreover, it was reported in a recent paper that serum cholesterol in a receptor-negative homozygous FH patient was reduced by 30% after treatment with simvastatin [34]. There may be a possibility that HMG-CoA reductase inhibitors could reduce

plasma level of cholesterol by decreasing the amount of apo B secretion in certain types of homozygous FH. The defect of the LDL receptor in the WHHL rabbit has been characterized and designated class 2 mutant. Namely, a small number of mutant LDL receptors are found on the cell surface. To rule out the possibilities that LDL taken up by these mutant receptors might affect cellular cholesteryl ester contents and apo B secretion, we examined the effect of methyl-LDL (Fig. 3). It has been demonstrated that methyl-LDL is taken up into tissues by mechanism(s) that do not depend upon the LDL receptor [18,35]. The effect of methyl-LDL on cellular cholesteryl ester contents and apo B secretion was the same as that of native LDL in WHHL rabbit hepatocytes (Fig. 3). Therefore, it was concluded that the uptake of LDL by mutant receptors could, if any, contribute minimally. In the next series of experiments, we investigated the intracellular mechanism whereby cellular cholesteryl ester contents regulate apo B secretion in WHHL rabbit hepatocytes. In a previous paper, we demonstrated that cellular cholesteryl ester regulates apo B secretion by changing intracellular apo B degradation rate in normal rabbit hepatocytes [18]. In WHHL rabbit hepatocytes, LDL at a concentration of 50 pg/ml had no effects on apo B intracellular turnover. On the other hand, 500 pg/ml of LDL slowed intra-

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cellular apo B degradation in WHHL rabbit hepatocytes (Fig. 5). It was conceivable that LDL taken up via LDL receptor-independent process(es) increased cellular cholesteryl ester contents and consequently slowed the intracellular apo B degradation rate in WHHL rabbit hepatocytes. This indicated that LDL receptor independent process(es) had the same regulatory effect on hepatic apo B secretion as did the LDL receptor pathway. We then investigated LDL degradation, cellular cholesteryl ester level, and apo B secretion in WHHL rabbit hepatocytes by incubating the hepatocytes with LDL at concentrations comparable to those in the plasma (Fig. 6). In WHHL rabbit hepatocytes incubated with LDL at a concentration of 1680 fig/ml, the amount of LDL degradation, cellular level of cholesteryl ester, and the amount of apo B secretion reached the same levels as those in normal rabbit hepatocytes incubated with 160 jig/ml of LDL. These results are consistent with the previous report that in transgenic mice overexpressing LDL receptors with lower concentration of total plasma cholesterol, cholesterol content in the liver was almost equal to that in normal mice [34]. Taken together, under any circumstance, plasma concentration of cholesterol might be set at a concentration where hepatic cholesterol contents could reach the same level. In this study, we designed experiments to differentiate apo B secreted by rabbit hepatocytes from human LDL apo B added to the culture media. For this purpose, we raised anti-rabbit apo B monoclonal antibody, which does not cross-react with human apo B. Therefore, we could not assess the effect of LDL from normal and WHHL rabbit. Also: it must be noted that this is an in vitro assay and that we need to be careful to apply all the results to the situation in vivo. In summary, when incubated with a high concentration of LDL, WHHL rabbit hepatocytes could take up a substantial amount of LDL through the receptor independent pathway. It was also indicated that cellular cholesteryl ester has the same regulatory effect on apo B secretion in WHHL rabbit hepatocytes as in normal rabbit hepatocytes. Therefore, it was suggested that at a high plasma concentration of LDL, the amount

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of apo B secretion in the WHHL the same as that in the normal words, in WHHL rabbits with cholesterol concentrations, LDL and cellular level of cholesteryl maintained the same as those in

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Acknowledgements

We would like to thank Dr. Mitsuyo Okazaki (Tokyo Medical and Dental Univ., Tokyo) for HPLC analysis and helpful discussion. This research was supported by Ministry of Education, Science and Culture of Japan Research Grants 04263104, 05404039, 05557052, and 04304051, International Scientific Research Program Grant 05044163 from the Japanese Ministry of Education, Science and Culture, a research grant for health sciences from the Japanese Ministry of Health and Welfare, Grant 5A-2 for cardiovascular diseases from the Japanese Ministry of Health and Welfare, the HMG-CoA Reductase Research Fund, and the Japanese Foundation of Metabolism and Diseases. References

[I] Goldstein JL, Brown MS, Regulation of the mevalonate pathway. Nature 1990;343:425. [2] Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34. [3] Kita T, Brown MS, Watanabe Y, Goldstein JL. Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA 1981;78:2268. [41 Yamamoto T, Bishop RW, Brown MS, Goldstein JL, Russell DW. Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science 1986;232:1230. [51 Bilheimer DW, Stone NJ, Grundy SM. Metabolic studies in familial hypercholesterolemia. J Clin Invest 1979;64:524. [61 Soutar AK, Myant NB, Thompson GR. The metabolism of very low density and intermediate density lipoproteins in patients with familial hypercholesterolemia. Atheroscrelosis 1982;43:217. [71 Kesaniemi YA, Witztum JL, Steinbrecher UP. Receptormediated catabolism of low density lipoprotein in man. J Clin Invest 1983;71:950. 181James RW, Martin B, Pometta D et al. Apolipoprotein B metabolism in homozygous familial hypercholesterolemia. J Lipid Res 1989:30:159.

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