Impaired Biosynthesis of Phosphatidylcholine Causes a Decrease in ...

Tm JOURNAL OF B I O ~ I CCHEMISTRY AL 0 1993 by The American Society for Biwhemistry and Molecular Biology, Inc

Vol. 268, No. 33, Issue of November 25, pp. 24990-24996, 1993 Printed in U S A .

Impaired Biosynthesis of Phosphatidylcholine Causes a Decrease in the Number of Very Low Density Lipoprotein Particles in the Golgi but Not in the Endoplasmic Reticulumof Rat Liver* (Received for publication, April 2, 1993, and in revised form, June 28, 1993)

Henkjan J. Verkade$Bfill, Darren G. Fast#Bn**,Antonio E. RusiiiolS $$, Douglas G. ScrabaB, and Dennis E. VanceSB $8 From the $Lipid and Lipoprotein Research Group and the §Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G2S2, Canada

We have investigated the mechanism by whichinhibition of phosphatidylcholine biosynthesis in rat hepatocytes by choline deprivation causes a reduction in the secretion of very lowdensity lipoprotein (VLDL) Cyao,Z., and Vance, D. E. (1988)J. BioL Chem 263, 2998-3004). Rats ingested a choline-deficient or control diet for 3 days, and subcellularfractions of liver were prepared. No change in the amount of apolipoprotein B in the lumina of the endoplasmic reticulum was observed, but there was a 4&6@% decrease of apolipoprotein B in the lumina of the Golgifrom choline-deficient compared with control rats. Incubation of microsomes, derived from choline-deficient and -supplementedhepatocytes, of apolipoprowith trypsin showed similar degradation tein B, indicating similar quantities of this protein are present on the surface andwithin the lumina. The VLDL particles in the Golgi of liver cells and in plasma, on average, were larger in samples derived from cholinedeficient compared with choline-supplemented animals. Incubation of plasma VLDL with proteasesdemonstrated that the apolipoprotein B of plasma VLDL particles from choline-deficientanimals had a different susceptibility to digestion than did VLDLfrom cholinesupplementedanimals.Thesedata indicate that the number of VLDL particles assembled inthe endoplasmic reticulum of liver is similar in choline-deficient and -supplemented rats, but the number of particles is decreased in the Golgi from choline-deficientanimals.

Previous studies have shown that the synthesis of apoB, triacylglycerol, cholesterol, cholesteryl ester, andphospholipid occurs on the ER, and nascent VLDL particles are assembled from these components (Vance and Vance, 1990; Gibbons, 1990; Davis, 1991; Boren et al., 1991; Dashti, 1991; Dixon and Ginsberg, 1993). Current information suggests that apoB combines with lipid components on the luminal sideof the ER withformation of full-sized VLDL particles (Rusifiol et al., 1993). The nascent VLDL particles are processed as they move through the Golgi where post-translationalmodifications of apoB occur, after which VLDL is secreted. Hepatocytes from humans secrete apoBlOO whereas rat liver secretes both apoBlOO and apoB48 (Davis, 1991). Phosphatidylcholine (PC) is the major phospholipid component of VLDL, and PC is located exclusively on the surface of the particle.We have been interested in the role of PC biosynthesis in the assembly and secretion of VLDLs from rat hepatocytes. Our approach has been to inhibit selectively PC biosynthesis by feeding rats a CD diet for 3 days. This regimen causes a 6.5-fold accumulation of triacylglycerol in the liver and a 60% reduction of VLDL in the plasma but has no effect on the amount of plasma high density lipoproteins (Yao and Vance, at which VLDL 1990). We haveinvestigatedthestage assembly/secretion is disrupted in hepatocytes cultured from CD rats. VLDL secretion was inhibited by 60-70% in CD hepatocytes that were maintained up to 24 h in a CD medium (Yao and Vance, 1988). ApoB synthesis and turnover were the same in CD and CS hepatocytes (CD hepatocytes that were supplemented with choline) even though the rate of PC synA majorfocus in lipoprotein research is the process by which thesis was inhibited by 70%. The requirement for active PC VLDL’ particles are assembled and secreted by hepatocytes. synthesis was shown to be highly specific since supplementaor monomethylethanoltion of the medium with ethanolamine * This work was supported by a grant from the Heart and Stroke amine (which is converted into phosphatidylmonomethylethaFoundation ofAlberta(to D. E. V.) and the Medical Research Council of nolamine) did not restore VLDL secretion (Yao and Vance, Canada (to D. G. S.). The costs of publication of this article were de- 1989). However, the addition of dimethylethanolamine (which frayed inpart by the payment of page charges. Thisarticle must there- is converted into phosphatidyldimethylethanolamine) partially fore be hereby marked “advertisement”in accordance with 18 U.S.C. restored the secretionof VLDL that contained apoB48 but did Section 1734 solely to indicate this fact. 1 Contributed equally to the experimental and theoretical aspects of not restore the secretion of particles that contained apoB100. this research. The biosynthetic origin of PC was notan important factor since 11 Supported by a postdoctoral fellowship from the Alberta Heritage normal VLDL secretion could be restored by the addition of Foundation for Medical Research.Present address: Dept.of Pediatrics, choline, methionine (which promotes PC biosynthesis via the KZ Groningen, The University of Groningen,Bloemsingel 10, 9712 methylation of phosphatidylethanolamine), or lyso-PC (which Netherlands. ** Supported by a studentship from the Alberta Heritage Foundation can be acylated to PC in hepatocytes)(Robinson et al., 1989). for Medical Research. The question we havenow addressed is: where is the defect $$ Supported by a postdoctoral fellowship from the Alberta Heritage in theprocess of assembly/secretion of VLDL from CD hepatoFoundation for Medical Research. $5 Medical Scientist of the Alberta Heritage Foundation for Medical cytes? As an initial approach to the question wehypothesized Research. lb whom correspondence should be addressed. Tel.: 403-492- that PC biosynthesis would be required for “budding“ of the 8286;Fax: 403-492-3383. VLDL particle into the lumenof the ER. If this were so, there The abbreviations used are: VLDL, very low density lipoprotein; apoB,apolipoproteinB;CD,choline-deficient;CS,choline-supplechloromethyl ketone. mented; ER, endoplasmic reticulum; PC, phosphatidylcholine; TPCK, ~-l-tosylamido-2-phenylethyl

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Phosphatidylcholine and VLDL Secretion

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should be fewer nascent VLDL particles in the lumina of the ER from CD, compared with CS, rat livers. On the other hand, if formation ofVLDL particles occurred at a normalrate in the livers from CD rats, there should be the same number of particles present in the lumina of ER in liver cells from CDand CS rats. The results of our study suggest that VLDL formation in the ER is not impaired in CD hepatocytes and provide new insight into the reason why fewer VLDL particles are secreted from CD hepatocytes. We have found that there is at least as much apoBin the lumina of the ER from livers of CD rats as in CS rats. In contrast, fewer particles are present in the Golgi lumina isolated from livers of CD rats versus control animals. Moreover, our studies suggest that impaired PC biosynthesis leads to the presence of slightly larger lipoproteins in the Golgi and plasma and with altered susceptibility of the apoB on the particles to protease digestion.

luminal contents of the subcellular fractions were isolated by treatment of the organelle vesicles with 100 m~ sodium carbonate (pH 11.5) for 30 min onice at a protein concentration below 2 mg/ml (Fujiki et al., 1982).)After incubation with sodium carbonate, bovine serum albumin (fatty acid free) was added to the incubation to a final concentration of 0.5% (wh), according to Bostrom et al. (1986). The contents and membranes were separated by centrifugation for 15 min at 200,000 x g. The luminal contents were diluted with an equal volume of NaHC0, buffer (200 m~ NaHCO,, 1 M NaCl, 2 mM EDTA, pH 8.3) and incubated with Sepharose 4B-anti-apolipoproteinB beads overnight at 4 "C in a rotary tumbler (Rusiiiol et al., 1993). The beads were washed four times with phosphate-buffered saline (pH 7.4), and lipid was extracted (Bligh and Dyer, 1959). The chloroform fraction was dried under nitrogen and stored a t -20 "C until analysis. Protein was determined according to Lowry et al. (1951) using bovine serum albumin as a standard. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis -Proteins were denatured in buffer (6.25 mM Tris-HC1,pH 6.8, 2% sodium dodecyl sulfate, 5% P-mercaptoethanol, 10% glycerol, 0.02% bromphenol blue) by boiling for5 min. Electrophoresis was on a 3-15% polyacrylamide gel that contained 0.1% sodium dodecyl sulfate. ProMATERIALSANDMETHODS teins were stained with either Coomassie Blue R-250or silver stained Chemical~-[~HILeucine (73 Ci/mmol),UDP-[6-3H]galactose (14.5 according to Hochstrasser et al. (1988). CUmmol), and an enhanced chemiluminescence detection kit were purQuantitation ofApolipoprotein %The amounts of apoB in the luchased f r o m h e r s h a m Corp. Boehringer Mannheim supplied NADPH, mina of isolated fractions were analyzed by both immunoblotting and trypsin, cathepsin D, sheep polyclonal antibody against human apoli- enzyme-linked immunosorbent assay. The proteins of the luminal con(ABTS), tents were electrophoresedas described above.The proteins were transpoprotein B, and 2,2'-azino-di-(3-ethylbenzthiazolinesulfonate~ the peroxidase substrate. The specificity of the antibody was confirmed ferred to a polyvinyl difluoridemembrane (18h, 70 V)in 62.5 mM boric by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and sub- acid buffer (pH 8.0) at 4 "C and subsequently reacted with rabbit polysequent immunoblotting of rat plasma VLDL.No bands other than clonal anti-rat-VLDL antiserum and peroxidase-linked anti-rabbit I&. apolipoprotein B48 and apolipoprotein BlOO were detected. Sepharose Visualization of immunoreactive species was viaenhanced chemilumi4B-CNBr was from Pharmacia Fine Chemicals, Sweden. The reagents nescence detection reagents. Densitometry was done with a Camag "LC for polyacrylamide gel electrophoresis were purchased from Bio-Rad. Scanner I1 a t 460 nm. L-1-Tosylamido-2-phenylethyl chloromethylketone (TPCK)-trypsinwas Enzyme-linked immunosorbent assays were performed as described from Pierce Chemical Co. Polyvinyl difluoride membranes (Immobilon byRusifiolet al. (1993). Luminal contents were coated on Immulon P) were from Millipore. Immulon microtiter immunoassay plates were microtiter plates. Polyclonalrabbit anti-rat apoB and peroxidase-linked from Dynatech Laboratories, Inc., Chantilly, VA. All other chemicals anti-sheep IgG were used as primary and secondary antibodies, respecwere from Sigma or Fisher. Antibody to rat apolipoprotein B was a tively. generous giR from Dr. Roger Davis, San Diego State University. Quantitation ofAlbumin-The amount of albumin was determined Animals and Diets-Choline-deficient diet (ICN Biochemicals, by enzyme-linked immunosorbent assays using rat albumin as stanCanada) consisted of 10% vitamin-free casein, 10%a-protein, 20% lard, dard and a rabbit anti-rat albumin primary antibody on Immulon mi56% sucrose, 4% salt mixture (Wesson), and ICN vitamin fortification crotiter plates using between 50 and 400 pg of total proteidwell. The mixture that lacked choline chloride. Male Sprague-Dawley rats, inisecond antibody was sheep anti-rabbit peroxidase conjugate. tially weighing 35-45g, were fed either thisdiet (CD group) or this diet Samples for ElectronMicroscopy-The ultrastructure of the density < with 0.4 d l 0 0 g choline chloride added (CS group). The liver and body 1.006 g/mlfraction from plasma and the luminal contents of Golgi from weights were identical in both CD and CS groups. CD and CS rats was studied. Isolated luminal contents were subjected Subcellular Fractionation-The CD and CS rats were anesthetized to sequential flotation to obtain the d < 1.006 fraction. VLDL was with diethyl ether and then by intraperitoneal injection of pentobarbitone ( 5 mg/100 g ofbody weight). In some instances the liver was isolated from plasma of CD and CS rats asfollows. Blood was collected perfused through the portal vein with cold phosphate-buffered saline and VLDL isolated by sequential flotation via ultracentrifugation in(pH 7.4) before excision. Typical liver weight of rats from either diet cluding initial centrifugation steps to remove chylomicrons.VLDL was stored under nitrogen, on ice, until fixation and negative staining for group was 2.0-2.5 g, and usually two livers were combined for the subcellular fractionation. The method of Croze and Mom6 (1984) was electron microscopy (usually within hours). The samples were allowed used for subcellular fractionation, with the modifications as described to adhere to hydrophilic carbon films and then washed with 2% sodium byVance (1990) and by Hamilton et al. (1991) in order to diminish phosphotungstate as a negative stain. Electron micrographs were obendosomal contamination of the Golgi fraction. These procedures re- tained with a Phillips EM420 electron microscope operated a t 100 kV. sulted in isolation of a heavy ER fraction (ER I), a light ER fraction (ER RESULTS II), and Golgi fromCD and CS livers. Membrane fractions were assayed for NADPH:cytochromec reductase (marker enzyme for ER)and UDPRecovery of Marker Enzymes from Subcellular Fractions of galactosexV-acetylglucosaminegalactosyltransferase (marker enzyme CD and CS Rat Livers Was Similar-Fig. 1 shows the specific for trans-Golgi) as previously described (Vanceand Vance, 1988). activity of NADPH-cytochromec reductase (ER marker) and of Preparation of Hepatocytes and Microsomes-Hepatocytes were isolated from the livers of CD rats aspreviously described (Yao and Vance, UDP-galactosew-acetylglucosamine galactosyltransferase 1988)and plated in 60-mm dishes at a density of 3 x 106 cellddish. The (Golgi marker) in the three fractions isolated from CD and CS cells were incubated for 4 h in choline- and methionine-deficient Dul- rat livers. No significant differences between CD and CS livers becco's modified Eagle's medium containing 20% delipidated fetal bo- were observed in the distribution of these enzymes among the vine serum. Subsequently, all dishes were incubated in the absence of ER and Golgi fractions. The specific activity of NADPH-cytoserum, but some dishes were supplemented with 100 p~ choline and chrome c reductase was enriched in fraction ER 11, which is 200 pM methionine (CS hepatocytes) (Yao and Vance, 1988). After an as a "smooth"ER fraction (Crozeand MoRB, 1984) designated overnight incubation (18 h) the cells were scraped into 2 ml of phosphate-buffered saline that contained 1 p~ phenylmethylsulfonyl fluo- relative to ER I (heavy ER fraction). The specific activity of the ride, 50 pg/ml leupeptin, and 1pg/ml aprotinin. The cells were pelleted trans-Golgi marker enzyme UDP-galactosew-acetylgluand resuspended in 10 mM Tris-HC1, pH7.4,0.25 M sucrose. Microsomes cosamine galactosyltransferase was high in the Golgi fractions were prepared and incubated with trypsin as described in the legend to compared with either of the ER fractions. According to comFig. 4 (Davis et al., 1990). parison of the specific activities of these marker enzymes, conImmunoaffinity Isolation of Apolipoprotein E-containing Particles from the Lumina ofSubcellular Fractions-The covalent binding ofthe tamination of the Golgifraction with NADPH:cytochromec human anti-apolipoprotein B antibody to Sepharose beads was per- reductase (ER marker) was 14.0 1.4%(CD) and 18.3 2.4% formed according to the instructions of the supplier (Pharmacia). The (CS).Contamination of ER I fractions with Golgi was 4.7 0.6%

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ER I ER II Golgi FIG.1. Specific activities of marker enzymes in subcellular fractions prepared from CD or CS livers. Subcellular fractions were isolated from rat liver and assayed for NADPH:cytochrome c reductase (top)and UDP-galactoses-acetylglucosamine galactosyltransferase (bottom) as described under “Materials and Methods.” ER I , heavy endoplasmic reticulum;ER I I , light endoplasmic reticulum.Values represent means S.E. from four to six fractionations.

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FIG.2. Immunoblot detection of apoB in lumina of subcellular fractions from CD and CS rat livers. Subcellular fractions were prepared from rat liver. Luminal contents corresponding to 50 pg of Golgi, ER I, or ER I1 protein were electrophoresed on a 3-15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The gel was electroblotted onto polyvinyl a difluoride membrane and incubated with rabbit anti-rat apoB antibody, followed by peroxidase-conjugated antirabbit IgG. The proteins were detected by enhanced chemiluminescence assay. No choline in the rat diet is indicated by -, and + indicates that there was choline in the diet.

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FIG.3. ApoB levels in lumina of ER and Golgi fractions from (CD) and 4.1 0.5% (CS). Similarly low contamination of ER I1 CD and CS livers and from CS and CD plasma. The luminal confractions wasobserved. Typically, the amountof protein recov- tents of ER and Golgi were prepared as described under “Materials and ered in thesefractions was 2.0-2.5 (ER I), 1.5-2.0 (ER 111, and Methods.” The amount of apoB was quantitated by a n enzyme-linked immunosorbent assay. The values represent the meansof three sepa0.4-0.6 (Golgi) mg/g of liver, respectively. Minimal endosomalcontamination of Golgi preparations was rate subcellular fractionations f S.D. **, p < 0.005;*, p < 0.05. Open bars, CD;closed bars, C S .

confirmed by the following experiment (Hamilton et al., 1991). Endogenously synthesized apoB was labeled by injection of [3H]leucine into a femoral vein 30 min before the rat was sacrificed. Fifteen min before sacrifice, the rat was injected in the other femoral vein with [12511VLDLas a label for endocytosed apoB. Subcellular fractions (Golgi, ER I, ER11)were prepared from the liver, and apoB-containing lipoproteins were isolated from the luminal contentsof each fraction by an immunoafinity procedure (Rusiiiol et al., 1993). The apolipoproteins from the homogenate were separated by polyacrylamide gel electrophoresis, and theapoB-containing bands were excised from the gels. The ratio of the radioactivity in apoB from 1251(endocytosed apoB) and 3H (endogenously synthesized apoB)was compared in the luminaof Golgi to that in theliver homogenate. The percentage of contamination in theGolgi of endogenously synthesized apoB with endocytosed apoB was 8.4% (CD) and 2.5% (CS). The contamination of ER fractions by endosomes was also low (CD ER I,3.2%; CDER II,2.8%; CS ER 4.4%; I, CS ER 11,6.3%). Thus, endosomal contamination was not a significant factor for our subsequent studies. Apolipoprotein B Is Decreased in the Lumina of the Golgi, but Not the ER, in CD Comparedwith CS Rat Livers-The levels of apoBlOO and apoB48 in the luminaof the isolated subcellular fractions from livers of CD and CS rats were determined by immunoblotting (Fig. 2). The content of both apoBlOO and

apoB48 in the lumina of ER I and ERI1 was somewhatenriched in CD compared with CS fractions. However, in the luminaof the Golgi from CD rats, the amountof both apoBs was significantly reduced. Immunoblots from six different preparations of Golgi and three different preparations of ER I and ER I1 from CD and CS rats were scanned by a densitometer (CamagTLC Scanner 11,460 nm). The ratioof the intensityof the apoBlOO band in the Golgi from CD rats to that from CS rats was 0.52 rt 0.29 (S.D., p < 0.02), and the ratio for apoB48 was 0.61 + 0.29 (S.D., p < 0.02). The ratiosof BlOO for ER I and ERI1 from CD rats to that from CS rats were 1.46 0.48 and 1.13 0.10. The ratios for B48 were 1.06 0.10 and 1.18 0.09, respectively. Quantitative analysis of apoB mass in the lumina wasperformed by enzyme-linked immunosorbent assays usinga rabbit polyclonal antibodydirected against rat apoB. Theresults showed minimal differences in the luminalapoB content of ER I and ER I1 fractions but a significant ( p < 0.05) decrease in the apoB content in the lumina of Golgi and plasmafrom CD compared with CS rats (Fig. 3). As a control, albumin levels were examinedin the luminaof the subcellular fractions using an enzyme-linked immunoassay. As shown in Table I, there are no significant changes in the levels of albumin in the lumina of the subcellular fractionsor in plasma.

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Phosphatidylcholine and VLDL Secretion Taken together, these data support the hypothesis that the initial stepsof VLDLassembly in theER are not quantitatively impaired in CD livers. The data also suggest that when PC biosynthesis is inhibited, there is an increased degradation of apoB-containing particles prior to arrival in, or within, the Golgi. Accessibility of ApoB to 12ypsin Digestion Is Similar in Microsomes from CD and CS Hepatocytes-When intact microsomes are prepared from homogenates of liver or cultured hepatocytes, more than half of the apoB is found associated with the membrane and is accessible to trypsin digestion (Davis et al., 1990; Wilkinson et al., 1992). If, as reported above, there were no major differences in the number of particles in the lumina of the ER from CD and CS rat livers and no effect on apoB synthesis (Yao and Vance, 19881,the accessibility to trypsin of apoB associated with the ER membrane would be expected to be similar in CS and CD hepatocyte samples. Therefore, intact microsomes, in which the proteins were labeled with [3Hlleucine, were isolated from CD and CS hepatocytes and incubated with trypsin for 30 min at 4 "C. It is evident from Fig. 4 that inintact microsomes from bothCS and CD cultured hepatocytes, approximately 70% of apoB48 and -B100 was digested with exogenously added trypsin. In contrast, only 2535% of albumin, the mature form of which is largely found in the lumina of microsomes, was degraded during the incubation with trypsin. Incubations in which trypsin was added immedi-

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ately followed by a mixture that contained trypsin inhibitor, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin gave the same results asthe control samples that contained no trypsin. Thus, the luminal contents of the microsomal membranes were largely protected from trypsin digestion. Since the trypsin susceptibility of both forms of apoB was similar in CD and CS microsomes, these experiments suggest that choline deficiency does not impair the translocation of either apoBlOO orapoB48 into the ER lumen. The experiment is consistent with the results thatapoBlOO and -B48 are found in the lumina of the ER from CD rat livers at concentrations similar to those found in CS rat livers. Larger ParticlesWere Detected in the d < 1.006glml Fraction from the Luminal Contentsof Golgi from Liver, and of VLDL in Plasma, from CD Compared with CS Rats-The luminal contents of Golgi from CD and CS livers were isolated aRer sequential flotation ultracentrifugation. The fraction with a density < 1.006 g/ml(VLDL) was usedfor negative staining electron microscopy (Fig. 5, A and B ) . There appeared to be more particles with larger diametersin the CD compared with CS samples. The diameters of the particles from three different isolations were measured and plotted into histograms (Fig. SA). The mean particle size S.D. in the Golgi fraction was greater in CD than in CS liver, 46.1 * 13.1 nm versus 40.0 * 13.7 nm, respectively ( p < 0.001, non-paired t test, n = 250). This difference in the average value was mostly due to a relative abundance of particles with a diameter between 50 and 80 TABLEI nm in the fractions from the CD liver. Effect of choline deficiency on albumin content in subcellular fractions With the same methods we determined the average particle and in plasma size of the VLDL fraction in plasma from CD and CS rats (Fig. Subcellular fractions were isolated from CD and CS rat livers (Croze and M o d , 1984), and the lumenal contents were isolated aRer treat- 5,C and D , and Fig. 6B). We have previously shownthat in CD ment with sodium carbonate as described under "Materials and Meth- rats, the amounts ofVLDL-apoB and triacylglycerols in the ods." Protein concentration of the content was measured and levels of plasma are reduced by 6040% compared with those in controls albumin assayed using an enzyme-linked immunoassay with rabbit (Yao and Vance, 1990). Interestingly, the VLDL particles that anti-albumin as the primary antibody. Detectionwas via a peroxidaseare present in CD plasma had a larger average diameter than conjugated second antibody and ABTS substrate (2,2'-axino-di-[3-ethylbenzthiazolinelsulfonate).Values for CS albumin levels per pg of lu- did plasma VLDL from CS rats, 44.6 * 15.1 nm versus 37.9 * menal organelle protein were 26.9 ng (ER I), 11.1ng (ER II), and 150 ng 12.4 nm, respectively ( p < 0.001, non-paired t test, n = 500).As (Golgi), and in plasma, 354 ng/pg of total plasma protein. The results was also the case in the d < 1.006g/ml fraction from Golgiof CD are f S.D. ( n = 3). rats, the histogram of plasma VLDL showed a relatively inRatio of albumin creased abundance of particles with a diameter larger than50 Fraction in CD/CS organelles nm, when compared with the plasma VLDL from CS rats (Fig. ER I 1.28 f 0.34 6A). Three different isolations of plasma VLDL from CD and ER I1 1.36 f 0.25 CS rats were performed, and the mean particle sizes found in Golgi 1.06 f 0.34 each were 44.2 * 16.3 (n = 200),43.7 16.8 ( n = EO), and 45.6 Plasma 0.99 f 0.01 f 12.7 (n = 150) for VLDL from CD rats, and 38.7 12.5 ( n =

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FIG.4. Accessibility of apoB to trypsin in microsomes from CD and CS hepatocytes. Microsomes were isolated fromCD and CS hepatocytes that had been labeledovernight (16 h) with [3H]leucine(20 pCi/dish). The labeled microsomes wereincubated with TPCK-trypsin (60 pg/400 pg of microsomal protein) or without (control)for 30 minat 4 "C. The reaction was stopped by the addition of a protease inhibitor mixture (1.25 mg/ml soybean trypsin inhibitor, 1 pg/ml aprotinin, 50 pg/ml leupeptin, and 1 p~ phenylmethylsulfonylfluoride) plus addition of an equal volume of 2 x sodium dodecyl sulfate-polyacrylamidegel sample buffer and immediately boiled for 5 min. The proteins were loaded ontoa 3-15% sodium dodecyl sulfate-polyacrylamidegel, electrophoresed, and stained with Coomassie Blue R250.Bands corresponding to apoB100, -B48,and albumin were cut from the gels and dissolved overnight in 0.25 ml of perchloric acid and 0.5 ml of H202(30%) a t 60 "C, and radioactivity was determined in a liquid scintillation counter. The results are from three separate experiments S.D.


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FIG.5. Electron microscopyof plasma VLDL and d e 1.006 glml fraction of lumina fromGolgi of CD and CS liver. The d < 1.006 g/ml fraction of Golgi lumina and VLDL from plasma were isolated by sequential flotation centrifugation and analyzedby negative staining electron microscopy as described under "Materials andMethods." Panel A, CD Golgi; panel B , CS Golgi; panel C , CD plasma; panel D,CS plasma.

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2001, 37.9 10.1 (n = 1501, and 36.9 14.3 (n = 150) nm for 460 nm) and calculated the amount of apoBlOOdegraded per pg VLDL from CS rats. of protease. Thus, from CS VLDL, 3.4 x densitometry ApoBs in PlasmaVLDL from CD and CS Rats HaveDifferent units of apoB were degraded per pg of trypsin. For apoBlOO Susceptibilities to Protease Digestion-The studies by electron from CD VLDL, the amount of apoB degraded was about 50% microscopy suggested that there were more large particles in less (1.73 x densitometryunitsperpg of trypsin).This plasma from CD than from CS rats. We investigated if addi- calculation could not be done for apoB48 since a breakdown tional differences might be detected in plasma VLDL from CD product ofapoBlOO migrated with apoB48. For digestion by and CS rats. The apolipoprotein compositions of the CS- and cathepsin D ofapoBlOO from CS VLDL, 1.1 x densitomCD-derived particles were similaras judged by silver staining etry unitsof apoBlOOwere degraded per of pgcathepsin D. The of proteins on sodiumdodecyl sulfate-polyacrylamide gels (data value for CD VLDL was %fold greater (3.8 densitometry units not shown). Next,we incubated VLDL from the plasmaof both per pgof cathepsin D). For digestion of apoB48 from CS VLDL, CD and CS animals with trypsin or cathepsin D a s a function 2.8 x densitometryunits of apoB48were degraded per gg of time and amount of protease. Some of the results are pre- of cathepsin D whereas for CD VLDL 1.0 x densitometry sented inFigs. 7and 8. The apoBlOO in plasma VLDL from CD units of apoB48 weredegraded per pgof cathepsin D. It, thererats was more resistant todigestion by trypsin than was that in fore, seems that notonly is the average size of the VLDL parVLDL from CS rats (Fig. 7). In contrast, cathepsinD appeared ticles larger in CD than in CS plasma but that theapoB may to degrade apoBlOO and 4348 more rapidly in the VLDL iso- have a different conformation on the surface of some of the lated from the CD compared with CS rats (Fig. 8). The major VLDL particles, which alters its susceptibility to proteases. peptides formed by digestion with trypsin were the same from DISCUSSION the CD and CS rats. Similarly, the peptides formed during cathepsin D incubations of plasma VLDL were similar for CD How Does Choline Deficiency Inhibit VLDL Secretion? New and CS rats. Thus, the differences in proteolysis of apoB were Hypotheses-When these studies were initiated our hypothesis a kinetic function. From thegels of the incubations, we scannedwas that decreased biosynthesis of PC, as a result of choline for the amounts of apoBlOOand -B48 (CAMAG TLC Scanner 11, deficiency, interferes with an early stageof VLDL assembly in

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Diameter (nm) FIG.6. Histograms of plasma VLDL and the d < 1.006 glml fraction isolated from the lumina of Golgi of livers from CD and CS rats. Measurements were madeof the diametersof 250 particles from CD and CS Golgi (A) and of 500 particles from CD and CS plasma( B ) . Examples of the corresponding images that were used measurements are shown in Fig. 5. ~

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B48 FIG.8.Cathepsin Ddigestion of VLDL from plasma of CD and CS rats. VLDL protein (20 pg) from either CD or CS plasma was incubated in the presence of 0-20 pg of cathepsin D in buffer a containing 37.5 mM sodium acetate (pH4.8) for 20 min at 30 "C. The reaction the legend was stopped and the samples analyzed as described in to Fig.

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B4X

CD CS Trypsin digestion of apoB in VLDL from the plasma of CD and CS rats. VLDL protein (20 pg) from eitherCD or CS plasma FIG.7.

was incubated in the presence of 0-500 ng of trypsin in a buffer that contained 10 mM Tris-HC1 (pH 7.4) for 10 min at 30 "C. The reaction was stopped by the addition of a n equal volume of sample buffer (12.5 mM Tris-HCI, pH 6.8,4% sodium dodecyl sulfate, 10% (v/v)P-mercaptoethanol, 20%glycerol, 0.04% bromphenol blue)and immediatelyboiled for 5 min. The samples were electrophoresed on a 3-15% polyacrylamide gel that contained 0.1% sodium dodecyl sulfate and silver stained.

the ER. We, therefore, expected to find fewer nascent VLDL particles in the lumen of the ER. The experimental results presented in this communication strongly suggest that this is not the case. There was no decrease in the number of apoBcontaining particles in the lumina of the ERfrom CD compared with CS rat livers. The lack of a translocation defect in the movement of apoB48 and apoBlOO into theER lumen wasalso evident from the studies with trypsin digestion of microsomes. If there had been fewer particles in the microsomes from CD, than from CS, hepatocytes, there should have been less luminal apoB present after trypsin digestion. Instead the amount of apoB that remained after trypsin digestion of intact micro-

somes was the same for CD- and CS-derived samples. In contrast, 40-50% fewer particles were present in the lumina of the Golgi from CD uersus CS livers. However, there wasno change in thelevels of albumin in the luminaof any subcellular fractions examined. In addition, there were more large VLDL particles in the Golgi from CD livers compared with CS livers. Similarly, the plasma VLDLs from CD rats were on average larger and showed different susceptibility to trypsin. These observations suggest that impaired PC synthesis, resulting from choline deficiency, does not limit the formation of nascent VLDL particles in the lumen of the ER. Instead, one hypothesis, consistent with the observations, is thatlimited PC biosynthesis results in theformation of particles that are different in structure. Since the number of particles in the ER lumina from CD rat liver is not diminished,it may be that some of the particles are degraded after leaving the ER. Digestion could be by proteasesinthelumen of the ER, transport vesicles, the "intermediate compartment" (LippincottSchwartz, 19931, or Golgi. Alternatively, transport vesicles that contained defective particles could be targeted to, and degraded in, lysosomes. Because VLDL particles with different protease sensitivity were found in the plasma of CD rats, it seems that not all of these altered particles are degraded. A different hypothesis, also consistent with the data, is that transport of the VLDL particles from the ER lumen is retardedin CD compared with CS livers. In thiscase a significant accumulation of VLDL in theER lumen would not be observed in CD livers if some of the defective particles were degraded in that compartment. Current Understanding of VLDL Secretion-The above proposals areconsistentwithwhat is presently known about VLDL assembly. ApoB is co-translationally inserted into the ER of the and containsa signal peptide that iscleaved in the lumen ER (Davis, 1991). Co-translationalinsertion (Chuck et al., 1990; Pease et al., 1991; Borkn et al., 1992) may involve pause transfer sequences (Chuck and Lingappa, 1992). The apoB is thought to interact with the luminal surface of the ER bilayer where triacylglycerol and phospholipid associate with theapoB and form VLDL particles, which bud into the lumenof the ER (Borkn et al., 1991). Recent studies implicate a luminal ER triacylglycerol transfer protein in the assembly of lipids into VLDL (Wetterau et al., 1992). Adefect in this transferactivity in CD hepatocytes seems unlikely since the VLDL particles in the ER have the same size and lipid composition as previously reported for apoB-containing particles in the ER lumenof normal liver cells (Rusiiiol et al., 1993). There ispublished evidence for degradation of apoB by cytosolic and ER luminal proteases. During theformation of VLDL particles, a large percentage of apoB is exposed to thecytosolic

Phosphatidylcholine and

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surface of the ER and is degraded rather than secreted (Davis et al., 1990;Thrift et al., 1992;Wilkinson et al., 1992).The apoB that is exposed on the cytosolic surface of the ER (Davis et al., 1990)is degraded by proteases that areinhibited by N-acetylleucyl-leucyl-norleucinal(Thrift et al., 1992). Our studies suggest that the action of such proteases on apoBis not affectedby choline deficiency. In addition to cytosolic proteases there is an abundance of evidence forluminal proteases in theER. Luminal ER proteins for example becauseof a missthat arenot assembled properly, ing subunit, are degraded in theER (Klausner and Sitia, 1990). Specific information about the nature of the ER-associated proteases is minimal (Klausner and Sitia, 1990).That theamount of apoB secreted is regulated by the luminal ER protease system has been suggested by studies with brefeldin A (Sato et al., 1990)and oleate (Dixon et al., 1991).More recent studies (Furukawa et al., 1992)confirm the existence of a luminal protease in the ER from Hep G2 cells that degrades apoB. In contrast, these workers did not find apoB proteolysis in theGolgi of Hep G2 cells as suggested by experiments with monensin nor in Golgi isolated from these cells (Furukawa et al., 1992).Assuming that these results are applicable to rat hepatocytes, the degradation of apoB that is enhanced in CD hepatocytes may, therefore, occur before the VLDL particles arrive in the Golgi. Acknowledgments-We are grateful to Susanne Lingrell and Roger Bradley for expert technical assistance and Dr. Jean Vance for helpful discussions and a critical reading of the manuscript.

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